5-ALA photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy

Hypertrophic scar (HS) is a somatopsychic disease that significantly affects quality of life. 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) holds promise for HS treatment, while challenges like poor transdermal delivery and rapid metabolism into non_xfffe_photosensitive heme restrict its effectiveness. Inspired by the natural phenomenon of a whale fall nourishing life, this study innovatively repurposes 5-ALA metabolic waste, heme, as a “whale-like” energy source to drive ferroptosis, thus establishing a zero-waste therapy. This is achieved by encapsulating 5-ALA and baicalin within human H-ferritin (HFn), subsequently incorporated into polyvinylpyrrolidone (PVP) microneedles (FAB@MN). The FAB@MN exhibits excellent targeting towards hypertrophic scar fibroblasts (HSFs) with pH-responsive programmed drug release. Upon application, 5-ALA is converted into PpIX to initiate PDT. Subsequently, baicalin is released, triggering ferroptosis while also synergizing with the ROS and heme accumulated during PDT to overstimulate the HO-1-heme metabolic axis. This activation releases Fe2+ and CO, further potentiating ferroptosis. Moreover, the enhanced ferroptotic response driven by FAB@MN induces mitophagy, leading to increased Fe²⁺ release from the mitochondria. Unlike conventional PDT only focuses on immediate effects, this approach uses 5-ALA photodynamic waste to fuel a sustained ferroptosis response post-PDT, offering new avenues for HS treatments. This research project has been approved by the Ethics Committee of Chongqing Medical University (CQHS-REC-2024-058). Nine HS tissues and the matched normal skin tissues were provided by patients undergoing surgical treatment, and each patient has voluntarily signed an informed consent form. All participants in this study did not have any known systemic diseases and were not receiving any treatments that could potentially affect the study results. In brief, tissues were first cut into 3 mm-thick slices, and then digested overnight with 0.25% neutral protease II. Subsequently, the epidermal layer was peeled off, and further digestion was carried out for 4-6 hours at 37°C with 0.2% Collagenase II. After digestion, cells were filtered and collected using a cell strainer with a 75μm pore size, then resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS and cultured at 37°C in an atmosphere of 5% CO2. The method of isolating fibroblasts from normal skin was similar to the aforementioned process for obtaining HSFs with minor modifications. HSFs were seeded at a density of 1 × 10^5 cells/well in a 24-well plate and cultured for 24 hours in DMEM containing 10% FBS. Subsequently, the cells were treated with FAB NPs for 4 hours, followed by 10 mins of illumination, and then incubated for another 24 hours. Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and the integrity of the RNA was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA samples were prepared using total RNA as a template. Initially, mRNA was extracted from total RNA using magnetic beads coated with poly-T oligonucleotides. This mRNA was then fragmented under high temperatures in the presence of divalent cations, using a 5X First Strand Synthesis Reaction Buffer. The synthesis of the first cDNA strand was achieved using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). This was followed by the synthesis of the second cDNA strand using DNA Polymerase I and RNase H. Exonuclease and polymerase activities were employed to transform remaining overhangs into blunt ends. Subsequent to the adenylation of the 3' ends of the DNA fragments, adapters containing hairpin loop structures were attached, setting the stage for hybridization. cDNA fragments ranging in size from 370-420 bp were isolated using the AMPure XP system (Beckman Coulter, Beverly, USA). Amplification of these fragments was then carried out using Phusion High-Fidelity DNA Polymerase, along with universal PCR primers and an Index (X) Primer. The PCR products were purified within the AMPure XP system, and the library's quality was evaluated using an Agilent Bioanalyzer 2100 system. Indexed samples were clustered on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's guidelines. Post-clustering, the library was sequenced on the Illumina Novaseq platform, producing 150 bp paired-end reads and thereby acquiring the sequence data of the fragments.