Burn injury is a common global threat that causes complex wounds that may affect the whole body. According to the World Health Organization (WHO), it is estimated that 11 million burn injuries occur worldwide, accompanied by 180,000 deaths a year [1]. For individuals with moderate to severe burns, challenges such as infection, inflammatory/immune response, metabolic changes, and distributive shock are all factors that can lead to multiple organ failure [2]. This is especially the case for individuals in severe conditions and/or combat settings where delayed care could result in a loss of viable tissue and increase chances of obtaining a life-threatening infection. Infections with multi-drug resistant biofilms are especially difficult to treat as they present challenges for post-burn therapeutic care. Biofilm refers to a complex community of adherent bacteria that consist of single or multiple bacterial strains, that attaches to a surface while colonizing within a matter of 2–3 days [3, 4]. Up to 80% of human bacterial infections are biofilm associated; such infections are most frequently caused by Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli [5].

Mechanisms of biofilm tolerance include mechanical protection due to encasement in extracellular polymeric substance (EPS) as well as protection from antibiotic clearance via metabolically dormant persister cells [6]. Current treatments for burns include topical antimicrobial therapies such as silver sulfadiazine (Silvadene®) or mafenide (Sulfamylon®) cream [7, 8] followed by the application of wound dressings. Although silver sulfadiazine dressings (SSD) are used commonly for second degree burns, they have been shown to potentially impair healing by exerting toxic effects on keratinocytes and fibroblasts [9].

Bacteria within biofilm release intrinsic signaling molecules, termed diffusible signal factors (DSF), to detach from the surface and allow for biofilm colonization throughout other areas of the body [10]. This cell-to-cell communication is known as quorum sensing, and it is performed by biofilm to assess and address the microenvironment it resides in [11]. We investigated cis-2-decenoic acid (C2DA), a short chain fatty acid that acts as a DSF involved with the regulation of biofilm growth, and virulence that can be used to disperse and inhibit biofilm formation [12, 13, 14]. Results from previous studies on the antimicrobial effects of C2DA show that it can prevent infection when acting alone and synergistically with other antibiotics [14, 15]. In addition, the cis conformation contributes to membrane permeability, allowing the passage of small molecule antibiotics into the cell [14].

Local anesthetics (LA) have long been used as a therapeutic strategy for post-burn care in the form of topical sprays or creams, due to their pain numbing effects and their lack of interference with the healing process [16]. Additional benefits of using LAs include increasing blood perfusion to pre-burn levels [17], modification of the inflammatory response [18] to calm inflammation, and reducing edemas that contribute to the conversion of burn wounds to deeper layers of tissue. An additional benefit from LAs, such as bupivacaine, is its slow onset and long-lasting effects [19]. Similar to C2DA, local anesthetics such as bupivacaine are hydrophobic, making these molecules difficult to deliver.

Chitosan is extensively researched due to its abundant supply, versatility, biocompatibility, and biodegradability that facilitate its fabrication into local antibiotic delivery systems [20]. The medical potential behind chitosan is ever-growing as it can be fabricated into hydrogels, chitosan sponge delivery systems [21], magnetic chitosan nanoparticles for targeted drug delivery [22], coatings for musculoskeletal implant fixation hardware, injectable chitosan paste for traumatic injuries [23], and electrospun chitosan membranes (ESCM) [24, 25]. Electrospinning is an inexpensive and reliable technique that entails dissolving a polymer and charging it to extrude the volatile solution onto a grounded plate where a multi-layered membrane is fabricated via nano-scale fiber collection. Previous work has shown chitosan nanofibrous membranes as well suited for tissue healing and drug delivery applications due to their increased surface area, high degree of biocompatibility and biodegradability, and ability to mimic the extracellular matrix [26, 27, 28, 29, 30, 31]. Preliminary evaluations of ESCM have revealed unique properties that make them particularly suited for delivering anesthetics, fatty acids, and antimicrobials [32]. Furthermore, Chitosan nanofibers have been studied for their applications in tissue regeneration but are limited in that aspect due to their hydrophilic properties which lead to swelling, and degeneration of the fibers. Acylating the membranes with carbon chains makes the material hydrophilic and prevents swelling from happening [33]. As-spun membranes are modified via acylation using fatty acids of different chain lengths to protect from swelling and dissolution of membrane fibers in aqueous solutions at low pH [32]. Modification via acylation surrounds the core chitosan nanofiber with a hydrophobic surface and minimizes adherence of the fibers to wounded tissue, which is ideal for patient care during dressing changes. Prior research has gone into evaluating modified ESCM with short chain fatty acids, with success of extended release of hydrophobic therapeutics [24, 25]. Preliminary studies assessed the cytocompatibility and release profile of therapeutics from ESCMs, and concluded that a concentration of 0.15 mg of C2DA and 0.5 mg of bupivacaine per 10 mm diameter ESCM is cytocompatible and allow for the controlled release of the drugs [25].

Electrospun chitosan membranes tailored with acyl lengths ranging from 6–10 carbons by reaction of fatty acid anhydride and loaded with both bupivacaine and C2DA may serve to (1) act as a physical barrier from microbial contamination, (2) release antimicrobial local anesthetic to reduce pain and modify the inflammatory response, (3) release natural antimicrobial fatty acids that prevent biofilm contamination, and (4) prolong delivery of these hydrophobic therapeutics for an extended period. These loaded membranes may be used as wound dressings for soft tissue wounds following 2nd and 3rd degree burns for prolonged prevention of infection and management of pain. In this study, we sought to determine release profiles of therapeutics from chitosan membranes and their ability to prevent S. aureus (UAMS-1; ATCC 21121), A. baumannii (BAA; 1710) and P. aeruginosa (PA; ATCC 27317) growth and biofilm formation.

Overall results of these studies indicate that anhydride modified ESCMs show promising potential to serve as a wound dressing for burn injury applications. Loading ESCMs with therapeutics via ethanol evaporation makes them readily useable in the context of combat-related wounds. Furthermore, local delivery of anesthetics from ESCMs would have the potential to alleviate the unfavorable dependency that follows the use of systemic opioid use and management [18, 19] for pain symptoms, while C2DA delivery may prevent infection post-burn. With minimal swelling of anhydride-treated fibers between the experimental groups, we can further tailor ESCMs to be more stable and maintain biological active loading capabilities [32]. Washing ESCMs after treatment successfully removed TFA salts, allowing for a decrease in cytotoxic effects while maintaining hydrophobic properties [24, 34]. The DOS calculation showed all of the experimental groups resulting in values less than 2 indicating more viable functional groups available on chitosan, possibly due to steric hindrance making it harder for anhydrides with longer carbon chains to react and bind to chitosan. In return, this allows for the potential loading of more or other therapeutics to further develop this novel burn wound dressing. The degree of substitution indicates how many carbon tails are attached to the electrospun membranes. Therefore, a higher degree of substitution correlates to higher hydrophobicity and larger capacity to encapsulate the drugs loaded. Unlike materials such as the chitosan sponge, the nanofibrous morphology of ESCMs resemble the extracellular matrix with high surface area to volume ratio with pores between the fibers to allow for the excretion of fluids from burn wounds, and reducing the risk of infection [35, 36]. Furthermore, it was reported that the electrospun material stimulates extracellular matrix which promotes epithelialization and angiogenesis [36].

Additionally, the intrinsic biofilm inhibitory properties and release of antimicrobial and anti-inflammatory agents may act as a potential burn wound dressing that cuts down on healing time and physiological scarring.

The release of bupivacaine from the dual-loaded experimental ESCMs was similar to another study in which ESCMs treated with hexanoic anhydride (different loading concentrations) resulted in a burst release [25]. Later the study inferred that this burst release is possibly due to excess bupivacaine drying on top of the membranes rather than being loaded in between the acylated fibers [25]. The reduced cumulative release of bupivacaine in control groups compared to experimental suggests that the hydrophobic carbon tails on modified chitosan allow it to capture and hold the hydrophobic drug within the structure. This extended release may provide the patient with pain relief for a longer amount of time and allow for an increase in time between dressing change in dire situations. Although the dual-loaded experimental ESCMs do not release their full payload, for a patient with a compromised immune system, this release may provide a more guided growth not hindered by high concentrations affecting immune/skin cells during the healing process than the release kinetics observed by the control groups.

C2DA release from all groups exhibited a burst release within the first 3 hours, but only the experimental groups eluted through the 24-hour time point suggesting that the hydrophobic interactions from the carbon chains resulted in the drugs being tightly bound within the modified matrix. Furthermore, the absence of C2DA at 12 hours could be attributed to the hydrophobic drugs having low solubility in aqueous environments such as PBS and bovine growth serum as the hydrophilic media outside the hydrophobic nanofibers impedes the diffusion of drugs. These results may be confirmed in future studies using another detection method, such as LC/MS. Our work, though somewhat contrary to similar studies of acylated chitosan membranes, is similar to another local drug delivery system that loaded C2DA onto phosphatidylcholine coatings, resulting in a burst release at first then tapering concentrations afterward [15]. The cumulative release of C2DA in all groups was less than the total loaded amount, suggesting that the samples are holding onto C2DA more tightly, possibly due to the chain length and configuration of the anhydride treatment on the hydroxyl group of chitosan allowing for attachment of long carbon chains dictating its time-dependent release. The long-tail carbon chains associate with the hydrophobic portions of the loaded drugs. Hydrophobic molecules aggregate in aqueous environments which restricts their contact with water and encapsulates the hydrophobic molecules within the chitosan matrix [37]. The retention of the drugs inside the matrix allows for a slower, controlled release as the drugs have to overcome the strong hydrophobic interactions to diffuse out [24].

Future studies will be performed to measure the C2DA within the samples more accurately via HPLC detection after chemically/physically destroying the samples for detecting therapeutics that may reside within the fibers. Additionally, although not fully understood, the synergistic effect [14] from loading C2DA with bupivacaine may play a role in release kinetics.

Activity against strains prevalent in burn injuries suggests these biomaterials would be useful in burn injury infection prevention. Similar antimicrobial activity against S. aureus was reported in a study performed by Harrison et al. [25], where the same therapeutics and method of delivery were used, but at much higher concentrations. In this study, nearly all groups had decreased growth within 24 hours, even groups not loaded with any therapeutic [25], indicating that materials themselves were inhibitory to bacteria, but not bactericidal at these concentrations. In contrast, our study looked at the response by 48 hours, where the only biomaterials with significant antibacterial properties were those loaded with both therapeutics, which suggests that the combination of therapeutics may have additive or synergistic effects for inhibition of biofilm. The absence of differences between the modified chitosan and control groups against planktonic A baumannii may be due to the lower susceptibility of this strain to both bupivacaine and C2DA. This is further confirmed by the reduced growth of planktonic bacteria at 48 hours only in the groups that received both drugs. The reduction however was not observed in P. aeruginosa planktonic data suggesting that the combination and concentration of both therapeutics seems to be more effective at inhibiting S. aureus and A. baumannii planktonic bacteria compared with planktonic P. aeruginosa. The decreased efficacy of the drugs is likely due to P. aeruginosa being one of the gram-negative strains that become harder to treat as time increases in comparison with other skin related infections from burn wounds [38]. Reports of high concentrations of C2DA have found that this agent increases cell membrane permeability [39], and P. aeruginosa among other strains produce and utilize these DSFs [12], which would explain the lack of inhibition of P. aeruginosa at such concentrations compared to that of other strains. It is likely that the antimicrobial activity exhibited by gauze is due to the processing method of the gauze, as they are bleached for color and sterility purposes. The lack of an inhibitory effect against the gram-negative strains may also be explained by the burst release seen in the elution studies, where a burst release of C2DA is followed by tapering concentration within the first day. The reduction of S. aureus and P. aeruginosa’s biofilm in the wells, below the samples, at 48 hours may be due to the increase in time resulting in more available therapeutics in the wells. In addition to more therapeutics being released, it is also important to note that most of these samples are hydrophobic and float within the TSB media, resulting in an increase of therapeutics reaching the bottoms of the wells via diffusion over time. The higher growth of S. aureus and A. baumannii biofilm on the chitosan sponge suggests that the material alone was not able to reduce the viability of biofilm for both time points as opposed to the experimental groups such as the decanoic anhydride modified ESCM, indicating it may have an inhibitory effect on its own. Groups loaded with both therapeutics reduced P. aeruginosa biofilm growth at 48 hours, which suggests that Bupivacaine and C2DA might have an additive effect on some strains of bacteria.

Future studies for this project entail a reconsideration of loading concentrations, in vivo studies, and exploring different loading strategies. Additional future studies include cytocompatibility studies using human dermal fibroblasts and keratinocytes. Other synthetic analogs of C2DA have been developed as well as new procedures for measuring therapeutics, so future studies may include incorporation of these analogs into chitosan membranes and evaluation of their potential to prevent and eradicate biofilm more effectively than C2DA. Although ESCMs loaded with C2DA and bupivacaine seem promising, there are still limitations; the antimicrobial efficacy of ESCMs modified with different chain lengths and the effects of different treatment methods on the DOS were not explored. Furthermore, different loading strategies to increase drug encapsulation capacity, and in vivo studies are needed to evaluate how the use of ESCMs as wound dressings could be translated to clinical use.

These in vitro evaluations investigated combination and individual loading of therapeutics for control and experimental groups to aid in pain relief and infection prevention as a burn wound dressing application. In austere locations or combat settings, where medical attention may be delayed, an antibacterial wound dressing may be advantageous for minimizing infections from further contamination as well as potential biofilm growth within the first 24 hours of administration, with local anesthetics remaining active over the course of 72 hours, until further care is available. Extending the alkyl chain length in modified ESCMs by using anhydrides with longer chains such as decanoic anhydride could increase hydrophobicity, promote better biocompatibility, and improve chemical and mechanical stability. Longer carbon chains also facilitate sustained drug release since they provide a diffusion barrier. Based on findings from the bacterial studies it is suggested that the combination of therapeutics play a major role in biofilm prevention for select strains such as S. aureus, A. baumannii, and P. aeruginosa, whether that is from direct contact or diffusion in aqueous solutions. To conclude, this in vitro study denotes the potential of varying acylated ESCMs, especially decanoic-modified, to be used as a burn wound dressing by releasing hydrophobic therapeutics for prevention of bacterial/biofilm growth and pain relief.

The data are available from the corresponding author upon request.

Research idea and study design, JDB, LRC and JAJ; bacterial growth and membrane treatments, LRC, ZH, EA, and ECM; data analysis and interpretation, TF, LRC, ZH, ECM, EA and JRB; supervision, JAJ, TF, JDB. All authors have been involved in drafting and revising the manuscript, and all authors have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

We thank Ashleigh Anderson, Alex Bryan, Brenna Ballard, Claire Abrey, and Bharat Jothlingham for assistance in membrane fabrication, imaging, sampling, and text editing.

Research is supported by the Military Burn Research Program (MBRP) under Award #: W81XWH-20-1-0430 and the University of Memphis/University of Tennessee Health Science Center Joint Program in Biomedical Engineering.

Given the role of Dr. Jessica Amber Jennings, and Dr. Josh Bush as Guest editors of Special Issue, they had no involvement in the peer-review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Viviana di Giacomo and Piergiorgio Gentile. The authors declare no conflict of interest.