Pullulan-based polymeric nanosystem for KEAP1 siRNA delivery: Mitigating ROS and promoting wound healing in vitro

Colloids Surf B Biointerfaces. 2026 Apr:260:115352. doi: 10.1016/j.colsurfb.2025.115352. Epub 2025 Dec 11.

Abstract

Recently, siRNA delivery has shown considerable potential for therapeutic use. While commercial options facilitate efficient delivery, they can be toxic to cells to a certain degree. Therefore, using relatively less toxic and safe delivery vehicles can increase the efficiency of therapy. This study examines the utilization of Pullulan-poly(ethyleneimine) nanoparticles (Pullulan-PEI-NPs) as an effective non-viral delivery system for Kelch-like ECH-associated protein 1 (KEAP1) siRNA, with the aims of mitigating oxidative stress and facilitating wound healing in HaCaT cells by enhancing cellular antioxidant mechanisms. The Pullulan-PEI-NPs were synthesized and characterized with Dynamic Light Scattering, Nanoparticle Tracking Analysis, Fourier Transform Infrared Spectroscopy, and electron microscopy techniques. The polyplexes were formed via electrostatic interaction between Pullulan-PEI-NPs and KEAP1 siRNA, and successful siRNA delivery was confirmed by cellular uptake analysis, mRNA expression studies, and KEAP1 and nuclear factor E2-related factor 2 (NRF2) protein level assessments. Pullulan-PEI-NPs had a silencing efficiency of 72 %, which is very close to the commercial transfection agent Lipofectamine 3000 (80 %). In following studies, KEAP1 siRNA delivery with Pullulan-PEI-NPs mitigates TBHP and H2O2 mediated ROS and facilitates wound healing with 98.9 % wound closure in a scratch assay on HaCaT cells. Obtained results highlight the therapeutic relevance of KEAP1 silencing in redox-regulated wound regeneration. This is the first report showing that Pullulan-PEI based cationic polymeric nanoparticles can be used to deliver KEAP1 siRNA and evaluate its role in mitigating ROS and increasing wound healing. These results highlight the promise of Pullulan-PEI-NPs as a safe and adaptable platform for non-viral gene therapy applications.

Keywords: KEAP1 siRNA; Polymeric nanoparticles; ROS; Wound healing; siRNA delivery.

MeSH terms

  • Glucans* / chemistry
  • HaCaT Cells
  • Humans
  • Kelch-Like ECH-Associated Protein 1* / antagonists & inhibitors
  • Kelch-Like ECH-Associated Protein 1* / genetics
  • Kelch-Like ECH-Associated Protein 1* / metabolism
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • Nanoparticles* / chemistry
  • Oxidative Stress / drug effects
  • Particle Size
  • Polyethyleneimine / chemistry
  • Polymers* / chemistry
  • RNA, Small Interfering* / administration & dosage
  • RNA, Small Interfering* / chemistry
  • RNA, Small Interfering* / genetics
  • Reactive Oxygen Species* / metabolism
  • Wound Healing* / drug effects

Substances

  • Glucans
  • pullulan
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Kelch-Like ECH-Associated Protein 1
  • KEAP1 protein, human
  • Polyethyleneimine
  • NF-E2-Related Factor 2
  • Polymers