Improved Cell-Penetrating Peptide Delivers Gene-Silencing Drugs Into Cells and Reduces Skin Inflammation in Mice | Research

The takeaway

An engineered version of the cell-penetrating peptide PepFect14, called PF14-Lys, outperformed the original in delivering therapeutic oligonucleotides into cells and successfully suppressed skin inflammation in a mouse model.

In vivo inflammation suppression
PF14-Lys carrying miR-146a suppressed inflammatory responses in a mouse dermatitis model when injected subcutaneously — demonstrating real-world therapeutic potential beyond cell culture

What the researchers found

PF14-Lys (a lysine-substituted analog of PepFect14) showed superior delivery of splicing-switching oligonucleotides and siRNA compared to the original PF14 in reporter cell lines. The alpha-helical structure of PF14 was found to be essential for delivery — mutations disrupting the hydrophobic or cationic face abolished nanoparticle formation and cell entry. PF14-Lys efficiently delivered miR-146a into human primary keratinocytes, downregulating target genes. Most importantly, subcutaneously administered PF14-Lys-miR-146a nanoparticles suppressed inflammatory responses in a mouse model of irritant contact dermatitis.

Why it matters

RNA-based therapeutics (siRNA, microRNA, antisense oligonucleotides) hold enormous potential for treating diseases from cancer to inflammatory conditions, but getting them inside cells efficiently and safely remains a major barrier. Cell-penetrating peptides offer a promising solution, and this study shows that rational peptide engineering — even simple amino acid substitutions — can meaningfully improve delivery performance. The successful in vivo demonstration in skin inflammation brings this technology closer to practical clinical applications.

How the study worked

Researchers introduced point mutations into PepFect14's peptide sequence and predicted modified CPP characteristics computationally. Biophysical methods analyzed peptide structure and ability to form nanoparticles with oligonucleotides. Delivery was tested using splicing-switching oligonucleotides and siRNA in reporter cell lines, and miR-146a in human primary keratinocytes. In vivo testing involved subcutaneous injection of PF14-Lys-miR-146a nanoparticles in a mouse model of irritant contact dermatitis.

What this study cannot tell us

The in vivo testing was limited to a single mouse model (irritant contact dermatitis) with subcutaneous injection. Bioavailability, toxicity profiling, and pharmacokinetics were not detailed. The study did not compare PF14-Lys to other state-of-the-art delivery systems like lipid nanoparticles. Long-term safety and immune responses to repeated peptide administration were not assessed. The mechanism by which lysine substitution improves delivery is not fully elucidated beyond structural observations.

How to read the evidence

This is a preclinical study combining in vitro peptide characterization, cell-based delivery assays, and a single in vivo mouse model. The methodology is thorough and includes both mechanistic (structure-activity) and functional (disease model) experiments, but the findings are early-stage and have not been tested in humans.

When this study was published

Published in 2025, this represents the current state of cell-penetrating peptide engineering for oligonucleotide delivery.

The bigger picture

This work advances the cell-penetrating peptide field by providing structure-activity insights (alpha-helix required, hydrophobic/cationic faces essential) and a tangible improved delivery vehicle. As RNA therapeutics move beyond liver-targeted delivery (where lipid nanoparticles dominate) toward skin, lungs, and other tissues, peptide-based carriers like PF14-Lys could fill a critical gap. The in vivo dermatitis result also intersects with the growing interest in topical RNA therapeutics for inflammatory skin diseases.

Questions still open

How does PF14-Lys compare to lipid nanoparticles and other delivery systems for RNA therapeutics in terms of efficiency, cost, and safety?
Could PF14-Lys be used for topical (skin surface) delivery of RNA drugs rather than requiring injection?
What is the safety and immunogenicity profile of PF14-Lys with repeated administration?

Common questions

What is a cell-penetrating peptide and how does it deliver drugs?

Cell-penetrating peptides are short chains of amino acids (typically 5-30 residues) that can cross cell membranes. They work like molecular delivery vehicles — they bind to therapeutic cargo like RNA drugs, form tiny nanoparticles, and carry the cargo across the cell membrane into the cell interior where it can work. Without a delivery vehicle, most RNA drugs cannot enter cells on their own.

Why did replacing ornithine with lysine make the peptide work better?

Both ornithine and lysine are positively charged amino acids, but lysine is more commonly found in natural proteins and may interact with cell membranes and RNA differently. The substitution maintained the peptide's essential alpha-helical structure and charge distribution while improving its ability to form nanoparticles and deliver RNA cargo into cells. The exact molecular mechanism is still being studied.

Read the original research

Engineered PepFect14 analog for efficient cellular delivery of oligonucleotides.

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 184, 117872

View on PubMed ↗Publisher’s paper ↗

Citation

Biswas, Abhijit; Periyasamy, Kapilraj; Maloverjan, Maria; Porosk, Ly; Arya, Geeta; Mehta, Sudhichan; Andla, Hanna; Raid, Raivo; Kisand, Vambola; Rätsep, Margus; Wengel, Jesper; Rebane, Ana; Pooga, Margus. (2025). Engineered PepFect14 analog for efficient cellular delivery of oligonucleotides.. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 184, 117872. https://doi.org/10.1016/j.biopha.2025.117872