Non-invasive transdermal delivery of biomacromolecules with fluorocarbon-modified chitosan for melanoma immunotherapy and viral vaccines

The study addresses the long-standing challenge of non-invasive transdermal delivery of hydrophilic biomacromolecules (proteins, peptides, nucleic acids), which are typically too large and polar to cross the skin barrier efficiently. While transdermal administration offers advantages (non-invasiveness, avoidance of first-pass metabolism, painless and self-administrable), current clinical enhancers mainly work for small hydrophobic drugs. Existing physical methods (ultrasound, electroporation, thermal ablation, microdermabrasion, microneedles) or chemical enhancers (cell-penetrating peptides, ionic liquids, hyaluronic acid-based approaches) have limitations such as skin damage, operator dependence, manufacturing complexity, or insufficient efficacy for large proteins. Motivated by prior success of fluorocarbon-modified chitosan (FCS) in enhancing transmucosal delivery, the authors hypothesize that FCS can form nanocomplexes with biomacromolecules to penetrate skin via paracellular and transappendageal routes, enabling efficient transdermal delivery for melanoma immunotherapy and vaccination (e.g., SARS-CoV-2).

The paper reviews transdermal enhancement strategies: chemical enhancers including membrane-penetrating peptides can deliver small proteins like insulin but with limited efficiency and poor performance for larger proteins. Physical devices (cavitational ultrasound, electroporation, sonophoresis) are difficult for self-operation and may damage skin. Microneedles show promise for drugs and vaccines but entail complex manufacturing/quality control and can still cause skin injury and infection risks. Newer non-invasive platforms (ionic liquids, hyaluronic acid-based systems) that open tight junctions have shown limited efficacy. Lipid nanocarriers (e.g., ethosomes) have been used against skin tumors but further improvements in safety and efficiency are desirable. Chitosan, a biodegradable cationic polymer with mucoadhesive and antibacterial properties, and its fluorocarbon-modified form (FCS) previously enhanced transmucosal delivery in bladder cancer therapy, motivating investigation for transdermal protein delivery.

• Materials and synthesis: Fluorocarbon-modified chitosan (FCS) was synthesized by grafting perfluoroalkyl carboxylic acid (perfluoroheptanoic acid) to chitosan via EDC/NHS-mediated amide coupling (fluorocarbon substitution ~4.9%). Purification by dialysis (MWCO 3.5 kDa) and lyophilization; substitution characterized by ninhydrin colorimetry and 19F NMR.
• Nanocomplex formation: FCS mixed with proteins (IgG, OVA, aPDL1, aCTLA4, S1) at various mass ratios (from 1:4 to 4:1) for 30 min to self-assemble into nanocomplexes.
• Characterization: Transmission electron microscopy (TEM) for morphology; dynamic light scattering (DLS) for hydrodynamic size and zeta potential; circular dichroism (CD) to assess protein secondary structure preservation; ELISA to confirm antibody binding affinity post-complexation.
• Ex vivo transdermal assessment: Franz diffusion cells using fresh mouse, rabbit, and porcine skins. FITC-labeled proteins measured over time in receptor chamber; dermal retention quantified by tissue digestion. Skin integrity monitored by resistance. Confocal imaging of skin slices to visualize penetration and colocalization.
• Mechanism studies: In vitro HaCaT keratinocyte monolayer model for transepithelial electrical resistance (TEER) monitoring before, during, and after FCS/protein exposure to assess reversible tight junction opening. Immunofluorescence for ZO-1 distribution; Western blots for ZO-1 and phosphorylated myosin light chain (p-MLC). TEM of skin epithelium to visualize tight junction opening. Assessment of transappendageal route via keratin 14 counterstaining of hair follicles/sweat glands and colocalization with FCS/protein. Transcellular contribution tested via apical exocytosis assay and clathrin inhibition (chlorpromazine) in Franz diffusion.
• Ointment formulation and in vivo administration: FCS/protein nanocomplex solution mixed 1:1 (w/w) with Aquaphor ointment to form a milk-like ointment; topically applied for 12 h and covered with 3M transparent film. Mouse treatment area ~3.14 cm²; rabbit ~50.24 cm².
• Melanoma therapy in mice: B16F10 tumors established in female C57BL/6 mice. Treatment groups included untreated, intravenous (i.v.) free aPDL1, transdermal CS/aPDL1, and transdermal FCS/aPDL1. Dosing: 20 µg antibody per mouse every 2 days, three times (topical for 12 h each). For combination therapy, bilateral tumors were established; groups received aPDL1 and/or aCTLA4 (20 µg each) by i.v. or transdermal FCS co-delivery on same schedule. Tumor growth and survival monitored; immune profiling by flow cytometry (CD4+, CD8+, Granzyme B+, Ki67+, IFN-γ+; Tregs).
• Biodistribution: 125I-labeled IgG in ointment for topical application; gamma counting to quantify tumor and organ accumulation over time; confocal imaging of IgG-Cy5.5 in tumors.
• Vaccine studies in mice: FCS mixed with SARS-CoV-2 S1 protein and PolyIC to form FCS/S1/PolyIC. Formulation optimization using OVA as model antigen at ratios 1:1:1 to 3:1:1 assessed by DLS, zeta potential, and Franz diffusion; 2:1:1 selected. Vaccination regimens: transdermal FCS/S1/PolyIC three administrations over 2 weeks (20 µg S1 and 20 µg PolyIC each time) vs subcutaneous (s.c.) S1/PolyIC twice (20 µg S1, 50 µg PolyIC per injection). Outcomes: anti-S1 IgG titers by ELISA over 60 days; splenic T cell subsets and IFN-γ+ CD4+/CD8+ at day 28; memory T cells at day 90 with single boost at day 75; cytokines (IL-12p40, IFN-γ). Antigen uptake by DCs in skin and lymph nodes assessed using OVA-Cy5.5; DC maturation (CD86+) and T cell activation (CD69+).
• Rabbit vaccination: Ex vivo Franz diffusion on rabbit skin (fat removed). In vivo vaccination with topical FCS/OVA/PolyIC vs intramuscular (i.m.) OVA/PolyIC at identical doses on days 0, 14, 30; anti-OVA IgG titers measured.
• Porcine ex vivo: Franz diffusion on porcine skin (fat removed); confocal imaging of IgG-Cy5.5 penetration.
• Statistics: Data as mean ± SD; one-way ANOVA with Tukey post-hoc for significance where indicated.

• FCS formed nanocomplexes with proteins (e.g., IgG, OVA) around ~200 nm at 1:1 mass ratio with positive zeta potentials that increased with FCS content; protein secondary structure and antibody binding affinity were largely preserved post-complexation.
• Ex vivo mouse skin: Optimal mass ratio 1:1 achieved highest penetration. FCS/IgG total dermal penetration (permeation + retention) reached ~36 µg/cm² (~6× free IgG). FCS/OVA reached ~55 µg/cm² (~11× free OVA). Confocal imaging showed colocalization of FCS and OVA in dermis; free OVA showed negligible penetration.
• Mechanism: FCS/protein nanocomplexes transiently decreased TEER in HaCaT monolayers, with recovery within 12 h after removal, indicating reversible tight junction opening. ZO-1 distribution became discontinuous without total expression change; p-MLC was upregulated, suggesting cytoskeletal rearrangement leading to paracellular transport. TEM showed enlarged intercellular spaces/tight junction openings. Colocalization with hair follicles and sweat glands indicated a significant transappendageal contribution. Transcellular route was negligible (apical exocytosis <2%; clathrin inhibition had no effect in Franz assays).
• In vivo melanoma delivery: Topical FCS/125I-IgG led to markedly higher tumor accumulation and lower off-target organ radioactivity compared to free IgG in ointment. Tumor accumulation of FCS/IgG peaked at >120 %ID/g at 12 h; signal decreased after ointment removal. FCS/aPDL1 topical treatment (20 µg ×3, q2d) significantly inhibited tumor growth and improved survival versus i.v. aPDL1 or CS/aPDL1, with increased intratumoral CD4+ and CD8+ T cells and elevated CD8+ granzyme B+, Ki67+, and IFN-γ+ fractions.
• Combination checkpoint therapy: Transdermal co-delivery of FCS/aPDL1/aCTLA4 outperformed single-antibody transdermal treatments, inhibiting both primary and distant (abscopal) tumors. In contrast, i.v. co-administration caused 50% mortality after the second injection (likely systemic toxicity). In distant tumors, CD8+ and Ki67+CD8+ T cells increased and Tregs decreased after FCS/aPDL1/aCTLA4 treatment.
• Vaccination in mice: FCS/OVA/PolyIC at 2:1:1 showed optimal skin permeability among tested ratios. Transdermal FCS/S1/PolyIC elicited anti-S1 IgG titers comparable to s.c. S1/PolyIC through 30–60 days and induced stronger IFN-γ secretion in both CD4+ and CD8+ T cells at day 28. Effector memory CD4+ and CD8+ T cells were higher at day 90 post-boost, and serum IL-12p40 and IFN-γ were elevated versus s.c. group. DCs beneath skin showed higher antigen (OVA) uptake after transdermal vaccination; DC maturation and T cell activation in lymph nodes were similar between transdermal and s.c. routes.
• Larger-animal feasibility: Rabbit ex vivo total penetration for FCS/IgG ~32 µg/cm²; in vivo transdermal FCS/OVA/PolyIC generated anti-OVA titers approaching i.m. vaccination (notably similar by days 30 and 60). Porcine ex vivo total penetration reached ~60 µg/cm²; confocal imaging confirmed deeper dermal penetration with FCS/IgG-Cy5.5 versus free IgG.
• Safety: Minimal skin irritation observed after repeated topical applications; transdermal treatments showed negligible abnormalities compared to systemic co-administration toxicity.

The findings demonstrate that fluorocarbon-modified chitosan enables efficient, needleless transdermal delivery of biomacromolecules by exploiting reversible paracellular opening (tight junction modulation via p-MLC upregulation) and transappendageal transport through hair follicles and sweat glands, while preserving protein structure and function. This mechanism allowed substantial dermal and intratumoral accumulation of antibodies, translating to potent local immune activation and tumor control. Compared to intravenous delivery, topical FCS/aPDL1 achieved higher tumor infiltration of effector T cells with reduced systemic exposure, mitigating toxicity. Co-delivery of aPDL1 and aCTLA4 via FCS induced abscopal effects, likely through enhanced antigen release and presentation, increased cytotoxic T cell priming, reduced Tregs, and effective checkpoint blockade within tumor-draining lymph nodes. For vaccination, despite lower absolute antigen deposition in lymph nodes relative to s.c. injection, transdermal FCS/S1/PolyIC directly engaged cutaneous DCs, resulting in humoral immunity comparable to s.c. vaccination and superior cellular responses and immune memory, which are critical for viral clearance. The successful ex vivo and in vivo performance in rabbit and ex vivo in porcine skin supports translational potential. Overall, FCS-based systems offer a practical, self-administrable platform for macromolecule delivery across skin barriers, relevant for cancer immunotherapy and viral vaccination.

This work introduces a versatile FCS-based nanocomplex platform that non-invasively delivers biomacromolecules across intact skin without auxiliary physical devices. The approach enables high intratumoral antibody accumulation and robust anti-tumor immunity, including abscopal control with dual checkpoint blockade, while reducing systemic toxicity relative to intravenous administration. As a transdermal vaccine carrier, FCS/S1/PolyIC elicits antibody titers comparable to subcutaneous injection and stronger cellular and memory responses, with evidence of direct activation of dermal DCs. The platform demonstrates feasibility in mouse, rabbit, and porcine skins, highlighting translational promise. Future studies should include viral challenge models for efficacy confirmation, detailed safety and pharmacokinetic profiling in larger animals, optimization of dosing/formulations for human skin, and extension to other therapeutic biomacromolecules (e.g., enzymes, nucleic acids).

• Viral challenge experiments for the SARS-CoV-2 vaccine were not performed due to facility constraints, limiting direct efficacy assessment against infection.
• Some permeability data for larger animals (porcine) are ex vivo; in vivo porcine/human translatability remains to be established.
• Long-term safety, local skin tolerability over extended dosing, and systemic pharmacokinetics were not comprehensively characterized.
• Mechanistic insights emphasize paracellular and transappendageal pathways but do not fully quantify their relative contributions in vivo across species.
• Optimization focused on select protein/adjuvant combinations and ratios; broader generalizability across diverse biomacromolecules needs further validation.