Multifunctional Bioactive Dual-Layered Nanofibrous Matrix for Effective Breast Cancer Therapy and Enhanced Wound Healing

Abstract

Surgical resection is the primary treatment for triple-negative breast cancer (TNBC). Post-operative complications, including tumor recurrence and bacterial infections, hinder complete remission and long-term recovery. To address these challenges, a multifunctional bioactive dual-layered nanofibrous matrix (MBDL-NanoMat) featuring adaptive shape control, excellent wound adherence, tunable drug release profiles, and superior biocompatibility for post-surgical applications is developed. The MBDL-NanoMat comprises a hydrophilic (HyPhil) layer and a hydrophobic (HyPhob) layer, offering distinct functionalities. The HyPhil layer, electrospun with gelatin and copper peroxide nanoparticles (Cu NPs), rapidly releases Cu NPs to induce anticancer effects through chemodynamic therapy (CDT), ferroptosis, and cuproptosis along with antibacterial action. Near-infrared laser irradiation enhances therapeutic efficacy through photothermal therapy (PTT). The HyPhob layer ensures prolonged therapeutic effects by releasing therapeutic molecules, such as rapamycin, enabling sustained chemotherapy (CT) and antibacterial activity. This synergistic therapeutic system integrates multiple mechanisms-CT, CDT, PTT, ferroptosis, and cuproptosis-suppressing tumor recurrence and accelerating wound healing. Preclinical results demonstrated enhanced angiogenesis, collagen deposition, and dermal regeneration without systemic safety. In short, the MBDL-NanoMat platform offers a unique advantage in post-surgical TNBC care by simultaneously targeting tumor recurrence and facilitating wound healing. Further investigation of this platform can significantly improve oncological and regenerative medicine strategies.

Keywords: bacteria‐infected wound treatment; chemodynamic therapy (CDT); dual‐layered nanofibrous matrix; ferroptosis and cuproptosis; multifunctional biomaterials; photothermal therapy (PTT); triple‐negative breast cancer (TNBC).

Figure 1

Illustration of the designed MBDL‐NanoMat for adjuvant cancer therapy and wound healing application. A) Schematic diagram of current limitations in breast cancer surgery. B) The manufacturing method of the MBDL‐NanoMat and characteristics of each layer. C) Application of the MBDL‐NanoMat in postoperative treatment of breast cancer. D) Complete wound healing with multiple anticancer/antibacterial functions of the MBDL‐NanoMat.

Figure 2

Physicochemical properties of MBDL‐NanoMat. A) Schematic illustration for the fabrication of MBDL‐NanoMat. B) WCA data of the HyPhil and HyPhob layers at predetermined times (0, 5, and 15 s). Each value represents mean ± SD (n = 3). C) FE‐SEM images of HyPhil and HyPhob layers as components of the MBDL‐NanoMat. The lateral side (scale bar for original image: 100 µm, scale bar for magnified image: 5 µm) and folded side (scale bar: 100 µm) are shown. D) FT‐IR data of the HyPhil and HyPhob groups. E) Schematic diagram of the stretchable, suturable, and adhesive properties of MBDL‐NanoMat. Stretchability test (tensile strength and elongation measurement) data of HyPhil, HyPhob, and MBDL‐NanoMat groups are shown. Each value represents mean ± SD (n = 3). *p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. The adhesive performance of the designed nanofibrous matrix (as an elongation‐load plot) is presented. F) Release profiles of Cu NPs from the HyPhil layer and RPM from the HyPhob layer. Each value represents mean ± SD (n = 3). G) Infrared thermal images of HyPhil + NIR, HyPhob + NIR, and MBDL‐NanoMat + NIR groups. Temperature change data are plotted. Each value represents mean ± SD (n = 3). H) TMB assay for assessing hydroxyl radical generation of HyPHil, HyPhob, and MBDL‐NanoMat groups. Each value represents mean ± SD (n = 4). I) ESR data of HyPHil, HyPhob, and MBDL‐NanoMat groups. J) Extracellular GSH assay. Absorbance profiles of DTNB, DTNB + GSH, HyPhob + DTNB + GSH, HyPhil + DTNB + GSH, MBDL‐NanoMat + DTNB + GSH, and Cu NPs + DTNB + GSH groups are plotted. Average values are plotted (n = 3).

Figure 3

In vitro anticancer and antibacterial efficacies. A) Schematic illustration of cellular experimental system design for evaluating MBDL‐NanoMat and multiple anticancer mechanisms. B) Antiproliferation efficacy test in 4T1 cells. Each point represents mean ± SD (n = 4). ^* p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. C) Intracellular ROS assay in 4T1 cells. Relative fluorescence intensity data of the control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat +NIR groups are displayed. Data are shown as mean ± SD (n = 3). ^* p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. D) Apoptosis assay in 4T1 cells. Population (%) values of lower left (LL), lower right (LR), upper left (UL), upper right (UR), and LR + UR panels are displayed. Data are shown as mean ± SD (n = 3). ^* p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. E) Intracellular GSH assay in 4T1 cells. Relative cellular GSH levels of the control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat +NIR groups are plotted. Each point represents mean ± SD (n = 3). One‐way ANOVA with Tukey's post hoc test. F) Lipid ROS and MDA assays in 4T1 cells. Relative lipid ROS and MDA levels of the control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat +NIR groups are plotted. Each point represents mean ± SD (n = 3–4). ^* p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. G) Western blot assay in 4T1 cells. Representative images of DLAT oligomer, DLAT, FDX1, LIAS, GPX4, and β‐actin bands are shown (n = 3).

Figure 4

In vivo antitumor efficacy of the multifunctional MBDL‐NanoMat system in 4T1 tumor‐resected mouse models. A) Schematic of the tumor resection and bacterial infection model development and corresponding time schedule. B) Tumor volume profiles of control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat + NIR groups (n = 7). C) Dissected tumor weight of control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat + NIR groups. Mean and SD values are shown (n = 7). D) Tumor incidence rate profiles according to the time (n = 7). E) Body weight profiles of control, HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat +NIR groups. Each point represents mean ± SD (n = 7). F) Whole‐body thermal images of HyPhil + NIR and MBDL‐NanoMat + NIR groups in tumor‐resected mouse models. Irradiation time‐dependent temperature change profiles are presented. Each point represents mean ± SD (n = 7). ^* p < 0.05, between indicated groups. Two‐tailed t‐test.

Figure 5

In vivo wound healing potential of nanofibrous systems in the bacteria‐infected mouse model. A) Rationales for the fabrication of MBDL‐NanoMat systems for anti‐recurrence and wound healing applications. B) Photographs of wounds treated with HyPhob, HyPhil, HyPhil + NIR, MBDL‐NanoMat, and MBDL‐NanoMat + NIR groups on days 0, 3, 7, and 14. C) Changes in wound area (%) after suturing with nanofibrous matrix. Each point represents mean ± SD (n = 7). ^* p < 0.05, between indicated groups. One‐way ANOVA with Tukey's post hoc test. D) Microscopic images of H&E, Masson's trichrome, and immunofluorescence staining (VEGF and CD31) in skin tissues. E) Images of colonies of S. aureus derived from wound tissues. The number of CFU is plotted. Each point represents mean ± SD (n = 3).