Advances in the Sensing and Treatment of Wound Biofilms

Abstract Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor‐based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point‐of‐care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound‐induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.


Introduction
Antibiotic-resistant infections are threatening to become one of the major health crises in the world, with enormous socio-economic consequences.P athogenic bacteria are gaining resistance to modern, last-resort antibiotics at an alarming rate.I nt his context, microbial biofilms represent ap articularly challenging problem. Biofilm-associated infections lead to as evere economic loss [1] and over ah alf million deaths annually.T he cost of treating biofilms is estimated at around USD 94 billion annually. [2] In 2007, the Center for Disease Control reported that around 1.7 million hospital-acquired infections were due to biofilms. [3] Biofilms are assemblages of one or more types of microorganisms that can grow on many different surfaces,r anging from packaging materials to soft tissues.B acteria in biofilms show distinctive differences to their planktonic counterparts,n amely the production of extracellular polymeric substances (EPS), up-or downregulation of genes,a nd slower growth rates. [4] Ab iofilm, in nature,i st ypically made up of several microbial species attached to EPS that surrounds and protects cells.Atypical EPS matrix contains polysaccharides,p roteins,l ipids,a nd extracellular DNA( eDNA). [5] Moreover,biofilms resist host immune responses and are much less susceptible to antibiotics.T hey are involved in numerous subacute and chronic infections and can cause persistent infections through microbial accumulation in the EPS of the biofilm. [6] One of the acute problems associated with biofilms is their formation on wound surfaces.B acteria in this form impede the healing of 60 %o fc hronic wounds and 6% of acute wounds. [7] Anti-biofilm wound dressings represent as ignificant part of the market, with USD 570.1 million in 2019 and acompound annual growth rate (CAGR) of 9.1 %from 2020 to 2027, reflecting its importance.A dditionally,i ncreasing antibiotic resistance and chronic wound infection rates are expected to increase demand for early diagnosis and treatment of chronic wound biofilms.
This review focuses on recent advances and current stateof-the-art in the field of wound biofilm detection and eradication. It focuses on novel, sensor-based approaches that show promise for early,a ccurate detection of biofilm formation on wound sites and that can be translated from laboratory applications to point-of-care settings.T hen, we discuss technologies inspired by new materials that show potential for efficient biofilm eradication. Theu ltrasoundinduced microbubbles and nanomaterials have the potential to both penetrate the biofilm and simultaneously carry active antimicrobials.N umerous detailed reviews describe the biology of biofilms and methods for their characterization and eradication thoroughly.However,only alimited number of reviews focus on detecting and eradicating biofilms that form on wound sites.T he focus of these reviews has been on small subsets of existing methods for eradication and diagnosis. [8] Our review focuses specifically on wound biofilms and their early detection through new technologies that can be used in point-of-care applications,a nd simultaneously,o n new technologies that can be used for efficient eradication of wound biofilms with the minimal patient inconvenience.With this review,w eh ope to draw the attention of scientists with expertise in sensor technology,d rug delivery,a nd micro-Wound biofilms represent ap articularly challenging problem in modern medicine.T hey are increasingly antibiotic resistant and can prevent the healing of chronic wounds.H owever,c urrent treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review,wepresent new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity,a nd biocompatibility.W ebriefly discuss existing methods for biofilm detection and focus on novel, sensor-based approaches that showpromise for early,accurate detection of biofilm formation on wound sites and that can be translated to point-ofcare settings.W ethen discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound-induced microbubblesand nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods. biology to the pressing issue of wound biofilms and, through discussing exciting new technologies and chemical avenues,to stimulate further work in these areas.

Chronic Wound Biofilms
All open wounds contain microorganisms from endogenous or exogenous sources because the innate protective covering of the skin is compromised. [9] Aw ound biofilm is formed when certain microorganisms,m ostly pathogenic bacteria, adhere to the wound surface.B iofilms are formed by at hree-dimensional (3D) matrix that provides protection and cohesion for bacteria growing in wound sites. [10] The hostsi mmune system clears microbes under normal physiological conditions,w hich leads to an ormal wound healing process.Microbes are more likely to attach to wound surfaces when the immune system is dysfunctional or dysregulated. With the secretion of EPS,abiofilm is formed. [10a] Macrophages and neutrophils are key players in protecting the bodysi nnate immune system from pathogens; [11] they are vital components of ah ighly effective defense mechanism against planktonic bacteria. However,bacteria trapped within biofilm structures are effectively protected from phagocytic attack by neutrophils and macrophages. [12] Ab iofilm also produces leukocyte-inactivating substances,resulting in acondition known as "frustrated phagocytosis". [13] Not all bacterial species found in wounds cause infection. [14] Detecting wound infections can be challenging, especially in wounds with pathogenic biofilms.T herefore,a n approach to detect specific bacterial species on the wound site is vital. Different in vitro and in vivo studies have investigated biofilms on wounds.B iofilms can be divided into two main categories:c ommensal and pathogenic.P athogenic biofilms differ from commensal ones in several ways,i ncluding the presence of more upregulated genes.This promotes excessive levels of degradation enzymes (matrix metalloproteinases (MMPs)), the growth of EPS,a nd increased microbial proliferation and dissemination (which characterize chronic wounds). [10a, 15] Theestablishment of pathogenic biofilms leads to an upregulation of the immune responses,l eading to chronic inflammation. Figure 1p rovides ac omparison between an acute healing wound and ac hronic nonhealing wound.
Thehostsimmune system controls or destroys pathogenic bacteria early in the development of chronic wounds.H owever,abiofilm matrix can develop when bacteria successfully attach to the wound surface. [16] Theb iofilm phenotype of bacteria is induced by complex intracellular signals that alter planktonic bacteria gene expression profiles.B yt his time, bacteria have formed microcolonies which become biofilms as they work in conjunction with quorum-sensing systems and bacterial small RNA-based systems.Abacterial small RNA (sRNA) is ar egulatory RNAt hat is approximately 40-500 nucleotides long. [17] There are two general mechanisms by which sRNAs form biofilms.T he first involves sRNAs interacting with other RNAs,a nd the second involves proteins binding to sRNAs.B ya ltering the accessibility of ribosome binding sites (RBS) or enhancing degradation by ribonuclease,base-pairing between sRNAs and their targeted mRNAs can alter mRNAt ranslation and stability and thus influence gene expression. [18] Themicrocolonies gradually mature into amatrix of EPS and inflammatory materials from the wound in ac ellular matrix surrounded by normal skin cells.The biofilm volume is dominated by EPS,w hich accounts for 90 %, while bacteria account for 10 %. [19] Biofilms eventually form stalk-like structures.T hen, the structure is dispersed and detached, Hubert Girault is Professor of Chemistry at the Ecole Polytechnique Federale de Lausanne (Switzerland). His research activities range from physical electrochemistry for the study of polarised interfaces to bioelectroanalytical chemistry with afocus on bacteria. He is also developing novel redox flow batteries and hydrogen production processes.
forming new colonies or incorporating existing biofilms in ac hronic wound. [8c] Wound biofilm formation is ad ynamic process,and it has been found that bacteria can form amature biofilm on aw ound bed within 24 h. [20] When the biofilm is well established, it will not be destroyed by the host immune system spontaneously.H owever, the formed biofilm can delay wound healing, require tissue amputation, and ultimately lead to sepsis and death. [2] Studies have shown that chronic wounds contain ap athogenic biofilm more than 60 %o ft he time, whereas only 6% of acute wounds contain biofilms. [7a] Ac hronic wound biofilm comprises multiple bacteria groups,g enerally with different genotypes,a nd which are further held together by EPS. [21] For instance,c hronic venous leg ulcers contain S. aureus (93.5 % of the investigated ulcers), Enterococcus faecalis (71.7 %), P. aeruginosa (52.2 %), coagulase-negative Staphylococci (45.7 %), Proteus species (41.3 %), and anaerobic bacteria (39.1 %). [22] Table 1s hows the participation of various bacterial groups presenting in chronic wound biofilms that cause associated diseases for the host body.I nm ost studies, S. aureus is present in chronic wounds as the most prevalent biofilm bacteria. [23] Asignificant portion of chronic wounds is colonized with P. aeruginosa in deep dermal tissues,a nd awound infected with P. aeruginosa causes significant wound area increases compared to aw ound not infected by the bacterium. [24] It is possible to delay or even prevent wound healing by introducing P. aeruginosa into the wound bed. [25] Consequently,preventing biofilm formation and then detecting aformed biofilm at the wound, especially at an early stage of formation, is vitally important.

Wound Biofilm Detection
Although some clinical symptoms of the formation of pathogenic biofilms,such as yellow exudate,pale wound bed, necrotic tissue,a nd clear tissue fluid, are distinguishable,t he bacterial aggregates in wound biofilms are not discernable by the unaided eye as they usually measure less than 100 mmi n size. [31] Therefore,different methods have been developed to detect microorganisms on the wound site ( Figure 2). Traditionally,b iofilm detection techniques are categorized into microbiology assays,m olecular assays,a nd imaging assays. While not the focus of this review,t hese methods are briefly described below: Microbiology culture-based techniques have been used to detect viable culturable bacteria in the wound. However, diagnosis of chronic wound infection using this method lacks accuracy and was demonstrated to considerably underesti-  mate the existence of bacteria in the wound. [31,32] Thebiofilm is sampled directly from the wound site through as urgical procedure or sonication. [33] Inaccuracies in the detection may arise from the fact that many bacteria do not form colonies in normal culturing conditions (slow-growing variants,d ormant persister cells,a nd anaerobic bacteria). [34] Fort hese reasons, some studies have been misconstrued and have underestimated bacterial populations in wounds. [35] DNA-and RNA-based analyses of biofilm bacterial species are more precise than culturing methods and can detect unculturable cells and samples with mixed species (anaerobic and aerobic bacteria). [36] Molecular sequencing methods such as denaturing gradient gel electrophoresis (DGGE) have also been used in biofilm detection. As part of the investigation of chronic wound biofilms,this method was used in conjunction with 16S rRNA-PCR to characterize biofilms that are complex and multispecies in nature. [37] Other sequencing methods for the detection of chronic wound biofilms include partial ribosomal amplification and pyrosequencing (PRAPS), whole ribosomal amplification, cloning Sanger sequencing (FRACS), partisan ribosomal amplification, denaturing gradient gel electrophoresis Sanger sequencing (PRADS), and PNA-FISH. [22,38] Nevertheless,some limitations are associated with DNAbased technologies.T he three primary concerns are the possibility of DNAc ontamination from the clinical environment, no cell viability information, and not distinguishing between biofilms and planktonic microbes.Combined DNAbased and messenger RNAanalyses,however, may be able to recognize the organismsgenotype. [33] Some bacterial species,  such as mycobacterium, cannot be detected by 16S rRNA sequencing. [39] Moreover,sequencing results may correspond to microorganisms not documented in existing databases, rendering the technique powerless to identify them. [40] Molecular assays,ingeneral, are accessible and cost-effective. By using these methods,wound biofilms can be analyzed, and the bacterial populations can be detected and quantified rapidly. [41] Wound biofilms can also be detected using microscopy.T o identify biofilms in wounds,c onfocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), which examine biofilms by imaging their polysaccharide matrix and bacterial morphologies,h ave also been used. [42] Through the use of am icroscope,i nterconnected fibrous networks of EPS are revealed as well as crosslinking between bacterial cells within the polymeric matrix, enzyme activity, species,a nd viability of microcolonies. [42][43] Electrochemical bioimaging is also an emerging readout for recording metabolic activity on biofilm that records biofilmss urface activity. [44] Nevertheless,t hese imaging techniques may require time-consuming staining of the biofilm components (bacteria or EPS), special equipment, and highly trained staff members.B ecause of this,t hey are not applicable in clinical settings. [8c] Biofilms can also be detected by microscopic techniques alone,w hich results in frequent false-negative results because bacteria tend to be patchy and dispersed rather than confluent, particularly on biotic surfaces. [45] While the methods described above have offered advances in biofilm detection, there is still an eed for precise and reproducible biofilm detection on the wound site.T his can be achieved through biosensor technologies that exploit specific characteristics of biofilm. In Table 2w ec ompare different traditional and sensor-based methods for wound biofilm detection.
Biofilms on wound beds produce quantifiable biomarkers and lend themselves as indicators for aw oundsn ormal or ·A ntigens of the pathogen and the host cannot be distinguished. ·P lanktonic bacteria can also possess DNA derived from extracellular bacteria. [38b] 16S rRNA PCR ·C an identify multiple VBNC states. ·R apid (< 24 h). ·C an identify pathogens that are difficult to culture.
·G enetic material may also be contributedb ynonviable bacteria. ·T he sensitivity of the pathogen to antibiotics cannot be determined. ·Abacterial biofilm is difficult to differentiate from planktonic bacteria. ·D NA derived from extracellularbacteria is also found in planktonic bacteria. [35] FRACS;P RADS;P RAPS ·C apable of identifyingm ultiple types of VBNC. ·P athogen identification within 24 hours by targeting the 16S rRNA gene.
·G enetic material may also be contributedb ynonviable bacteria. ·T he pathogen'ss ensitivity to antibiotics cannot be determined. [47] Imaging assays CLSM and SEM ·T his method is most reliable for detecting biofilms on biopsy tissues. ·S urface biofilms can be accurately diagnosedusing non-invasive methods.
·D ifficult to perform routinely in clinical practice. [48] Sensors Bacterial species and wound biofilm EPS sensors ·R apid method to detect bacteria and biofilm. ·S pecific for ap articular species of bacteria.
·U nable to detect the type of bacteria and can be influenced by physical and environmental conditions. ·U sually unable to detect the bacterial infection at early stages. [50] Enzymes sensors ·R apid detection with high sensitivity (< 1h).
·U sually unable to discriminate active/inactive states of the enzyme. ·U nable to monitor dynamicp rocesses. [51] pathological state. [52] Biomarkers are classified into predictive,d iagnostic,a nd indicative.P redictive biomarkers are used to report the likelihood of benefit from treatment. They can be ap owerful tool in designing personalized treatment options according to the needs of specific patient populations. Diagnostic biomarkers can be employed to recognize with high precision single or multiple pathogenic bacteria present in infection sites and facilitate the choice of potential treatment, therefore improving clinical outcomes.Anindicative biomarker can be used to assess disease progression or response to therapy in real-time. [52] Table 3presents different biomarkers in wound biofilms or the infected host body. Apart from specific biomarkers,p H, transepidermal water loss from peri-wound skin, nutritional factors (e.g.,z inc, glutamine,a nd vitamins), reactive oxygen species,a nd temperature are other indicators of wound biofilm establishment. [53] There is an increasing body of work on developing sensors that show potential for the precise detection of different wound biofilm-associated markers and the provision of reliable information about wound status.T his field of study has progressed vigorously,and examples abound. Thefollowing classifications illustrate recently developed sensors for the detection of different wound biofilm-associated markers.

Specific Bacterial Species and Wound Biofilm EPS Sensors
Thef irst category of studies used in wound biofilms indicates whole bacteria in the wound site rather than specific biomarkers produced by them.
Thet et al. [14] describe as ensor for detecting skin pathogenic bacteria at an early stage during the formation of biofilms.Afluorescent dye is encapsulated within liposomes. Toxins from infecting bacteria cause liposomes to burst, trigger dye activation, and cause the sensor color to turn from yellow to green, demonstrating infection ( Figure 3a). The biofilms were swabbed and mixed in the liposome suspension, and the colorimetric response characterized the population density of pathogens in the biofilm model. Ngernpimai et al. [49b] synthesized as ensor made of poly(oxanorborneneimide) (PONI) polymers for rapid identification of biofilms. Thei nteractions between these polymers with the biofilm matrix caused differential fluorescent profile changes,p roviding as pecies-based characterization of the biofilm (Figure 3b). PONI was incorporated into two elements,t he recognition element based on cations and one sensitive transducer.S elective binding is an important characteristic of the cationic recognition elements.F rom as ingle-well measurement, each of the PONI polymers provided four characteristic excitation/emission peaks and two effective FRET signals.T his sensor shows the ability to discriminate between bacterial species,t hree of which are pathogenic clinical isolates: P. aeruginosa, Bacillus subtilis (B. subtilis), E. coli, Enterobacter cloacae complex, and methicillin-resistant S. aureus.
Jones et al. [66] focused on the visualization of polymicrobial populations based on porphyrin fluorescence.I nt his study,32bacteria and 4yeast species were plated on agar and tested for red fluorescence.According to the findings,28of32 bacteria studied and one in four yeast species and monomicrobial biofilms produce red fluorescence when they produce porphyrin ( Figure 4). Thus,p orphyrin production is [64] Pyocyanina nd uric acid Wound fluid Potential indicators of infection and wound healing progress. [65] ap rimary biological source of red fluorescence,i maged by fluorescence imaging upon excitation with violet light. These sensors offer precise detection for pathogenic bacteria and particularly for chronic nonhealing infected wounds.

Sensors to Indicate Environmental Parameters (e.g.,pHand Temperature)
Different studies have demonstrated that the pH profiles of chronic, acute,a nd healthy skin differ significantly. [67] Chronic wounds have an alkaline pH, whereas healthy skin has aslightly acidic pH ( Figure 5). [68] When an infected wound has ahigh pH, it inhibits microorganism growth and invasion. This pH change affects matrix metalloproteinases,f ibroblast activity,k eratinocyte proliferation, microbial proliferation, and immunologic reactions in the wound. [69] Since pH changes can modulate biological and biochemical processes in wound healing, various pH sensors have been developed to monitor wound status.D ifferent methods are available to measure wound pH, including optical and electrochemical approaches. [70] Thew avelengths of absorption and emission of apH-sensitive dye used in optical methods are in the visible range. [71] Electrochemical pH sensors mainly measure the pHsensitive potential, current, or impedance. [71] In as tudy by Rahimi et al., [72] ap otentiometric pH sensor was developed for wound infection detection. Thewound model was infected with Gram-positive cocci, Staphylococcus epidermidis (S. epidermidis). In addition to their high sensitivity,t he sensors are optically transparent, so the tissue beneath the sensor can be imaged. pH changes were monitored successfully by the sensor in simulated in vitro wounds.Alaser ablation process was used to produce transparent and flexible electrical substrates from commercial indium tin oxide (ITO) films (Figure 6a,b).  Under violet light illumination, 28 of 32 bacterial species emitted red fluorescence, while the four known non-porphyrin-producing species did not produce the signal. Red fluorescence was observed from porphyrin-producing bacterial species grown in abiofilm. [66] Wound inflammation and infection are closely linked to wound temperature. [73] It is possible to predict infection by observing abnormal wound temperature changes before any secondary symptoms appear. [74] As such, temperature is an important indicator of wound biofilms.With the emergence of flexible electronics,different innovative wound dressings with integrated temperature sensors have been designed to provide precise real-time information about the wound environment. [15a] Thel ocal temperature of wounds lies between 33 8 8Ca nd 41 8 8C ( Figure 7). [75] Wound repair is hampered when the temperature of the wound is less than 33 8 8C. [76] Local wound temperatures may exceed 37 8 8Cdue to local congestion and inflammation;h owever, sudden increases in wound temperatures are an indication of infection. [77] Furthermore,h igher local wound temperatures were associated with ag reater risk for Gram-positive infection. Fori nstance,Z hang et al. [78] showed that wounds with P. aeruginosa and K. pneumoniae were about 37.5-38 8 8C, while wounds with S. aureus were 38.5 8 8C.
Pang et al. [79] developed adouble-layer structure including polydimethylsiloxane-encapsulated flexible electronics integrated with at emperature sensor and ultraviolet (UV) lightemitting diodes (upper layer) and aU V-responsive antibacterial hydrogel (lower layer). Through the use of an integrated temperature sensor,w ound temperatures were continuously monitored and transmitted in real-time to as martphone via Bluetooth communication. UV-LEDs were used to control the release of antibiotics in situ (Figure 8a,b). Tissue compatibility,high sensitivity,and durability were all device features. Also,the results in vivo in amodel of infection in Bama mini pigs showed that the device could detect infection at an early stage and provide an indication of treatment.
Recently,t he integration of flexible sensors in abandage or so-called innovative wound dressing has attracted much attention. Smart bandages have been engineered to provide precise real-time information about wound conditions,including pH, temperature,m oisture,a nd oxygen, to personalize individual patients clinical treatment. [15a] Smart bandages have been reviewed in detail by Derakhshandeh et al. [80] Although sensor-integrated wound dressings can provide information about the wound environment, some detectable symptoms may stem from other reactions in the body,leading to an incorrect diagnosis.M ultiple sensors have been incorporated simultaneously in wound dressings to provide

Angewandte Chemie
Reviews orthogonal information from various wound parameters to counteract this problem. Fori nstance,M ostafalu et al. [81] designed an automated, flexible wound dressing incorporating potentiometric pH and temperature sensors to provide information on both bacterial infection type and inflammation level. In addition, at hermo-responsive drug carrier was deployed to provide ac ontrollable drug release system in response to temperature changes.T he integration of more than one sensor can give even more accurate information about the wound environment and wound biofilm formation status.H owever, engineering and integration of am ultitude of sensors are still too challenging to be economically viable at present.

Enzyme Sensors
Va rious studies have demonstrated the role of enzymes, precisely that of matrix metalloproteinases (MMPs), in the healing process of chronic wounds.M etalloproteinases are ag roup of endopeptidases classified according to their primary catalytic substrate:c ollagenases,g elatinases,m atrilysins,stromelysins,and membrane-type MMPs. [82] There are more than 20 structurally related zinc-dependent endopeptidases in the MMP family. [83] Some of these enzymes are excessively released and activated in chronic cutaneous wounds,w hich lead to long-term healing.E xtracellular material can be digested by MMPs,a llowing an influx of reparative keratinocytes,fibroblasts,and endothelial cells.For awound to heal, MMPs should be at an appropriate level and in the correct location for ap recise duration of time.T IMPs (tissue inhibitors of metalloproteinases)a re generally produced in excessive amounts during wound healing,a nd their levels are concomitantly downregulated, resulting in decreased production of MMPs.I nr esponse,t he balance between MMP and TIMP is altered. [84] MMPs are chronically elevated in chronic wounds,a nd TIMPS are reduced, such that aberrations in their ratios can serve as potential diagnostic biomarkers to detect wound biofilms.Intwo different recent review studies by Kirchhain et al. [85] and Lei et al., [86] different approaches were reviewed to quantify the level of MMPs as am arker of many pathological conditions,together with their strengths and weaknesses.T able 4p rovides asummary of different technologies for MMP detection.

Wound Biofilm Therapy
Wound biofilms can pose severe challenges for therapeutic treatment. Chronic or hard-to-heal wounds are becoming a" silent epidemic,"a ffecting up to 2% of the population in the middle-to high-income countries. [92] Prevalence can reach between 3and 5% in the senior population as the wound healing processes are slowing down. [93] With an estimated annual cost of USD 20 billion in the United States alone,chronic wound management also has am assive economic impact, burdening patients and healthcare systems. [94] There are few treatment options for clinicians against biofilm infections that efficiently disrupt biofilms without being toxic to the host tissue.H ence,a lternative treatment is urgently needed.
Mature biofilms are intractable to treatment. Their resistance to antimicrobial agents,d isinfectants,a nd the hostsi mmune system can be up to 1000 times higher. [95] Thec urrent standard of care (SOC) for wound biofilm includes frequent biofilm eradication through conventional treatments.T he biofilm eradication comprises physical and chemical debridement, and application of topical and systemic antimicrobials and dressings (Figure 9). [96] Here,w e review recent developments on the novel treatments based on ultrasonic debridement and nanotechnology,which are more specific and more effective against chronic wound biofilms.
Chronic wound infections caused by biofilms are minimally treatable with topical and systemic antibiotics currently available. [97] EPS acts as ap rotective barrier inhibiting diffusion and penetration of antimicrobials,r esulting in slower or incomplete penetration. [98] It is also possible that antimicrobial agents react chemically with the extracellular components of the biofilm, rendering them ineffective,o r they can stick to the anionic polysaccharides without reaching the target bacterium. [99] Agrowing movement is under way to find non-antibiotic alternatives to antibiotics due to the emergence of drug-resistant bacterial strains. [100]

Biofilm Eradication
Mechanical debridement or sharp debridement of wounds can interrupt biofilm growth and cause faster wound healing. [101] However,t his process can cause damage to healthy skin tissue and the release of bacteria in the area of the wound, which can cause secondary infections.Asignificant disadvantage of mechanical debridement is that it is often not enough to eradicate all bacteria. Thedevelopment of resistant biofilms after debridement indicates impaired treatment efficacya nd delayed wound healing. [102] Another major disadvantage to sharp debridement is that it causes pain and discomfort to the patient, decreasing compliance and treatment effectiveness.
EPS from biofilms can also be chemically degraded. Manganese and iron are essential for the metabolism of bacteria and the bacterial cell wall structure.C alcium and magnesium crosslink polysaccharides within EPS. [103] The competition for these ions and their removal (chelation) will affect biofilm formation. There is aw ide range of metal [91] Figure 9. Methods for therapy of chronic wound biofilm.

Angewandte Chemie
Reviews chelating agents.H owever,b iocompatibility and safety considerations limit their application to polyanions,s uch as phosphates and citrates,and ethylenediamine tetraacetic acid (EDTA). One of the most common anti-biofilm agents is EDTA, which has the best calcium and magnesium ion affinity. An umber of extracellular proteins,s uch as structural proteins and enzymes,m ay even be present at higher concentrations than polysaccharides within the biofilm matrix. By connecting cells to the extracellular matrix, structural proteins stabilize biofilm architecture. [104] Polysaccharides (e.g., dispersin B), matrix proteins (e.g.,p roteases), and eDNA( e.g.,D Nases) are all degraded by enzymes. [104] Recent research used ah ybrid method of combining proteases,antiseptics,and EDTAtoinhibit the growth of biofilms of S. aureus and P. aeruginosa in chronic wounds. [105] The destabilizing effect of EDTAc omes from chelating cations and blocking the activity of matrix metalloproteases. [106] In addition, it potentiates the action of antiseptics when combined with enzymes,m aking them more effective when administered at lower doses. [105] Lefebvre et al. [105] reported the broad-spectrum effect of several bacterial strains using non-specific enzymes.T he combination treatment with synergistic effects significantly reduced bacterial viability.H owever,t here is an eed for efficient methods of delivering the molecules into the biofilm. [105]

Ultrasound Debridement
Ultrasound debridement has been reported to treat wounds infected with biofilms and potentiate antibiotics while promoting the healing process. [8b, 107] Researchers have observed bacterial counts and wound size decreasing after ultrasound debridement for diabetic foot ulcers and lowerextremity wounds. [107d, 108] Thea dvantages of ultrasound debridement are that it is non-invasive,l ess painful, and less invasive than sharp debridement. [109] There have been prom-ising developments in the use of ultrasound-induced micro/ nanobubbles in ultrasound-mediated therapy.A ctivating nanobubbles and microbubbles with acoustic waves delivers drugs and mechanically disrupts biofilms at the same time ( Figure 10). [8b] Gas-filled bubbles may have various architectures but commonly include as hell comprising polymer, surfactant, protein, or phospholipid encapsulating ag aseous core. [110] Theacoustic response to ultrasound stimulation relies on the mechanical stability of the bubbles,w hich can be controlled by their composition and size. [111] So the size of bubbles plays ak ey role in acoustic properties,d rug loading capacity,longevity,and ultimately,the safety of bubbles in in vivo application. [112] During long-term storage,m icrobubbles generally become bigger,with adiameter distribution ranging from 1t o1 0mm. [113] Nanobubbles,o nt he other hand, vary from 50 nm to 1 mmi ns ize,w ith as table range of 50 nm to 300 nm. [114] Thes ize of nanobubbles is determined by the amount of gas dissolved in the solution. Low concentrations of gas result in smaller nanobubbles,whereas high concentrations produce larger ones. [115] Microbubble shells commonly contain phospholipids.Aninterface between hydrophilic and hydrophobic lipid molecules forms am onolayer,a llowing the hydrophilic tails to expose themselves to aqueous environments.I n contrast, the hydrophobic heads remain in gaseous environments to stabilize the gas core.T he permeability of the microbubble shell depends on the length of the acyl chain of al ipid. [116] Lipids with longer hydrophobic chains are more cohesive and pack well, [117] thus preventing gas entry into microbubble shells during storage and increasing stability throughout the therapeutic period. [118] Surfactants can be added to microbubbles and are especially beneficial for biomedical applications. [119] Recent research has focused on adapting composition, size,a nd fabrication process,a nd optimizing biocompatibility. [8b, 110b,111c, 112, 120] Target applications have mainly been directed towards cancer therapies;however,research on their application for biofilm treatment is increasing.Microbubbles exert biophysical effects by developing localized pushing and pulling forces on cell membranes when subjected to systematic expansion and contraction. [121] Alternatively,fluid can be continuously streamed by oscillating microbubbles (also called cavitation microstreaming). When microbubbles oscillate,d ivergent (i.e., radial) flows result. When shear stress is increased over nearby cells on as urface that interacts differently with blood flow (e.g.,atarget tissue), transmembrane pores can form. [113] Thestreaming flow field can also be exploited to accelerate the shedding of constituents from amicrobubble,such as therapeutic compounds. [122] Using this technique,t herapeutic material can be deposited (or "printed") over cell membranes. [123] Using ultrasound-targeted microbubble destruction, He et al. [124] (Figure 11) demonstrated as ignificant enhancement of the effect of vancomycin in killing S. epidermidis RP62A. Biofilms were treated in vitro for 12 hwith vancomycin in combination with ultrasound microbubbles.F ollowing ultrasound exposure, biofilms were cultured for another 12 hours and were found to contain many micropores,a nd both the film density and viable count of S. epidermidis were significantly lower than the controls (Figure 11). [124] Another study by Hu et al. [125] reported that the biofilm produced by ac linical strain of S. epidermidis was more sensitive to ultrasound microbubble and vancomycin treatment. Several treated cells showed apparent cell wall damage, and visible cell fragments were observed around damaged cells. ( Figure 12).

Nanotechnology
Forchronic wounds,itwould be ideal to use asystem that is effective against biofilms but does not necessarily involve mechanical disruption. Nanoparticles (NPs) can prevent wound infections caused by biofilms in an ew and promising way.N anoparticle-based approaches have been developing over the last decade to design nanoparticles with specific chemical and physical properties that prevent and inhibit biofilm infections. [126] Nanoparticle-based strategies have recently been proposed as potential antimicrobial therapeu-tics for wounds infected by biofilms.Aschematic illustrating how different nanoparticle-based systems interact with biofilms can be seen in Figure 13. Further details about the design and synthesis of these antimicrobial nanoparticles can be found in recently published review articles. [127]

Metal/Metal Oxide Based nanoparticles
Several types of nanoparticles have been shown to possess antimicrobial properties against wound biofilms,i ncluding silver,c opper, gold, titanium, and zinc. Nanoparticles based on silver have particularly attracted attention. In order to exert their antimicrobial action, silver ions need to interact with sulfhydryl groups. [128] Therefore,t hey disrupt the integrity of bacterial membranes,r espiratory chains,a nd enzyme activities. [129] As ar esult, silver ions compromise intermolecular forces and destabilize the biofilm matrix. [130] Wound proteins and other cellular components can readily sequester silver ions,which reduces their bioavailability and antimicrobial effectiveness. [131] In ar ecent study,P ermana et al. [132] reported the selective delivery of silver nanoparticles utilizing dissolving microneedles to improve biofilm skin infection treatment (for more on microneedles see Section 4.2.6). Silver nanoparticles synthesized using green tea extract have been examined as antibiofilm agents against S. aureus and P. aeruginosa biofilms.T he release of silver nanoparticles from microparticles in S. aureus and P. aeruginosa increased ninefold, demonstrating the selectivity of this approach. It has been shown that dissolving microneedles containing silver nanoparticles improved dermatokinetic profiles more than dissolving microneedles without microparticles.Furthermore, Figure 11. Ultrasounddebridement: The LIVE/DEADv iability stain (SYTO9/PI)s hows viable cells green and dead cells red in confocal laser scanning microscopy(CLSM) images. Adapted with permission from ref. [124].C opyright 2011 AmericanS ociety for Microbiology. Adapted with permissionf rom ref. [125].C opyright2 018 Nature.
100 %o ft he bacterial bioburden was eradicated following administration of this system for 60 hours in an ex vivo biofilm model in rat skin. This study confirmed that silver nanoparticles could be loaded into responsive microparticles for improved antibiofilm performance when delivered with dissolving microneedles. [132] An anti-biofilm dressing using silver nanoparticles was described in another recent study by Katas et al. [133] Using them may be an effective strategy for reducing wound exacerbations.Silver nanoparticles were produced on amushroom substrate using chitosan as the stabilizing agent. Gramnegative bacteria responded more sensitively to nanoparticles with high antibacterial and anti-biofilm activities.G elatin hydrogels were formulated from genipin-crosslinked silver nanoparticles for wound dressings.The antibacterial and antibiofilm effect of the hydrogels against S. aureus, B. subtilis, P. aeruginosa,and E. coli effectively inhibited the growth of the selected bacteria with the minimum inhibitory concentration of 63 mgmL À1 .Ananoparticle-loaded gelatin hydrogel crosslinked with genipin appears to be an effective antimicrobial wound dressing to combat biofilms involved in wound infections.
Other nanoparticles have also been proposed in recent studies as antibiofilm reagents.F or example,M irzahosseinipoura et al. [134] examined the photodynamic effect of curcumin silica nanoparticles and free curcumin on planktonic and biofilm forms of P. aeruginosa and S. aureus. Curcumin-silica nanoparticles were found to decrease bacterial biofilm production and number in planktonic conditions when used as photosensitizers.A dditionally,c urcumin-silica nanoparticles did not have any significant cytotoxic effect on normal human fibroblasts and showed wound healing properties in an in vitro scratch test. Thus,curcumin-silica nanoparticles could be used to perform antimicrobial photodynamic therapy to treat chronic wound infections caused by multidrug-resistant bacteria (Figure 14 a).
Qiu et al. [135] investigated the antibacterial properties of gold nanoparticles.A P-AuNPs (antibacterial photodynamic gold nanoparticles) are synthesized by coupling ap hotodynamic peptide with poly(ethylene glycol) (PEG)-stabilized AuNPs.F urthermore,i na ddition to aqueous and light stability and ar emarkable antibacterial effect on S. aureus and E. coli upon light irradiation, the AP-AuNPs demonstrated the significant generation of reactive oxygen species (ROS). Additionally,t he synthesized nanocomposites inhibited bacterial growth in vitro and prevented biofilm formation. In S. aureus infections,p hotodynamic antibacterial therapy accelerated wound healing,similar to Staphylococcal skin infections.The combination of abactericidal peptide,the photodynamic effect of ap hotosensitizer,a nd multivalent clustering on AuNPs results in amaximal antibacterial effect (Figure 14 b). [135] Metal oxides are believed to have antibacterial properties, so the development of metal oxide nanoparticles has garnered considerable interest. These oxides include zinc oxide (ZnO), magnesium oxide (MgO), iron oxide,a luminum oxide,a nd copper oxide (CuO). One of the most widely used nanoparticles is ZnO nanoparticles. [127a] An improvement in wound healing and areduced bacterial growth rate were observed in ar at wound infection model when ZnO nanoparticles were combined with chitin dressing. [136] In ac omparative study, ZnO and CuO nanoparticles were investigated for their respective antimicrobial properties.T he antibacterial ability of these products has been proven against Gram-positive S. aureus and B. subtilis and Gram-negative E. coli and P. aeruginosa bacteria. [137] Additionally,ZnO nanoparticles were found to have antimicrobial properties against biofilms formed by P. aeruginosa [138] and S. aureus. [139] Though it is unclear how ZnO nanoparticles work as an antibacterial agent, there has been speculation that hydrogen peroxide production [140] and damage to the cell membrane [141] may be responsible.I narecent study,M ahamuni-Badiger et al. [142] investigated ZnO NPs incorporated into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/polyethylene oxide (PEO) microfibers for antibacterial, antibiofilm, and wound dressing applications.Acomposite of ZnO NPs was prepared in chloroform using PHBV and PEO polymer (4:1) solutions. Thea ntibacterial and antibiofilm activities of the prepared microfibers revealed that ZnO NPs incorporated at different concentrations (1 %, 3%,a nd 5%)d isplayed different degrees of antibacterial activity against Gram-positive S. aureus and Gram-negative P. aeruginosa (Figure 15 a,b). Results indicated that PHBV-PEO ZnO (5 %) has am aximum percentage of biofilm inhibition (28.17 %) in S. aureus as compared with P. aeruginosa (24.51 %). ZnO content in the PHBV-PEO microfibers increased the percentage of biofilm inhibition. Following the incorporation of ZnO NPs,t he PHBV-PEO-ZnO microfibers showed excellent hemocompatibility and swelling characteristics.P HBV-PEO-ZnO microfibers prepared in this manner were nontoxic as determined by in vitro cytotoxicity assays.A na dditional focus of this study was the potential effect of PHBV-PEO-ZnO microfibers on antibacterial and antibiofilm mechanisms (Figure 15 c).

Polymeric Nanoparticles
In addition to offering high structural integrity,s torage stability,e ase of preparation and functionalization, and controlled release,p olymeric nanoparticles are excellent drug delivery vehicles. [143] Polymeric nanoparticles are made from biodegradable polymers or copolymers,i nw hich the drugs can be dissolved, entrapped, encapsulated, or attached. They can be composed of natural, synthetic, and semisynthetic polymers,l ike gelatin, albumin, alginate,c hitosan, poly(glycolic acid), copolymers,P LGA, and PCL poly alkylcyanoacrylate.T hey have several advantages,i ncluding controlled/sustained release,encapsulation degree,enhanced bioavailability,a nd biocompatibility.A sw ound dressings or delivery vectors,p olymeric nanoparticles (such as chitosan, alginate,c ellulose,a nd hyaluronic acid) exhibit good antibacterial and re-epithelializing abilities. [144] As ignificant role for hydrogels in wound healing has been demonstrated among different kinds of polymers.Specifically,wound dressings are utilized to optimize wound bed moisture content by either donating fluid, absorbing excess exudate,o rc ontrolling moisture loss. [145] Fori nstance,i nar ecent study,T avakolian et al. [146] developed carboxyl-modified cellulosic hydrogel as the base material for wound dressings.T he hydrogel was covalently linked to e-poly-L-lysine,anatural polyamide.The antibacterial efficacy of the hydrogel was tested against two model bacteria, S. aureus, and P. aeruginosa. Thel ive/dead assay was performed to measure the number of compromised bacteria. Results show that 99 %ofthe exposed bacteria were killed by the antibacterial hydrogel after three hours (Figure 16 a). Antibacterial hydrogels developed in this investigation are light, have high water-uptake capacity,a nd are biocompatible with mammalian cells.A ss uch, they are apotential candidate for wound dressings (Figure 16 b).
Abdalla et al. [147] developed ad ual-antibiotic dressing using gelatin hydrogels incorporated with nanosilver and lactoferrin (Ag-LTF). An in vitro analysis of the hydrogelsa nti-biofilm and antibacterial properties against S. aureus and P. aeruginosa,a swella si ts cytotoxicity,w as conducted. Primary wound healing gene expression of HDFs was also examined. Hydrogels formulated with AgNPs and LTFw ere released at adequate concentrations,d emonstrating antimicrobial activity against two bacteria strains.I n addition, the cellular functions were not significantly altered by the Ag-LTF-loaded hydrogel.
These findings indicate that Ag-LTF and the hydrogels do not affect cell viability,m igration rates,o rgene expression. Chitosan is one of the best hydrogel candidates because it is biodegradable,n on-toxic,a nd antimicrobial. Amajor benefit of chitosan is that it exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. [148] Due to its loose cationic nature and low solubility at ap Ha bove 6.5, practical applications are limited. In order to overcome this problem, the backbone chain of chitosan can be modified. Antimicrobial activity was also enhanced by modified chitosan. [149] Chitosan derivatives that are highlighted in the literature include quaternized chitosan, [150] carboxyalkylated chitosan, [150] sulfonated and sulfobenzoyl chitosan, [151] carbohydrate-branched chitosan, [152] and chitosanamino acid conjugates. [153] Another strategy to enhance the properties of chitosan is its combination with metals (oxide). [154] Several studies have used metal oxides (oxides) such as ZnO, TiO 2 ,and Ag NPs with chitosan. [155] Examples are mentioned in Table 5.

Liposome Nanoparticles
Nanocarriers protect drugs from degradation, enhance intracellular absorption, offer controlled and sustained delivery,a nd optimize the location of active compounds. [156] Liposomes are excellent carriers of hydrophilic molecules like hydrophilic peptides [157] and macromolecules. [158] Due to their lipid bilayer structure that mimics cell membranes and is biocompatible,l iposome nanoparticles are widely used as drug delivery vehicles. [158,159] Hydrophilic drugs can be encapsulated in their aqueous interior, and hydrophobic drugs can be contained in their phospholipid membranes. [127b,160] To prevent rejection by the reticulo-endothelial system [161] and allow penetration through water channels [162] in infectious biofilms,l iposomes for infection control should have diameters in the range of 100-200 nm. [163] There are four types of liposomes:c ationic,a nionic, zwitterionic, and fusogenic.Ingeneral, negatively charged bacteria are more likely to react with cationic liposomes. [164] Theu se of liposomes in conjunction with hydrogels could prevent rapid drug release. [165] Hemmingsen et al. [166] developed al iposomes-in-chitosan hydrogel to boost the effectiveness of chlorhexidine for eradicating biofilms in vitro. Electrostatic interactions between negatively charged phospholipids and the positively charged amino groups of chitosan cause chitosan to coat the surface of the negatively charged liposomes. [167] Chitosan is an excellent biopolymer for coating liposomes,asit increases the stability of liposomes and prevents leakage.I na ddition to increase efficacy,t he chitosan coating helps minimize drug release in undesirable locations.I tp romotes the cellular uptake of the liposomes by cells due to its positive charge. [167b] Figure 17 illustrates the synthesis (Figure 17 a) and effect of chlorhexidineliposome-in-hydrogel against biofilms (Figure 17 b). In lipopolysaccharide (LPS)-induced macrophages,c hlorhexidine-liposomes-in-hydrogels ignificantly inhibited nitric oxide (NO) production and reduced the adherent bacterial cells in biofilm by 64.2 %-98.1 %. Chlorhexidinesa ntimicrobial and anti-inflammatory effects were improved by chitosan hydrogels (Figure 17 c).

Carbon-Based Nanoparticles
Carbon nanomaterials are highly biocompatible and exhibit strong antibacterial properties. [168] It is possible to use carbon nanomaterial biomolecules alone or in combination with other materials as antibacterial agents. [169] Carbon dots (CDs) are anew type of nanomaterial that have attracted considerable attention because of their unique properties, including optical properties,g ood water solubility,l ow toxicity,b iocompatibility,a nd cell permeability. [170] As ar esult of their exceptional chemical and photoelectric properties,C Ds are great candidates for antibacterial theranostic applications. [171] Recently,L ie tal. [172] prepared CDs with gentamicin on ammonium citrate through thermal decomposition. Based on the in vivo wound healing models conducted on the backs of rats infected with S. aureus,t he CD-hydrogel showed better skin healing capabilities than the commercially available hydrogels and demonstrated good biocompatibility.  Curettage was used to scrape away the underlying film and manage the pathophysiology gently. [192] Severely contaminated wounds Cold atmospheric plasma treatments. Argon-based Maxium electrosurgery unit with Maxium beamer and beam electrode (Gebrüder Martin GmbH + Co.K.G.) [193] Venous leg ulcer Continual debridement and negative-pressure wound therapy and split-thickness graft [192] Lower limb traumaticw ound in apatient with peripherala rterial disease Biofilm-based wound care was used. The wound healed in 6months. [2] Venous leg ulcers Ultrasounddebridementp atients. Fewer treatments and faster healing than patients treated with sharp debridement. [109] Periprosthetic joint infections Ultrasoundsonication for eradicatingbiofilms;only effective when used in conjunctionw ith antibiotics. [194] S. aureus biofilm Acoustically activated nanodropletswith vancomycin decreased biofilm viability and metabolic activity. [195] 2. Chemical debridement Venous leg ulcer Wound cleaned with sodium hypochloritebetween dressing changes. [192] P. aeruginosa and S. epidermidis in vitro Awater-solublegel formulation that contains0.1% EDTA, acetic acid, citric acid, and carbopol. [196] Mature biofilm TetrasodiumEDTA (tEDTA) [103] S. aureus P. aeruginosa Eugenol as an antimicrobial agent in combination with EDTA. [197] 3. Antibiotics Acinetobacter baumannii in both the planktonic and biofilm phenotypes CZ-01179.CZcompounds are first-in-class series of antibiofilm antibiotics. The name of this class is condensed from the company name, CŪ RZA. These compounds are inspired by the antimicrobial potentialofnaturally occurring peptides and aminosterols, including magainin and squalamine. [198] S. aureus and P. aeruginosa biofilms Combinatorial effects of antibiotics and enzymes:meropenem and amikacin with the combinationo ftrypsin, b-glucosidase, and DNase Ienzymes [199] 4. Nanotechnology E. coli and S. aureus Scaffolds of chitosan + ZnO NPs + silk sericin. Higher antimicrobial activity increased HaCaT cells' proliferation and viability compared with chitosan/silk sericin/acidlauric. [155a] S. aureus and E. coli Films of chitosan + polyaniline + montmorillonite + ZnO NPs. High antimicrobial activity against S. aureus and E. coli.

Reviews
Thee xceptional mechanical strength, thermal conductivity,p hotoluminescence properties,a nd structural stability of CNTs make them an excellent material. [173] Several different therapeutic molecules can be absorbed on the surface of CNTs and transported directly into cells without being metabolized by the body. [174] Ac oating of CNTs prevents bacterial adhesion and subsequent biofilm formation on medical devices and prosthetic implants. [175] There is evidence that long immobilized nanotubes create unstable substrates as ac onsequence of their mobility which prevents bacterial settlement and biofilm formation. [176] Fori nstance,H e et al. [177] developed photothermal antibacterial nanocomposite hydrogels of Pluronic F127/carbon nanotubes with conductive self-healing and an adhesive surface.A saresult of the addition of CNTs,t he hydrogel exhibited promising antimicrobial activity and excellent conductivity in vitro and in vivo.
Nanostructured graphene and derivatives of graphene have antibiofilm properties due to both the presence of sharp edges and oxidative stress. [178] Indirectly,t hey inhibit biofilm formation by damaging bacterial cell membranes and causing the loss of proteins, RNA, and other intracellular species when they contact bacterial cells directly. [179] GO has been reported to have antibacterial activity only when ab iofilm has reached aspecific maturation stage. [180] In ar ecent study, based on the boronic acid functionalized graphene quaternary ammonium salt (B-CG-QAS), Wang et al. [181] reported an ew dual-targeted antibacterial platform. Adual effect of electrostatic adhesion and covalent coupling enabled B-CG-QAS to specifically bind to bacteria and their biofilms at the sites of infection caused by Gramnegative bacteria, resulting in superior targeting ability ( Figure 18).
Further improving the antimicrobial effect could be achieved by near-infrared laser irradiation in synergy with hyperthermia. Moreover, B-CG-QAS could be used effectively to treat multidrug-resistant Gram-negative bacteria and their biofilms,as well as to speed healing of wounds that are infected with bacteria ( Figure 18).

Nanoemulsions
Nanoemulsions are nanosized emulsions designed to deliver drugs directly and efficiently to target sites under physiological conditions with al ong-term therapeutic effect. Using nanoemulsions as carriers of essential oils could result in lower concentrations being required to achieve equal levels of microbial inactivation compared to conventional emulsions or bulk oils. [182] Both Gram-positive and Gram-negative bacterial biofilms were eradicated in vitro by nanoemulsions. Based on am urine wound biofilm model, it was found that nanoemulsions could reduce bacterial loads in wounds and accelerate wound healing. In ar ecent study,L ie tal. [183] reported on ab acterial biofilm in vivo treatment that accelerated wound healing through the use of all-natural materials.G elatin was stabilized by photo-crosslinking with riboflavin (vitamin B2) and carvacrol (the primary component in oregano oil) as the active antimicrobial component (Figure 19 a). Thee ngineered nanoemulsions were demonstrated to have broad-spectrum antimicrobial activity against drug-resistant bacterial biofilms in an in vivo murine wound biofilm model. These nanoemulsions show antimicrobial activity,w ound healing promotion, and biosafety characteristics to manage wound infections.Amurine wound biofilm model was used to evaluate the in vivo activity of the nanoemulsions after their in vitro evaluation. (Figure 19 b). Nanoemulsions significantly reduced the number of bacteria in wounds compared to PBS controls (Figure 19 c). After treatment with nanoemulsions,t he wounds were significantly smaller than those treated with vancomycin or PBS (Figure 19 d). The wound beds of mice treated with nanoemulsions had normal-looking healed wounds and zero purulence scores in contrast to the vancomycin group (Figure 19 e).

Microneedles
Microneedles (MNs) serve as carriers for therapeutic agents,a s the MN structure is able to penetrate the top layer (biofilm and cellular debris) and dissolve upon contact with biological fluid thus releasing therapeutics into the wound. In addition to bypassing the stratum corneum without hypodermic needles,M Ns deliver drugs more effectively than hypodermic needles as they inject directly into the bloodstream rather than muscle tissue. [184] According to Woodhouse et al., [185] ap olymer composite microneedle array can penetrate physicochemical barriers (such as bacterial biofilms) to deliver oxygen and bactericidal agents to chronic wounds.T he microneedles were found to have strong bactericidal effects on both liquid and biofilm cultures of Gram-positive (S. aureus)a nd Gram-negative (P. aeruginosa)b acterial strains (Figure 20 a-c). Calcium peroxide (CPO) alone did not affect colony number (Figure 20 d,e). By contrast to CPO powder, MNs loaded with CPO significantly reduced bacteria in mature biofilms formed by S. aureus and P. aeruginosa (Figure 20 d,e). Thef lexible microneedle array improves the effectiveness of topical oxygenation as well as the treatment of wounds infected with intrinsically antibiotic-resistant biofilms.S ue tal. [186] developed ab iphasic scaffold as an antimicrobial delivery system by combining nanofiber mats and dissolvable microneedle arrays.Avariety of antimicrobial agents,i ncluding AgNO 3 ,G a(NO 3 ) 3 ,a nd vancomycin, were electrospun into nanofiber mats,which allowed for sustained delivery.Integrated antimicrobial agents provide direct access to drugs within biofilms through dissolvable microneedle arrays.C ombining nanofiber mats with microneedle arrays can deliver multiple antimicrobial agents to wound sites effectively with ac ombination of nanofiber mats and microneedles.
Apart from the approaches discussed above,t here are other examples of emerging technologies that show promise against planktonic bacteria and biofilms,such as nanocompo- Figure 18. Graphene nanoparticles for wound healing:Anillustration of how BCG-QAS acts against biofilm. Adapted with permission from ref. [181].C opyright 2020 Elsevier.  (Table 5), DNAn anotechnology [187] micelles, [188] and dendrimers. [189] Each of the biofilm treatment strategies discussed above present advantages and disadvantages.F or example,n anoparticles exhibit high stability and ease of production and functionalization but are hampered by potential side effects and cytotoxicity.U ltrasound-mediated approaches show great promise through the combination of drug delivery and mechanical disinfection. However,t he complicated implementation of the technique means that it is not yet clinically viable,and its success is very highly dependent on the size and shape of the wound. Finally,microneedles are safe and can be used to deliver avariety of substances but this approach might be difficult to scale up for use in clinical settings.T he next breakthrough in drug delivery for wound infections will likely come from ac ombination of recent innovations rather than from one single field.

Conclusion and Perspectives
Microbial control in open wounds is still an unsolved problem in modern medicine.T he complexity of biofilms and their increased resistance to traditional disinfection processes make them av ery challenging target. Early,accurate sensing of biofilm establishment in the wound can offer opportunities for early intervention, thereby increasing the chances for efficient treatment. Moreover, advances in biofilm sensing have the potential to improve our understanding of the crucial factors that affect the establishment and severity of biofilm infections,t hereby enabling the development of more sophisticated treatment options.T here has been promising progress in biofilm sensing in recent years, with sensors becoming more accurate and precise.T he significant growth of studies on the early detection of biofilms within the past decade indicates the importance and demand for further research and development of such technologies. While most studies on biofilm sensing remain mostly laboratory proof-of-concept studies, several in vivo applications have been translated into clinical settings.I no rder to establish tools that succeed in the laboratory and become widely available,itisc rucial that work is continued in this direction.
Eradicating biofilms remains ac hallenge.N ovel approaches have shown promise both in the areas of mechanical and chemical debridement and also through nanotechnology-based therapies.I nt he future,t he development and establishment of innovative solutions to treat biofilms in open wounds should be an area of significant research focus.T he combination of reliable sensing with efficient antimicrobial delivery in biofilms has the potential to provide am uch-needed breakthrough in wound biofilm treatment. Even though great progress is being made on in vitro studies and in vivo on animal models,t here is still very limited work on human subjects.H owever,r ecent calls for action and the corresponding actions from governments both in the EU and the U.S. promise to incentivize antimicrobial development and translation of new technologies to the clinic, through economic,l egislative,and regulatory actions. [190]