Pathogenic bacterial infections pose a serious threat to public health, especially in chronic and acute wounds where bacterial colonization and biofilm formation make wound healing more difficult. The therapy based on reactive oxygen species (ROS) has the advantages of broad-spectrum antibacterial activity and low drug resistance, and has become a non antibiotic strategy for treating infected wounds. However, its clinical application is still limited by issues such as low drug delivery efficiency, poor bacterial targeting, and uncontrolled inflammation after treatment.

To this end, Professor Li Wei's team from the School of Pharmacy at Wuhan University has designed a multifunctional double-layer microneedle patch (LFT/S-MN) aimed at effectively sterilizing and promoting wound healing. The patch combines positively charged lactoferrin based nanoparticles (LFT NPs) with porphyrin acoustic sensitizer (TAPPFe), placed in a soluble substrate layer, and loaded with anti-inflammatory drug spermidine (SPD) in a pH responsive needle tip layer. When microneedles are inserted into infected tissue, the released LFT NPs can effectively target and aggregate negatively charged bacteria, generating a large amount of ROS through synergistic chemodynamic and sonodynamic therapy (CDT/SDT) under ultrasound stimulation. At the same time, the acidic microenvironment of the wound triggers the gradual degradation of the needle tip layer, thereby achieving controlled release of SPD and immune regulation. In a mouse wound model infected with Pseudomonas aeruginosa, LFT/S-MN patch significantly reduced bacterial load, inhibited inflammatory response, promoted collagen deposition and neovascularization, and accelerated wound closure.

This work proposes a promising non antibiotic strategy that achieves wound antibacterial and tissue regeneration through synergistic CDT/SDT and inflammation regulation. This microneedle patch is made using Mofang Precision Surface Projection Micro Stereoscopic Lithography (P μ SL) technology (microArch) ® S240， Precision: 10 μ m) 3D printed molds were prepared by PDMS flip molding, and the related research results were published in the journal "Journal of Controlled Release" under the title "Three in one bilayer microneedle patch for ultrasound activated antibacterial therapy and immune modulation in wound healing". Jiang Peng, a doctoral student from the School of Pharmacy at Wuhan University in 2023, is the author of the paper, Professor Li Wei is the corresponding author of the paper, and the School of Pharmacy at Wuhan University is the completion unit of the paper. The above research work has been supported by the National Natural Science Foundation of China project and the PI funding of Wuhan University Taikang Life Medicine Center.

As shown in Figure 1, the patch integrates positively charged lactoferrin nanoparticles (LFT NP) coupled with porphyrin acoustic sensitizer (TAPPFe) in a soluble substrate, as well as a pH responsive apex layer loaded with anti-inflammatory agent spermidine (SPD). After insertion into infected tissue, the released LFT NPs effectively target and aggregate negatively charged bacteria, thereby generating powerful ROS through synergistic chemodynamic and sonodynamic therapy (CDT/SDT) under ultrasound stimulation. At the same time, the acidic wound microenvironment triggers the gradual degradation of the apical layer, and the controllable release of SPD is used for immune regulation. In a mouse wound model infected with Pseudomonas aeruginosa, LFT/S-MN patch significantly reduced bacterial load, inhibited inflammation, promoted collagen deposition and neovascularization, and accelerated wound closure.

Figure 1. Schematic diagram of the structure and treatment mechanism of LFT/S-MN patch for treating infected wounds. (A) The design and preparation of double-layer LFT/S-MN patches consist of a soluble substrate layer loaded with positively charged LFT NPs and a pH responsive tip layer encapsulating SPD. (B) After insertion into the infected wound, the basal layer rapidly dissolves and releases LFT NPs, which actively target negatively charged bacteria and generate ROS through synergistic SDT and CDT under US, achieving effective bacterial removal. Meanwhile, the acidic wound microenvironment triggers sharp layer degradation, leading to sustained release of SPD for inflammation regulation. This joint strategy promotes bacterial clearance, immune regulation, and tissue regeneration, ultimately accelerating wound healing.

In order to achieve site specificity and control drug release, a pH responsive bilayer MN patch, called LFT/S-MN patch, was prepared using a sequential molding strategy. For visualization and structural validation, two fluorescent probes, Rhodamine B-labeled bovine serum albumin (RhB BSA) and fluorescein isothiocyanate (FITC), were loaded onto the tip and substrate layers of the nanoparticles, respectively (Figure 2A and B). The sharp layer is synthesized by UV induced free radical polymerization and Schiff base crosslinking between methyl methacrylate chitosan (CSMA) and SPDTA. The base layer is composed of 18 wt% polyvinyl alcohol (PVA) and sucrose solution, and the backing layer is composed of 20 wt% polystyrene (PS). Energy dispersive X-ray spectroscopy (EDS) analysis (Figure 2C) shows that the iron element from LFT NPs is uniformly distributed and confined to the substrate layer, while the carbon element distribution in the pointed layer is uniform, indicating significant spatial separation. The obtained LFT/S-MN patch consists of a 10 × 10 MN array with a span of approximately 0.5 cm2 (Figure 2D). Each needle has a clear conical shape, with a height of approximately 850 μ m and a distinct bilayer structure (Figure 2E and F). Mechanical testing shows that the breaking force of each needle is about 4N (Figure 2G), which is sufficient for effective percutaneous insertion and potential clinical applications.

Figure 2. Characterization of LFT/S-MN patch. The representative bright field and fluorescence images of (A-B) LFT/S-MN patch demonstrate the structural integrity and fluorescence labeling of the doped components. (C) The energy spectrum confirms the distribution of elements within MNs. (D) Optical images and (E, F) SEM images show the morphology of LFT/S-MNs. The yellow dashed line represents the interface between the pH responsive peak layer and the soluble substrate. (G) The force displacement curve of LFT/S-MNs indicates that its mechanical strength is suitable for skin penetration.

Evaluate the skin penetration ability of LFT/S-MN patch using ex vivo pig skin. After application, MNs successfully broke through the stratum corneum and delivered encapsulated fluorescent dyes into the skin, as shown by fluorescence imaging (Figure 3A) and confirmed by histological cross-section (Figure 3B). Observe the dissolution behavior and subsequent release of LFT NPs through fluorescence microscopy. As shown in Figure 3C, the substrate dissolved in PBS (pH 7.4) within 80 seconds, promoting rapid release of LFT NPs from the MN substrate. Considering that the microenvironment of infected wounds is usually acidic, the authors further investigated the pH responsive behavior of MN tips. In vitro release studies have shown that the apical layer gradually degrades under mild acidic conditions (pH 5.6) and almost decomposes by day 15, while minimal degradation occurs under neutral conditions (pH 7.4) (Figure 3D), indicating the acid sensitive behavior of the apical layer. Quantitatively measure drug loading and delivery efficiency (Figure 3E and F). The LFT is 145.43 ± 2.76 μ g, and the SPD is 14.96 ± 0.08 μ g. After transdermal administration, the dosage of LFT via MNs was 132.83 ± 2.91 μ g, and the dosage of SPD via MNs was 14.38 ± 0.22 μ g. The efficacy rates of both drugs exceeded 90%. In addition, the cumulative release of SPD under acidic conditions (pH 5.6) reached 88.87 ± 3.58% on day 15, significantly higher than PBS (pH 7.4), further confirming the acid responsive release profile of MN tip (Figure 3G).

Figure 3. Drug release evaluation of LFT/S-MN patch. (A) Representative bright field and fluorescence microscopy images of pig skin in vitro after LFT/S-MN patch implantation. (B) Histological sections of pig skin after MN patch implantation. (C) The optical image showed rapid dissolution of the basal layer and separation of the apical layer in PBS within 80 seconds. (D) Inverted fluorescence microscopy images showed that MN degraded within 15 days under different pH conditions (pH 7.4 and 5.6), simulating neutral and mildly acidic wound environments. (E) Quantification of LFT loading and delivery for LFT/S-MN patch. (F) Quantification of LFT/S-MN patch SPD loading and delivery. (G) The in vitro cumulative release curve of SPD over time in LFT/S-MNs. The data is expressed as mean ± SD (n=3 independent samples).

The author systematically evaluated the in vitro antibacterial effect of LFT/S-MN patch. As shown in Figure 4D, the minimum inhibitory concentration (MIC) of LFT under ultrasound irradiation (3 MHz, 0.97 W/cm2, 50% duty cycle, 30 min) is 128 μ g/mL. The LFT/S-MN patch alone exhibited moderate antibacterial activity, with an inhibition rate of 36.41 ± 0.75% against Staphylococcus aureus and 35.87 ± 1.48% against Pseudomonas aeruginosa (Figure 4A and E). In contrast, after ultrasound activation, the antibacterial rate of LFT/S-MN patches significantly increased, reaching 99.95 ± 0.04% and 99.96 ± 0.04%, respectively (Figure 4E), indicating that the bacteria were almost inactivated. SYTO 9/PI staining further validated these results, with the LFT/S-MN US group showing predominant red fluorescence, indicating widespread bacterial death (Figure 4B and F). Scanning electron microscopy (SEM) provides additional morphological evidence; The bacteria in the untreated group maintained intact and smooth membranes, while the bacteria exposed to ultrasound activated LFT/S-MN showed significant structural damage and membrane rupture (Figure 4C). Overall, these findings confirm that after ultrasound activation, LFT/S-MN patches can generate a large amount of local ROS, effectively eliminating bacteria at the lesion site.

Figure 4. In vitro antibacterial performance study of LFT/S-MN patch. (A) Representative photos of agar plates showing the antibacterial effects of LFT/S-MNs against Staphylococcus aureus and Pseudomonas aeruginosa. (B) Fluorescence microscopy images show oxidative stress caused by the production of ROS by Staphylococcus aureus and Pseudomonas aeruginosa under different treatment conditions. (C) SEM images of bacterial morphology after processing show membrane disruption and structural deformation of Staphylococcus aureus and Pseudomonas aeruginosa. (D) The minimum inhibitory concentration (MIC) of LFT against Staphylococcus aureus in LFT/S-MNs showed dose-dependent antibacterial activity. The red arrow represents the MIC value of LFT. (E) Quantitative analysis of bacterial viability after different treatments, evaluated by live/dead staining. (F) The corresponding green fluorescence intensity (FI) measurement results in the live/dead experiment further confirmed the antibacterial effect. The data is displayed as mean ± SD (n=3 independent samples). ** p < 0.01，** p < 0.001，**** p < 0.0001。 Ns indicates no significance.

To verify the in vivo therapeutic effect of LFT/S-MN patch, the authors utilized Pseudomonas aeruginosa（P. aeruginosa）Establish a mouse model of skin wound infection (Figure 5A). Infected mice were randomly divided into 8 groups (n=5 in each group), including non ultrasound group (Blank, S-MN, LF/S-MN, LFT/S-MN) and ultrasound treatment group (Blank+US, S-MN+US, LF/S-MN+US, LFT/S-MN+US). The ultrasound protocol includes 3 MHz daily irradiation, 0.97 W/cm2, 50% duty cycle, 30 minutes, for 3 consecutive days. On days 0, 1, 3, 5, and 9 after treatment, wound progression was monitored by taking photos (Figure 5B). On the first day, the LFT/S-MN+US group showed a significant reduction in purulent exudate, reflecting the combined antibacterial effect of CDT and SDT. In contrast, the non ultrasound group showed more extensive sepsis and inflammation. On the third day, after the completion of ultrasound administration, wound bed samples were collected for bacterial culture to evaluate residual bacterial load (Figure 5E). Plate counting showed that the LFT/S-MN+US group had the least colony formation, indicating stronger antibacterial activity in vivo (Figure 5F). On the 9th day, the wound healing rate of mice treated with LFT/S-MN+US was high, and tissue regeneration was significant (Figure 5C). Throughout the entire treatment process, weight monitoring showed no significant fluctuations between the groups (Figure 5D), indicating that the MN patch has good systemic biocompatibility. In summary, these results indicate that the combination of LFT/S-MN and ultrasound can effectively eliminate bacterial infections and significantly accelerate wound healing in vivo.

Figure 5. In vivo antibacterial effect and wound healing performance of LFT/S-MN patch. (A) Schematic diagram of a mouse wound model infected with Pseudomonas aeruginosa and treatment strategy using MN patch with or without US stimulation. (B) Representative photos of the wound site on days 0, 1, 3, 5, and 9 after treatment. The black dashed circle represents the initial wound boundary. (C) Quantitative analysis of wound healing over time in each treatment group (n=5). (D) Body weight monitoring of mice during treatment (n=5). (E) Agar plate photos of bacterial colonies cultured in wound beds on the third day of each group. (F) Quantification of bacterial vitality based on colony forming unit (CFU) analysis (n=3). The data is expressed as mean ± SD. *** p < 0.001，**** p < 0.0001。 Ns indicates no significance.

Conclusion:This study successfully developed a pH responsive bilayer microneedle patch (LFT/S-MN) that integrates ultrasound activated synergistic antimicrobial therapy (CDT/SDT) with immune regulation triggered by acidic microenvironment, achieving efficient treatment of infected wounds. This patch can not only rapidly release LFT NPs targeting bacteria and generate a large amount of ROS under ultrasound to clear bacteria and biofilms, but also continuously release SPD under acidic wound conditions, regulate macrophage polarization, and inhibit excessive inflammation. In vivo experiments have confirmed that this strategy can significantly accelerate wound closure, promote tissue regeneration, and has excellent biocompatibility. This work provides a new strategy with clinical translational potential for the treatment and tissue regeneration of non antibiotic dependent infected wounds.