Simultaneous Delivery of Multiple Antimicrobial Agents by Biphasic Scaffolds for Effective Treatment of Wound Biofilms

Abstract

Biofilms pose a major challenge to control wound-associated infections. Due to biofilm impenetrability, traditional antimicrobial agents are often ineffective in combating biofilms. Herein, we reported a biphasic scaffold as an antimicrobial delivery system by integrating nanofiber mats with dissolvable microneedle arrays for the effective treatment of bacterial biofilms. Different combinations of antimicrobial agents, including AgNO₃, Ga(NO₃)₃, and vancomycin, were incorporated into nanofiber mats by co-axial electrospinning, which enabled sustained delivery of these drugs. The antimicrobial agents-incorporated dissolvable microneedle arrays allowed direct penetration of drugs into biofilms. By optimizing the administration strategies, drug combinations, and microneedle densities, biphasic scaffolds were able to eradicate both methicillin-resistant Staphylococcus aureus (MRSA) and MRSA/Pseudomonas aeruginosa blend biofilms in an ex vivo human skin wound infection model without necessitating surgical debridement. Taken together, the combinatorial system comprised of nanofiber mats and microneedle arrays can provide an efficacious delivery of multiple antimicrobial agents for the treatment of bacterial biofilms in wounds.

Graphical Abstract

Biofilms harbor complex structural and biological attributes, such as the presence of an extracellular polymeric matrix, physical and chemical heterogeneity, and drug tolerance/resistance, all of which provide remarkable therapeutic challenges.^[1] Biofilms are often found in chronic wounds and evidence suggests that biofilms contribute to the non-healing environment of chronic wounds.^[2] Normally, bacterial biofilms are characterized as highly resistant to antibiotic treatment and immune responses.^[3] The current biofilm management strategies in clinics are largely based on aggressive debridement and local treatment with high doses of antibiotics or other antimicrobial agents.^[2b,4] However, mechanical debridement is invasive, can cause pain in patients, and allows for biofilm reformation. Thus, treatment of biofilm infections remains challenging and warrants significant scientific efforts.^[5]

To overcome the drug resistance and impenetrability, two main strategies are under development for management of biofilms. One focuses on the disruption of biofilms based on different physical principles including cold plasmas, ultrasound, electrical currents, photothermal, photodynamic, and alternating magnetic fields (AMFs).^[6] Potential problems with these approaches include inadequate efficacy, insufficient breadth of susceptible pathogens, and difficulty of in vivo implementation. Additionally, these approaches require expensive equipment and extensive training prior to use. Another strategy is to develop new drug formulations that can overcome drug resistance and effectively penetrate biofilms.^[1c,7] Given the attrition rate within the existing drug discovery model, the process is costly and time consuming.^[8] Therefore, reformulating existing antimicrobial agents for effective management of biofilms becomes an attractive approach. Different antimicrobial agents may target microorganisms through different modes of action. For example, Ag⁺ binds to membrane proteins responsible for transport of substances in and out of bacterial cells and the DNA to block cell division.^[9] Ag⁺ also interferes with bacterial cell respiration and destroys energy production, initiating a bacterial cell death pathway.^[9b] Additionally, Ga³⁺ disrupts ferric iron-dependent metabolic pathways, thereby inhibiting microbial growth due to its ionic radius, which is identical to Fe³⁺.^[10] Vancomycin, a glycopeptide antibiotic, displays its antimicrobial activity through binding bacterial cell wall mucopeptide precursors that terminate in the sequence l-Lys-d-Ala-d-Ala.^[11] Simultaneous delivery of multiple antimicrobial agents is able to initiate complementary killing mechanisms, which may exhibit synergistic antimicrobial efficacy. Therefore, the objective of this work was to develop a biphasic scaffold consisting of dissolvable microneedles and nanofiber mats for concurrent delivery of different combinations of three above-mentioned antimicrobial agents. We hypothesized that these drugs can readily penetrate to the inside of biofilms following microneedle penetration, where their synergistic antimicrobial activities can contribute to the eradication of biofilms and inhibition of their regrowth.

To test our hypothesis, we first developed the biphasic scaffold by combining polyvinylpyrrolidone (PVP) microneedle arrays with electrospun pluronic F-127-poly(ε- caprolactone) (F127-PCL) nanofiber mats with incorporation of different combinations of antimicrobial agents. The biphasic scaffold served as an antimicrobial agent delivery system for eradication of mature biofilms on artificial wounds created on ex vivo human skin explants. Due to their hydrophilic nature, antimicrobial agents were encapsulated in nanofiber cores using co-axial electrospinning, following our previous studies (Figure 1A).^[12] Figure 1B shows the fabrication process of biphasic scaffolds. Briefly, the 20% w/v PVP solution containing certain combination of antimicrobial agents was poured into a customized polydimethylsiloxane (PDMS) mold, and a vacuum pump was applied for 30 min to allow the PVP solution to fully infiltrate into the mold. Then, the biphasic scaffold was achieved by stacking the barely dry PVP microneedle patch on the top of the F127/antimicrobial agents-PCL core-sheath nanofiber mat and drying at ambient conditions for 24 h.

Figure 1. Schematic illustrating the fabrication of biphasic scaffolds.

(A) Preparation of antimicrobial agents/F127-PCL core-sheath nanofiber mats using co-axial electrospinning. (B) Procedures for fabrication of biphasic scaffolds including (1) placing antimicrobial agents containing PVP solutions to the mold for microneedle array formation, (2) immobilizing antimicrobial agents containing PVP microneedle arrays to the surface of antimicrobial agents/F127-PCL core-sheath nanofiber membranes, and (3) removing the mold to form biphasic scaffolds.

Figure 2, A–D shows SEM images of the immobilized AgGaVan containing PVP microneedle arrays of biphasic scaffolds with regular (100 needles on a 6-mm diameter disc) and high (150 needles on a disc of 6-mm diameter) microneedle densities. Figure 2E shows a typical SEM image of the F127/AgGaVan- PCL core-sheath nanofiber mat, indicating a fibrous and porous structure. Figure 2F shows the in vitro release kinetics of Ag⁺, Ga³⁺, and vancomycin from F127/antimicrobial agent-PCL nanofiber mats, suggesting the duration of sustained release for these antimicrobial agents can surpass 4 weeks. The encapsulation efficiencies of AgNO₃, Ga(NO₃)₃ or vancomycin for F127/AgNO₃-PCL, F127/Ga(NO₃)₃-PCL and F127/vancomycin-PCL core-sheath nanofiber samples were 94.4±4.1%, 93.1±2.7%, and 92.7±3.6%, respectively. Corresponding loadings of AgNO₃, Ga(NO₃)₃, and vancomycin in F127-PCL nanofibers were 23.59 ± 1.02, 23.28 ± 0.68. and 23.18 ± 0.90 mg/g. We also tested the in vitro release kinetics of Ag⁺, Ga³⁺, and vancomycin from F127/AgNO₃/Ga(NO₃)₃/vancomycin-PCL nanofiber membranes, finding each active ingredient had a continuous release over 28 days (Figure S1). Besides, microneedle arrays were made of PVP which is highly water-soluble. The PVP microneedles would dissolve completely within one minute after immersing in aqueous solutions, and all the antimicrobial agents incorporated would release instantaneously. Therefore, the drug release profiles were only measured for drug-loaded nanofiber membranes and not for the entire biphasic scaffolds containing the drug-loaded microneedle arrays.

Figure 2. Characterizations of biphasic scaffolds.

(A) A representative SEM image showing the immobilized AgGaVan containing regular-density PVP microneedle arrays of biphasic scaffolds. (B) high-magnification of image of (A). (C) A representative SEM image showing the immobilized AgGaVan containing high-density PVP microneedle arrays of biphasic scaffolds. (D) High-magnification image of (C). (E) A representative SEM image showing a F127/AgGaVan-PCL core-sheath nanofiber mat serving as the substrate for immobilization of microneedle arrays. (F) In vitro release profiles of silver ions, vancomycin, and gallium ions from F127/AgNO₃, Ga(NO₃)₃, or vancomycin-PCL nanofiber membranes, indicating each antimicrobial agent (e.g., vancomycin, Ag⁺, and Ga³⁺) had a sustained release over 28 days from single antimicrobial agent-loaded nanofiber membranes. All data are presented as mean ± SD (n = 6).

To assess the efficacy of biphasic scaffolds against biofilms, we first established methicillin-resistant Staphylococcus aureus (MRSA) USA300 biofilms in excisional wounds created using ex vivo human skin explants following our established protocols.^[12b] We then applied the AgNO₃, Ga(NO₃)₃ or vancomycin-containing biphasic scaffolds with regular microneedle density to the biofilm-containing wounds for 72 h, replacing with new scaffolds every 24 h. No treatment, F127-PCL nanofiber mats, F127/single antimicrobial agent-PCL nanofiber mats, F127/single antimicrobial agent-PCL nanofiber mats plus single antimicrobial agent free drug were used as controls for comparison. Finally, we quantified the bacterial load through CFU quantification. The treatment with biphasic scaffolds (F127/single antimicrobial agent-PCL nanofiber mats integrated with single antimicrobial agent containing PVP microneedle arrays) showed the best efficacy (7.56, 7.07 and 7.62 Log reduction, respectively) against MRSA USA300 biofilms among all the treatment groups (Figure 3 A–C). It is worth mentioning that, compared to treatment with AgNO₃, Ga(NO₃)₃, or vancomycin/F127-PCL nanofiber mats plus AgNO₃, Ga(NO₃)₃, or vancomycin free drugs, the administration of biphasic scaffolds with the same dose of antimicrobial agents showed a significantly higher efficacy (3.99, 3.66 and 2.42 Log reduction, respectively) against MRSA USA300 biofilms (Figure 3 A–C). This result indicates that the dissolvable microneedle arrays were critical in the delivery of AgNO₃, Ga(NO₃)₃ or vancomycin to the inside biofilms due to their direct physical penetration.

Figure 3.

Efficacy of single antimicrobial agent containing biphasic scaffolds against MRSA biofilms using different administration strategies (Each panel was labeled with antimicrobial agent/time interval between changes of biphasic scaffolds/density of microneedles.). (A-C) Change scaffolds containing regular density of microneedle arrays 3 times within 72 h. (D-F) Change scaffolds containing regular density of microneedle arrays 3 times within 108 h. (G-I) Change scaffolds containing high density of microneedle arrays 3 times within 108 h. (J-L) Change scaffolds containing high density of microneedle arrays 3 times within 144 h. F127-PCL: pluronic F127-PCL core-sheath nanofiber membranes. F127/Ag (or Ga, Van)-PCL: AgNO₃, Ga(NO₃)₃ or vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/Ag (or Ga, Van) -PCL + aqueous Ag (or Ga, Van): AgNO₃, Ga(NO₃)₃, or vancomycin/pluronic F127-PCL core-sheath nanofiber membranes and AgNO₃, Ga(NO₃)₃, or vancomycin aqueous solution. F127/Ag (or Ga, Van) -PCL-PCL + PVP/Ag (or Ga, Van) - PCL MN: biphasic scaffolds composed of AgNO₃, Ga(NO₃)₃ or vancomycin containing PVP microneedle arrays and AgNO₃, Ga(NO₃)₃ or vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. All data are presented as mean ± SD (n = 6).

In order to further reduce the bacterial load, we explored a different administration strategy by applying the biphasic scaffold three times for 36 h each time. It was found that the bacterial load was not detectable after applying the biphasic scaffold three times, indicating the complete removal of MRSA biofilms (Figure 3 D–F). In contrast, using the same strategy, there were still up to 2.4 ×10⁷, 1.6 × 10⁷ and 3.0 ×10⁷ CFU/g bacteria remaining on the wounds treated by F127/AgNO₃, Ga(NO₃)₃, or vancomycin-PCL nanofiber mats alone, and after combination with AgNO₃, Ga(NO₃)₃, or vancomycin free drugs, there were about ~10⁶ CFU/g bacteria remaining on the wounds (Figure 3 D–F). To enhance the antibiofilm efficacy, we further increased the density of microneedles on the surface from 100 to 150 per biphasic scaffold and used the same administration method (i.e., apply three times for 36 h each time) (Figure 3 G–I). Interestingly, we could not detect the bacteria after applying the AgNO₃ or vancomycin containing biphasic scaffolds twice and three times (Figure 3 G, I). Merely ~200 CFU/g bacteria remained after the second administration of Ga(NO₃)₃ containing biphasic scaffolds and no colonies were detected after the third change (Figure 3 H). By contrast, there were ~10⁶ to ~10⁷ CFU/g bacteria remaining on the wounds treated by AgNO₃, Ga(NO₃)₃ or vancomycin-loaded nanofiber mats without and with incorporation of free drugs twice and three times (Figure 3 G–I). From the above results, it seems that appropriately extending the intervals between administrating scaffolds may result in reduction of administration times. Therefore, we further extended the interval to 48 h. After the first administration, the number of CFU for the biphasic scaffold treatment group was smaller than the change intervals of 24 h and 36 h. Interestingly, we could not detect any colonies after the second administration using a single antimicrobial agent containing biphasic scaffolds (Figure 3 J–L). These results indicate reducing the distance between the adjacent microneedles can promote the anti-biofilm efficacy, likely due to the increase of the diffusion area of antimicrobial agents. In addition, optimizing the intervals between administrations of biphasic scaffolds could be an effective way to reduce the number of scaffold changes.

It is known that Ag⁺, Ga³⁺, and vancomycin have different bactericidal mechanisms.^[9–11] Subsequently, we explored the antibiofilm efficacy of biphasic scaffolds by incorporating different combinations of the three aforementioned antimicrobial agents. In this case, we used high density microneedle arrays in biphasic scaffolds, as they proved more efficacious. The theoretical loading for each drug (25 mg/g) was maintained as it was for biphasic scaffolds containing single antimicrobial agent. We changed the biphasic scaffold every 36 h after administration a total of 3 times (108 h total). The MRSA biofilm could be completely eradicated after the second change of biphasic scaffolds containing two or three different types of antimicrobial agents (Figure 4). Compared with single antimicrobial agent-loaded biphasic scaffolds, the scaffolds incorporating two or three antimicrobial agents showed improved antibiofilm efficacy (Figure 3 and Figure 4). It is worth mentioning that we could not detect any bacteria on the wounds after the third change of biphasic scaffolds containing Ga(NO₃)₃ alone (Figure 3 H). However, bacteria was not detectable after the second change of biphasic scaffolds containing Ga(NO₃)₃ together with AgNO₃ and/or vancomycin (Figure 4 B–D). Our ultimate goal was to minimize the number of scaffold changes to minimize cost and patient discomfort. Remarkably, after the wounds were treated once by biphasic scaffolds containing three antimicrobial agents for 48 h, bacteria were undetectable, suggesting the biofilms were eliminated after a single treatment. In addition, we also incorporated three different antimicrobial agents with each at 8.33 mg/g into microneedles to form a total concentration of 25 mg/g. We found that the combinatorial loading of three antimicrobial agents was indeed more effective than a single agent loading when using biphasic scaffolds for the treatment of biofilms (Figure S2). Thus, we concluded that the three antimicrobial agents had a synergistic effect when working together due to different acting mechanisms. These results support our hypothesis that the biphasic scaffolds containing multiple antimicrobial agents with distinct mechanisms of action showed synergistic antimicrobial effects.

Figure 4.

Efficacy of biphasic scaffolds containing different combinations of antimicrobial agents against MRSA biofilms using different administration strategies (Each panel was labeled with different combinations of antimicrobial agents/time interval between changes of biphasic scaffolds.). (A, B) AgNO₃/vancomycin. (C, D) AgNO₃/Ga(NO₃)₃. (E, F) Ga(NO₃)₃/vancomycin. (G, H) AgNO₃/Ga(NO₃)₃/vancomycin. (A, C, E, G) Change scaffolds containing high density of microneedle arrays 3 times within 108 h. (B, D, F, H) Change scaffolds containing high density of microneedle arrays 3 times within 144 h. F127-PCL: pluronic F127-PCL core-sheath nanofiber membranes. F127/AgVan-PCL + aqueous AgVan: AgNO₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes and AgNO₃ and vancomycin aqueous solution. F127/AgGa-PCL + aqueous AgGa: AgNO₃ and Ga(NO₃)₃, /pluronic F127-PCL core-sheath nanofiber membranes and AgNO₃ and Ga(NO₃)₃ aqueous solution. F127/GaVan-PCL + aqueous GaVan: Ga(NO₃)₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes and Ga(NO₃)₃ and vancomycin aqueous solution. F127/AgGaVan-PCL + aqueous AgGaVan: AgNO₃, Ga(NO₃)₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes and AgNO₃, Ga(NO₃)₃ and vancomycin aqueous solution. F127/AgVan-PCL+PVP/AgVan MN: biphasic scaffolds composed of vancomycin containing PVP microneedle arrays and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/GaVan-PCL+PVP/GaVan MN: biphasic scaffolds composed of Ga(NO₃)₃/vancomycin containing PVP microneedle arrays and Ga(NO₃)₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/AgGa-PCL+PVP/AgGa MN: biphasic scaffolds composed of AgNO₃/Ga(NO₃)₃ containing PVP microneedle arrays and AgNO₃ and Ga(NO₃)₃/pluronic F127-PCL core-sheath nanofiber membranes. F127/AgGaVan-PCL +PVP/AgGaVan MN: biphasic scaffolds composed of AgNO₃ and Ga(NO₃)₃ containing PVP microneedle arrays and AgNO₃ and Ga(NO₃)₃/pluronic F127-PCL core-sheath nanofiber membranes. All data are presented as mean ± SD (n = 6).

Bacterial biofilms in wounds often contain more than one type of bacterial species.^[13] To more closely emulate a clinical scenario, we established a Pseudomonas aeruginosa (P. aeruginosa)(Gram−) and MRSA (Gram+) blend biofilm-infected wound model using ex vivo human skin explants by co-inoculating both bacterial strains for 72 h.^[12b] We then tested the anti-biofilm efficacy of biphasic scaffolds with high microneedle density containing single and multiple antimicrobial agents against the blend biofilm. No colonies were detected after the second administration of biphasic scaffolds containing two or three distinct antimicrobial agents within 72 h (Figure 5A). We extended the interval between scaffold changes to 48 h, and no colonies were detected after the second administration when the biofilms were treated by biphasic scaffolds containing two or three distinct antimicrobial agents within 96 h. In addition, after the wounds were treated with three different types of antimicrobial agents containing biphasic scaffolds for 48 h, only ~300 bacterial colonies were detected. This indicates that our biphasic scaffolds incorporating different combinations of antimicrobial agents were not only effective in treating biofilms composed of a single type of bacteria, but also had outstanding performance in managing biofilms composed of multiple types of bacteria (Figure 5B). In contrast, there were still about 2.1×10², 2.2×10² and 1.6×10⁵ CFU/g bacteria remaining on the wounds treated by AgNO₃, Ga(NO₃)₃ or vancomycin only-loaded scaffolds, even after the third administration within 108 h. Further, there were still about 1.1×10², 1.3×10² and 1.3×10⁵ CFU/g bacteria remaining on the wounds treated by AgNO₃, Ga(NO₃)₃ or vancomycin only-loaded scaffolds even after the third change within 144 h. These results were very encouraging as the biphasic scaffolds incorporating different combinations of antimicrobial agents proved to be effective treatment modalities for eliminating complex, multi-strain biofilms, and demonstrated great potential for applications in management of biofilm-infected wounds. To further investigate the efficacy of biphasic scaffolds in relevant animal models (e.g., a biofilm containing type II diabetic mouse wound model) in the future,^[12b,14] we tested their in vitro cytotoxicity. No significant difference in proliferation or activity was observed for HaCaT and U937 cells within 5 days of incubation with the tissue culture polystyrene plates (TCPS) and biphasic scaffolds, indicating no evident scaffold-induced cytotoxicity (Figure S3). It is worth mentioning that the cells were exposed to comparable concentrations of the antimicrobial agents to the in vitro wound model used in the bacterial assay. Based on this, a 6-mm diameter biphasic scaffold contained 10 mg of nanofibers and 5 mg of microneedles. When a biphasic scaffold was placed in 1 ml of cell culture medium, the microneedles would dissolve and release all the incorporated antimicrobial agents within around 1 min, making the concentration in the culture medium reach 125 μg/ml. Based on the release curve, the amount of antimicrobial agent released from the nanofiber membrane on the first, third, and fifth days was 70, 90, and 120 μg. Therefore, the total antimicrobial agent concentrations in the medium on day 1, 3, and 5 were 195, 215, and 245 μg/ml, respectively, which were higher than their minimum inhibitory concentrations (MIC). Our results also demonstrated that these concentrations of antimicrobial agents were not toxic to cells.

Figure 5.

Efficacy of single and multiple antimicrobial agents containing biphasic scaffolds against mixed-MRSA/P. aeruginosa biofilms using different administration strategies. Scaffolds containing high density of microneedle arrays were changed 3 times within 108 h (A) and 144 h (B). F127-PCL: pluronic F127-PCL core-sheath nanofiber membranes. F127/Ag-PCL+PVP/Ag MN: biphasic scaffolds composed of AgNO₃ containing PVP microneedle arrays and AgNO₃/pluronic F127-PCL core-sheath nanofiber membranes. F127/Ga-PCL+PVP/Ga MN: biphasic scaffolds composed of Ga(NO₃)₃ containing PVP microneedle arrays and Ga(NO₃)₃/pluronic F127-PCL core-sheath nanofiber membranes. F127/Van-PCL+PVP/Van MN: biphasic scaffolds composed of vancomycin containing PVP microneedle arrays and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/AgGa-PCL +PVP/AgGa MN: biphasic scaffolds composed of AgNO₃ and Ga(NO₃)₃ containing PVP microneedle arrays and AgNO₃ and Ga(NO₃)₃/pluronic F127-PCL core-sheath nanofiber membranes. F127/GaVan-PCL +PVP/GaVan MN: biphasic scaffolds composed of Ga(NO₃)₃, and vancomycin containing PVP microneedle arrays and Ga(NO₃)₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/AgVan-PCL +PVP/AgVan MN: biphasic scaffolds composed of AgNO₃ and vancomycin containing PVP microneedle arrays and AgNO₃ and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. F127/AgGaVan-PCL +PVP/AgGaVan MN: biphasic scaffolds composed of AgNO₃, Ga(NO₃)₃, and vancomycin containing PVP microneedle arrays and AgNO₃, Ga(NO₃)₃, and vancomycin/pluronic F127-PCL core-sheath nanofiber membranes. All data are presented as mean ± SD (n=6).

Human skin tissues obtained during plastic surgeries have been used to investigate skin barrier repair, wound healing, chemical toxicity, chronic inflammatory diseases, DNA vaccination and fungal infection.^[15] The availability of human skin explants also offers an opportunity to create a biofilm-containing wound model. Previous studies described different methodologies and applications of the human skin explant model.^[16] In this study, we identified the most frequently used methods and assays based on previously reported protocols^[12b,17] and established an ex vivo biofilm-infected human skin wound model for testing the antibiofilm efficacy of biphasic scaffolds with incorporation of different combinations of antimicrobial agents.

The major challenges for eradication of biofilms are the drug resistance and penetration barrier due to extracellular polymeric substances.^[18] To overcome these challenges, we recently demonstrated the antibiofilm efficacy of a wound dressing capable of delivering a molecularly-engineered antimicrobial peptide with broad-spectrum antimicrobial activity, including multi-drug resistant bacteria, to the inside and surface of biofilms.^[12b] Novel engineered peptides are often considered as new molecular entities, according to the U.S. Food and Drug Administration (FDA), which could make clinical translation time-consuming and costly. Here, we proposed a different biofilm treatment strategy by developing biphasic scaffolds consisting of dissolvable microneedle arrays and nanofiber mats for simultaneous delivery of multiple FDA-approved antimicrobial agents for combating biofilms. We chose PCL as raw materials for the nanofiber mats as it is biodegradable, biocompatible, and has been used in the preparation of FDA-approved medical devices.^[19] In the biphasic scaffold, the F127-PCL nanofiber mat retained fibrous and porous structures capable of mimicking native extracellular matrix (ECM) architecture and could serve as an artificial ECM suitable for wound healing. Moreover, the core-sheath nanofiber structure can protect the encapsulated biological agents from a hostile microenvironment. Finally, PVP was used to prepare dissolvable microneedle arrays because of its biocompatibility, high hydrophilicity, and broad use as a binder in pharmaceutical tablets.^[20]

We incorporated different combinations of three therapeutics with distinct antimicrobial mechanisms: AgNO₃, Ga(NO₃)₃, and vancomycin. Among them, silver ions have been widely studied and used for prevention and treatment of bacterial infections in wounds from burns or trauma. Silver ions are effective antimicrobial agents and show a broad antimicrobial spectrum against many different pathogens, including multi-drug resistant pathogens.^[21] Ga (III), in the form of citrate-buffered gallium nitrate, has been approved by the FDA (Ganite®) for the treatment of cancer-associated hypercalcemia, indicating there is no concern regarding the safety of this drug.^[22] Therefore, Ga³⁺ have been repurposed in many studies for treatment against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) in nosocomial infections by interfering with iron-dependent metabolic pathway.^[23] Vancomycin hydrochloride is a narrow-spectrum antibiotic, only sensitive and effective to gram-positive bacteria, such as Streptococcus (especially MRSA), Pneumococcus and Enterococcus, to name a few.^[24] Based on our results, it appeared that biphasic scaffolds incorporating two or three antimicrobial agents (e.g., AgNO₃, Ga(NO₃)₃, and vancomycin) resulted in higher efficacy against both MRSA biofilms and MRSA/P. aeruginosa blend biofilms compared to the scaffolds loaded with a single antimicrobial agent, indicating a synergistic antibactericidal mechanism by combined delivery of antimicrobial agents. It is known that vancomycin has limited antibacterial efficacy against most clinically relevant gram-negative bacteria like P. aeruginosa.^[25] Thus, biphasic scaffolds containing vancomycin alone may not be able to eradicate the MRSA/P. aeruginosa blend biofilms (Figure 5). Interestingly, after incorporation of Ag⁺ and/or Ga³⁺, the blend biofilms were eradicated completely after two administrations of biphasic scaffolds (Figure 5). Compared to the MRSA biofilm treatment, the efficacy of biphasic scaffolds containing vancomycin and Ag⁺ and/or Ga³⁺ against blend biofilms was profound. This is of great significance as biofilms in wounds often consist of multiple types of bacterial strains.^[26]

In summary, we have fabricated biphasic scaffolds for simultaneous delivery of different combinations of therapeutic agents for effective treatment of multi-species biofilms in excisional wounds created in human skin explants without debridement. The microneedle arrays can physically assist the penetration of antimicrobial agents into biofilms for improved efficacy. We demonstrated that biphasic scaffolds with incorporation of multiple antimicrobial agents with different mechanisms of action showed enhanced antibacterial efficacy. The approach developed in this work may provide an efficacious intervention with great potential for translation into the clinics that could effectively treat biofilms, in particular, multi-drug resistant bacteria and pathogens blend biofilms, and improve the quality of wound care.