New Fluorinated Cationic Cell-Penetrating Peptide FR6: Achieving Dual Breakthroughs in Intracellular Delivery and Transdermal Absorption of Peptide Therapeutics

Today, we share important research led by the team of Yiyun Cheng/Jingjing Hu from East China Normal University, published in Bioactive Materials. Addressing key delivery bottlenecks for therapeutic peptides, such as poor cell membrane permeability and difficult tissue penetration, this study developed a novel class of enhanced fluorinated cationic cell-penetrating peptides (eFPPs). Through systematic library construction and screening, the research team successfully identified fluorinated hexa-arginine (FR6) as the optimal "super-enhancer". FR6 not only efficiently promotes the intracellular delivery and endosomal escape of various peptides via a unique synergistic effect between its fluoroalkyl chains and arginine residues, but also demonstrates exceptional transdermal delivery capabilities. In animal models, FR6 significantly enhanced the anti-tumor efficacy of the pro-apoptotic peptide KLA and successfully enabled effective treatment of UVB-induced skin photoaging with the anti-aging peptide AHP-8. This work provides a novel, versatile delivery platform to overcome the biological barriers of peptide drugs.

Fig. 1. Screening of the super-enhancer from a library of eFPPs for improved cell membrane, tissue and transdermal penetration. The lead peptide FR6 exhibited the highest cellular uptake and was used to deliver pro-apoptotic peptide for cancer therapy or anti-aging peptide for treating UVB-induced skin photoaging.

01 Research Background

Peptide drugs hold immense potential for treating cancer, infections, and metabolic diseases due to their high specificity, potency, and good biocompatibility. However, their clinical application faces three core challenges: low bioavailability, weak cell membrane permeability, and poor tissue penetration. Particularly for transdermal delivery, peptides must traverse the dense stratum corneum structure, presenting a significant barrier for hydrophilic macromolecules. Traditional cell-penetrating peptides (CPPs, e.g., TAT, oligoarginines) can improve endocytosis but primarily rely on the endosomal pathway, often leading to drug degradation and reduced efficiency. While existing strategies (e.g., chemical modification, nanocarriers, physical methods) have progressed, a universal solution that efficiently overcomes multiple physiological barriers at both cellular and tissue levels is still lacking. Fluorination tag technology has previously been shown to enhance intracellular delivery, but its application has mostly been limited to overcoming a single barrier. This study aimed to develop a superior class of enhanced fluorinated penetrating peptides (eFPPs) capable of simultaneously improving the intracellular delivery, tumor tissue penetration, and transdermal absorption of peptides.

02 Innovative Highlights

Construction of a Modular Fluorinated Cationic Peptide Library:

A structurally defined peptide library was designed and synthesized by conjugating a fluorinated tag containing 13 fluorine atoms (13F) via a disulfide bond to the C-terminal cysteine of oligoarginine (Rn) or oligolysine (Kn). This systematic approach evaluated the impact of peptide chain length (n=4, 6, 8, 10, 12) and cationic type (arginine vs. lysine) on delivery efficiency. This modular design enabled precise identification of the optimal structure.

Identification of the Lead Molecule FR6 with High Efficiency and Versatility:

Through systematic screening of cellular uptake efficiency (flow cytometry, confocal microscopy), fluorinated hexa-arginine (FR6) was identified as the top performer in the library. It achieved an optimal balance between the membrane-insertion driving force of the fluorinated tag and the initial electrostatic binding and endosomal escape functions of the arginine residues. Longer or shorter chains, or other cationic types (e.g., the lysine series), were less efficient than FR6.

Elucidation of FR6's Unique Endocytosis and Endosomal Escape Capabilities:

Studies found that FR6 can be internalized via multiple pathways, and its fluorinated tag grants it far superior endosomal membrane disruption ability compared to ordinary oligoarginine (R6). Gal8-YFP endosomal damage assays and lysosomal co-localization analysis indicated that FR6 escapes acidic organelles more rapidly and effectively, ensuring active peptides are delivered to the cytoplasm. Molecular dynamics simulations further revealed that FR6's fluoroalkyl chain inserts deeper into and perturbs the lipid bilayer more than ordinary alkyl chains.

Demonstration of FR6's Dual Efficacy in Intracellular and Transdermal Delivery:

This study is the first to simultaneously apply the super-enhancing effect of a fluorinated peptide to two vastly different biological barrier scenarios: intracellular delivery (e.g., cancer therapy) and transdermal delivery (e.g., skin disease therapy). This proves the broad applicability of FR6 as a "super-enhancer," surpassing the single-function limitations of traditional delivery systems.

03 Results and Discussion

3.1 Screening and Basic Characterization of FR6

In HeLa cells, FR6 exhibited significantly higher cellular uptake efficiency than non-fluorinated R6. Notably, longer chains were not necessarily better, as FR8 efficiency decreased, indicating an optimal length (n=6). The overall efficiency of fluorinated lysine series (FKn) was lower than the fluorinated arginine series (FRn), highlighting the importance of arginine residues. Synthesis of a non-fluorinated control peptide with the same carbon chain length (CR6) confirmed its efficiency was far lower than FR6, directly proving the crucial role of the "fluorine effect" (Fig. 2).

Fig. 2. Intracellular delivery efficiency of fluorous cationic peptides.

3.2 In-depth Investigation of FR6's Mechanism of Action

Gal8-YFP endosomal damage assays showed that FR6 treatment caused more and larger Gal8-YFP fluorescent puncta, indicating its endosomal membrane disruption ability was significantly stronger than R6. Confocal microscopy time-lapse observations revealed that FR6 rapidly escaped from lysosomes (LysoTracker Red labeled), whereas R6 remained trapped for extended periods. Furthermore, endocytosis inhibitor experiments indicated FR6 can enter cells via multiple pathways. Molecular dynamics simulations provided a molecular-level explanation for these phenomena: FR6's fluoroalkyl chain interacts more strongly with and inserts deeper into the lipid bilayer (Fig. 3).

Fig. 3. Endocytosis and endosome escape behaviors of FR6.

3.3 FR6 Enhances Intracellular Delivery and Anti-tumor Efficacy of Therapeutic Peptides

Simply mixing FR6 with 12 different therapeutic peptides (including the pro-apoptotic peptide KLA) formed complexes that significantly enhanced their cellular uptake. Notably, the combination of FR6 with fluorinated KLA (FKLA) induced the strongest tumor cell apoptosis in vitro. In a 3D tumor spheroid model, the FR6/FKLA group showed the deepest tumor penetration. In a mouse model of 143B osteosarcoma, FR6/FKLA treatment most effectively inhibited tumor growth, with the FKLA dose reduced to one-third of that used in previous studies, demonstrating both high efficiency and safety (Fig. 4).

Fig. 4. FR6 promotes cytosolic and in vivo delivery of fluorous tagged peptides.

3.4 Validation of FR6 as a Super-enhancer for Transdermal Delivery

Franz diffusion cell experiments found that FR6 significantly increased the skin permeation rate and apparent permeability coefficient of the model peptide KLA in mouse skin. In vivoexperiments further confirmed that topical application of the FR6/Cy5.5KLA complex resulted in fluorescence signals penetrating to a depth of approximately 600 μm after 12 hours, an effect far superior to KLA alone or complexes with other control peptides (R6, CR6). FR6 also demonstrated universal transdermal enhancement for several other peptides (p13, p14, p15).

3.5 Application of FR6/AHP-8 in Treating Skin Photoaging

Finally, the anti-aging peptide AHP-8 (Acetyl Hexapeptide-8) was selected as the model drug. In a UVB-induced mouse skin photoaging model, topical application of FR6/AHP-8 for six consecutive days effectively alleviated skin erythema, edema, and scabbing, significantly improving the skin damage score. Histological analysis showed that FR6/AHP-8 treatment effectively restored normal epidermal thickness and significantly promoted collagen deposition (increased HYP content) while reducing oxidative stress (decreased ROS and MDA levels). Long-term safety experiments indicated FR6/AHP-8 caused no skin irritation or systemic toxicity.

04 Conclusion and Future Perspectives

This study successfully developed and validated fluorinated hexa-arginine (FR6) as a highly efficient, versatile "super-enhancer" capable of simultaneously overcoming multiple peptide drug delivery barriers at both cellular and skin levels. Its core advantage lies in the clever fusion of the strong membrane-perturbing properties of the fluorinated tag with the membrane-penetrating and endosomal escape functions of arginine residues, achieving a synergistic 1+1>2 effect. In summary, the success of FR6 marks an important evolution in peptide delivery strategies from "single-function" to "multi-functional synergy." Its simple "mix-and-use" mode of operation holds significant potential for clinical translation. Future research could focus on: ① Further resolving the fine atomic-level mechanisms of FR6's interaction with different biological membranes to provide a blueprint for rational optimization; ② Exploring FR6's application in other delivery scenarios (e.g., blood-brain barrier penetration, mucosal delivery); ③ Developing next-generation intelligent delivery systems with enhanced targeting based on the modular design concept of FR6. This work not only provides a powerful new tool for addressing the delivery challenges of peptide drugs but also opens new avenues for designing multifunctional biomaterials.

Original Article：

Rong G, Fan Q, Chen K, Cheng Y, Hu J. Fluorous oligoarginines as supra-enhancers for intracellular and transdermal peptide delivery. Bioact Mater. 2025 Dec 30;59:305-316. doi: 10.1016/j.bioactmat.2025.12.027. PMID: 41551769; PMCID: PMC12804165.