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Pullulan-based polymeric nanosystem for KEAP1 siRNA delivery: Mitigating ROS and promoting wound healing in vitro
Gülşen Bayrak1, Dilara Karaguzel1, Cagatay Karaaslan1
1Hacettepe University, Faculty of Science, Department of Biology, Molecular Biology Section, Beytepe Campus, Ankara 06800, Turkiye.
Colloids and Surfaces. B, Biointerfaces
|December 19, 2025
Summary
New Pullulan-poly(ethyleneimine) nanoparticles (Pullulan-PEI-NPs) effectively deliver Kelch-like ECH-associated protein 1 (KEAP1) siRNA. This safe, non-viral gene therapy approach mitigates oxidative stress and enhances wound healing in cells.
Area of Science:
- Biomaterials Science
- Gene Therapy
- Cell Biology
Background:
- Current siRNA delivery methods face challenges with cellular toxicity.
- Developing safe and efficient non-viral vectors is crucial for therapeutic applications.
- Oxidative stress plays a significant role in impaired wound healing.
Purpose of the Study:
- To evaluate Pullulan-poly(ethyleneimine) nanoparticles (Pullulan-PEI-NPs) as a non-viral delivery system for Kelch-like ECH-associated protein 1 (KEAP1) siRNA.
- To investigate the potential of KEAP1 siRNA delivery via Pullulan-PEI-NPs in mitigating oxidative stress and promoting wound healing in HaCaT cells.
- To assess the cellular antioxidant mechanisms influenced by KEAP1 silencing.
Main Methods:
- Synthesis and characterization of Pullulan-PEI-NPs using Dynamic Light Scattering, Nanoparticle Tracking Analysis, FTIR, and electron microscopy.
- Formation of polyplexes between Pullulan-PEI-NPs and KEAP1 siRNA via electrostatic interactions.
- Assessment of siRNA delivery efficacy through cellular uptake, mRNA expression, and protein level analysis (KEAP1, NRF2).
- Evaluation of ROS mitigation and wound healing capacity using scratch assays in HaCaT cells.
Main Results:
- Pullulan-PEI-NPs demonstrated a high siRNA silencing efficiency (72%), comparable to commercial agents (Lipofectamine 3000 at 80%).
- KEAP1 siRNA delivered by Pullulan-PEI-NPs effectively mitigated reactive oxygen species (ROS) induced by TBHP and H2O2.
- Significant facilitation of wound healing was observed, with 98.9% wound closure in HaCaT cells via scratch assay.
- Successful cellular uptake and modulation of KEAP1 and NRF2 protein levels confirmed effective gene silencing.
Conclusions:
- Pullulan-PEI-NPs serve as a promising, safe, and adaptable non-viral platform for gene therapy.
- KEAP1 siRNA delivery using Pullulan-PEI-NPs is effective in reducing oxidative stress and promoting wound regeneration.
- This study establishes the therapeutic relevance of KEAP1 silencing in redox-regulated wound healing and highlights the potential of this nanocarrier system.
