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

Abstract

Transdermal drug delivery has been regarded as an alternative to oral delivery and subcutaneous injection. However, needleless transdermal delivery of biomacromolecules remains a challenge. Herein, a transdermal delivery platform based on biocompatible fluorocarbon modified chitosan (FCS) is developed to achieve highly efficient non-invasive delivery of biomacromolecules including antibodies and antigens. The formed nanocomplexes exhibits effective transdermal penetration ability via both intercellular and transappendageal routes. Non-invasive transdermal delivery of immune checkpoint blockade antibodies induces stronger immune responses for melanoma in female mice and reduces systemic toxicity compared to intravenous injection. Moreover, transdermal delivery of a SARS-CoV-2 vaccine in female mice results in comparable humoral immunity as well as improved cellular immunity and immune memory compared to that achieved with subcutaneous vaccine injection. Additionally, FCS-based protein delivery systems demonstrate transdermal ability for rabbit and porcine skins. Thus, FCS-based transdermal delivery systems may provide a compelling opportunity to overcome the skin barrier for efficient transdermal delivery of bio-therapeutics.

Fig. 1. The characterization of FCS-containing nanocomplexes.

a The schematic image of FCS-containing nanocomplexes for transdermal delivery. b Representative TEM images of FCS/IgG and d FCS/OVA. c, e Size distribution and zeta potential of FCS-containing nanocomplexes including c FCS/IgG and e FCS/OVA (n = 3). f, g Circular Dichroism (CD) spectra of f IgG and g OVA pre and post FCS coating. h The relative binding affinity of aPDL1 with or without FCS measured by the standard indirect ELISA (iELISA) assay (n = 2). i Schematic illustration of Franz diffusion cell system used for the skin permeation study. j Cumulative permeation and k retention of FCS/IgG-FITC and l, m FCS/OVA-FITC permeated across the mouse skin after incubation with different FCS-containing formulations over time (n = 4 for IgG and n = 3 for OVA). Total dosage: 200 μg/cm². n Representative confocal images of mice skin treated with FITC-FCS/OVA-Cy5.5 for 12 h (n = 3). Scale bar: 200 μm. All illustrations were created with BioRender.com. Data are presented as mean ± standard deviation. Source data are provided as a Source Data file.

Fig. 2. The transdermal mechanism of FCS-containing nanocomplexes.

a Illustration of the HACAT monolayer cell model. b Effects of FCS/IgG on the TEER of the HACAT monolayer cell model (n = 3, each TEER was tested 3 times). c Immunofluorescence images of the distribution of tight junction-related protein ZO-1 on the HACAT cell membrane after being treated with FCS/IgG (n = 3). The white arrows indicated the allocation change of ZO-1. Scale bar: 10 μm. d, e Western blotting images showing ZO-1 (n = 4) and the phosphorylated level of MLC (p-MLC, n = 1) in cells after incubation with FCS/IgG. The raw figures were provided in Figs. S27 and S28. f The graphical representations of the relative intensity of MLC/pMLC with the addition of FCS/IgG (n = 1). g Representative TEM image of skin epithelium after being treated with FCS/IgG. The white arrows indicated the tight junctions (TJs) and the opening of TJs (n = 3). h Representative immunofluorescence images exhibiting the colocalization of keratin 14 and FCS/IgG-Cy5.5 (white arrows, n = 3). Scale bar: 200 μm. i The schematic image of the transdermal mechanisms. FCS-containing nanocomplexes could penetrate the skin epidermis through both paracellular and transappendageal routes. By the paracellular route, FCS could stimulate the phosphorylation of MLC and thus open the tight junction between epidermis cells by sealing strands of tight junction proteins. By the transappendageal route, FCS-containing nanocomplexes could cross the epidermis through hair follicles and sweat glands. All illustrations were created with BioRender.com. Data are presented as mean ± standard deviation. Source data are provided as a Source Data file.

Fig. 3. Transdermal delivery of aPDL1 for the treatment of B16F10 melanoma tumors.

a Schematic illusions illustrating the localized transdermal administration of FCS/aPDL1 for the treatment of B16F10 melanoma tumors. b The accumulation of FCS/¹²⁵I-IgG in the tumor at different time intervals (n = 3). c Biodistribution of FCS/¹²⁵I-IgG at 12 h based on radioactivity measurement. The total accumulation and biodistribution analysis was illustrated in Fig. S10 (n = 3). d Representative confocal images showing the accumulation of FCS/IgG-Cy5.5 in the tumor at different time intervals (n = 3). Scale bar: 500 μm. e Tumor growth curves of mice in different groups (n = 5). Growth curves were stopped when the first mouse in the related group was dead, or its tumor size exceeded 1000 mm³. f Quantification of CD4⁺ T cells and CD8⁺ T cells in the tumor after different treatments (n = 6). The representative flow cytometric plots were illustrated in Fig. S15. g–i Quantification of granzyme B⁺ (CD3⁺CD8⁺Granzyme B⁺), Ki67⁺ (CD3⁺CD8⁺Ki67⁺), and IFN-γ⁺ (CD3⁺CD8⁺ IFN-γ⁺) T cells in the tumor after different treatments (n = 4). The representative flow cytometric plots were illustrated in Figs. S16–S18. All illustrations were created with BioRender.com. Data are presented as mean ± standard deviation. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.

Fig. 4. The abscopal effect induced by transdermal delivery of combined immune checkpoint antibodies.

a Schematic illustration of transdermal co-delivery of aPDL1 and aCTLA4 to inhibit the growth of both primary and distant tumors. b–e Tumor growth curves of primary and distant tumors after different treatments (n = 5). Growth curves were stopped when the first mouse in the related group was dead, or the first mouse’s tumor size exceeded 1000 mm³. f–h Representative flow cytometry plots (f) and the related quantification of g CD4⁺ T cells and h CD8⁺ T cells in distant tumors after different treatments (n = 5). i, j Quantification of Ki67⁺ (CD3⁺CD8⁺Ki67⁺) T cells (i) and Tregs (CD3⁺CD4⁺Foxp3⁺) (j) in distant tumors after different treatments (n = 5). The representative flow cytometric plots were illustrated in Fig. S21. Data are presented as mean ± standard deviation. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.

Fig. 5. Transdermal delivery of SARS-CoV-2 vaccine.

a A schematic illustration for transdermal delivery of the SARS-CoV-2 vaccine and the triggered immune responses. After transdermal delivery, such SARS-CoV-2 nano-vaccines could activate immune cells such as DCs in the dermis, or migrate to the nearby lymph nodes for immune activation. b Schematic illustration of the experimental design showing transdermal delivery of SARS-CoV-2 vaccine. c, d DLS (c) and zeta potential (d) of FCS-based transdermal vaccines with different mass ratios from 1:1:1 to 3:1:1 (n = 3). e, f The skin penetration ability of FCS-based transdermal vaccine with different mass ratios (n = 3). Total dosage: 200 μg/cm² (g) SARS-CoV-2 specific IgG antibody titers at different time intervals determined by ELISA (n = 4). h Quantification of CD4⁺ T cells, CD8⁺ T cells in the spleen at day 28 (n = 6). i Quantification of IFN-γ⁺ secreting CD4⁺ T cells (CD3⁺CD4⁺ IFN-γ⁺) and j CD8⁺ T cells (CD3⁺CD8⁺ IFN-γ⁺) in the spleen at day 28 (n = 6). k, l Quantification of OVA-Cy5.5⁺ (CD45⁺CD11c⁺Cy5.5⁺) in DCs in k lymph nodes and l skin (n = 4). m, n Quantification of m DC maturation (CD45⁺CD11c⁺CD86⁺) and n T cell receptor (TCR) activation (CD45⁺CD3⁺CD8⁺CD69⁺) in lymph nodes (n = 4). All illustrations were created with BioRender.com. The representative flow cytometric plots were illustrated in Figs. S22 & S23. Data are presented as mean ± standard deviation. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.

Fig. 6. Evaluation of transdermal protein ability on rabbit and porcine models.

a Schematic illustration of in vivo vaccination on the rabbit model. b, c Cumulative percentages of penetration (b) and skin retention (c) of FCS/IgG-FITC permeated across the rabbit skin over time (n = 3). Total dosage: 200 μg/cm². d Schematic illustration of the experimental design showing transdermal delivery of OVA vaccination in a rabbit model. e OVA-specific IgG antibody titers in rabbit sera at different time intervals determined by ELISA (n = 3). f Schematic illustration of ex vivo skin penetration on the porcine model. g, h Cumulative percentages of penetration (g) and skin retention (h) of FCS/IgG-FITC permeated across the porcine skin over time (n = 4). Total dosage: 200 μg/cm². i Representative confocal fluorescence images and j statistical analysis to show the permeation depth FCS/IgG-Cy5.5 through the porcine skin in 12 h. Free IgG-Cy5.5 was used as a control in those experiments (n = 4). Scale bar: 100 μm. All illustrations were created with BioRender.com. Data are presented as mean ± standard deviation. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.