Harnessing Exosomes from Human Dermal Fibroblasts and Pirfenidone-exosomes as Innovative Strategies for Scarless Tissue Repair in Wound Healing

Abstract

The wound healing process often leads to scar formation that can negatively affect patients both physically and psychologically. The management and treatment of scars also place a considerable financial burden on healthcare systems. Significant efforts are being made to improve wound healing outcomes by accelerating closure while simultaneously minimizing scar formation. To facilitate scarless wound healing, developing an anti-scarring treatment that modulates dermal fibroblast activity is a promising strategy, with pirfenidone (PFD) showing potential due to its anti-fibrotic properties by targeting intracellular pathways that regulate collagen disposition. PFD, particularly when delivered via dermal fibroblast-derived exosomes, may further enhance therapeutic effectiveness and promote scarless healing. To achieve this goal, we began by isolating high-purity exosomes from in vitro cultured human dermal fibroblasts. Two common isolation methods—PEG precipitation and affinity-based techniques—were compared to identify the most efficient approach for obtaining high-purity and relatively homogenous exosomes. A range of characterization techniques, including transmission electron microscopy (TEM), atomic force microscopy (AFM), antibody arrays, and enzyme-linked immunosorbent assays (ELISA), confirmed the successful isolation of high-purity exosomes. The affinity-based method demonstrated superior performance, yielding well-dispersed and highly pure exosomes. Due to the difficulties in achieving efficient drug encapsulation in exosomes, the following chapter specifically focused on the encapsulation and formulation optimization of the antifibrotic compound PFD and explored the use of exosomes as a drug delivery platform. We optimized an active drug loading method using sonication to enhance encapsulation efficiency (EE%) and loading efficiency (LE%), ensuring that careful control of the sonication process maintained exosome integrity. The optimal formulation of PFD-exosomes achieved an EE% of 11.14% ± 1.27% and an LE of 10.01% ± 1.03%, with a particle recovery rate of exosomes at 64.21% ± 8.49%. Then, we investigated how to harness exosomes from dermal fibroblasts and PFD-exosomes as innovative strategies for achieving scarless tissue repair in wound healing. Our findings showed that exosomes enhanced fibroblast migration and proliferation through an autocrine mechanism, highlighting their potential as a stand-alone cell-free therapy for wound healing. Additionally, this study was ground-breaking in demonstrating that exosomes can improve the efficacy of PFD as a drug carrier, amplifying its anti-fibrotic effects in both in vitro and in vivo models. The in vivo results indicated that PFD-exosomes accelerated wound healing while organizing the extracellular matrix (ECM) by reducing excessive collagen deposition. Overall, PFD-exosomes present an innovative strategy for pre-scarring interventions, offering benefits of enhanced wound healing outcomes while minimizing scarring.