Transcutaneous Immunotherapy for RNAi: A Cascade-Responsive Decomposable Nanocomplex Based on Polyphenol-Mediated Framework Nucleic Acid in Psoriasis

Abstract

Skin is the first barrier against external threats, and skin immune dysfunction leads to multiple diseases. Psoriasis is an inflammatory, chronic, common, immune-related skin disease that affects more than 125 million people worldwide. RNA interference (RNAi) therapy is superior to traditional therapies, but rapid degradation and poor cell uptake are the greatest obstacles to its clinical transformation. The transdermal delivery of siRNA and controllable assembly/disassembly of nanodrug delivery systems can maximize the therapeutic effect. Tetrahedral framework nucleic acid (tFNA) is undoubtedly the best carrier for the transdermal transport of genes due to its excellent noninvasive transdermal effect and editability. The authors combine acid-responsive tannic acid (TA), RNase H-responsive sequences, siRNA, and tFNA into a novel transdermal RNAi drug with controllable assembly and disassembly: STT. STT has heightened resistance to enzyme, serum, and lysosomal degradation, and its size is similar to that of tFNA, enabling easy transdermal transport. After transdermal administration, STT can specifically silence nuclear factor kappa-B (NF-κB) p65, thereby maintaining the stability of the skin's microenvironment and reshaping normal skin immune defense. This work demonstrates the advantages of STT in RNAi therapy and the potential for future treatment of skin-related diseases.

Keywords: DNA nanotechnology; lysosomal escape; psoriasis; siRNA delivery; tetrahedral frame nucleic acid.

Figure 1

Schematic illustration showing the cascade‐responsive decomposition of STT and transcutaneous immunotherapy of psoriasis through silencing NF‐κB p65.

Figure 2

Synthesis, characterization, and stability of STT. (a) Schematic diagram showing the composition of ST and STT. (b) PAGE graph showing the successful synthesis of ST (1: ST, 2: tFNA`, 3: S1`+S2`+S3`, 4: S1`+S2`, 5: S4`, 6: S3`, 7: S2`, 8: S1` and 9: Cy5‐loaded siRNA). (c) CE graph showing the successful synthesis of ST (1: ST, 2: siRNA, 3: tFNA`, 4: S1`+S2`+S3`, 5: S1`+S2`, 6: S4`, 7: S3`, 8: S2`, and 9: S1`). (d) UV absorption spectroscopy of TA, siRNAs, tFNAs, STs and STTs. (e) Molecular size of tFNAs, STs and STTs measured by DLS. (f) TEM images of tFNAs and STTs. Scale bars are 50 nm. (g) AFM images of tFNAs and STTs. Scale bars are 25 nm and 200 nm. (h) Images of AGE showing Cy5 loaded siRNAs, STs and STTs after incubation with FBS in a concentration gradient (0%, 1%, 2%, 4%, 6%, 8%, and 10%) at 37°C for 24 h. (i) Images of AGE showing Cy5 loaded siRNAs, STs and STTs after incubation with RNase A at a concentration gradient (0, 0.1, 0.5, 1, 2, 4, 8, and 16 U/mL) at 37°C for 24 h. (j) Statistical chart showing the relative concentration of the undegraded siRNA shown in the images presented in (h) and (i). (k) Images of AGE showing Cy5 loaded STTs after incubation at room temperature and 4 °C for 0, 1, 2, 3, 4, 5, 6 and 7 days.

Figure 3

Transdermal delivery and responsive decomposition of STTs. (a) Schematic diagram showing the transdermal delivery and responsive decomposition of STTs. (b) and (c) Cellular uptakes of Cy5‐loaded siRNAs, STs and STTs in DCs and HaCaTs detected by flow cytometry. (d) Confocal fluorescence images showing the uptake of Cy5‐loaded siRNAs, tFNAs, STs, and STTs in DCs and HaCaTs for 24 h. (cytoskeleton: green; Cy5: red; nuclear: blue; 3D: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of Cy5). Scale bars are 15 µm and 10 µm. (e) Confocal fluorescence images showing the colocalization of Cy5‐loaded siRNAs, tFNAs, STs, STTs and lysosomes stained by Lysotracker in living DCs and HaCaTs after treatment for 24 h (Lysotracker: green; Cy5: red; Hoechst: blue). Scale bars are 3 µm and 10 µm. (f) i: Images of AGE showing the decomposition of Cy5‐loaded STTs under 500 U/mL RNase H for 0, 1, 3, and 6 h at pH 5.5. ii: Images of AGE showing the decomposition of Cy5‐loaded STs and STTs under RNase H in a concentration gradient (0, 50, 100, 200 and 500 U/mL, pH: 7.4) for 1 h at 37 °C. (g) Confocal fluorescence images showing the transdermal delivery of Cy5‐loaded siRNAs, tFNAs, STs, and STTs for 1 and 6 days. Scale bars are 200 µm and 50 µm.1: siRNAs, 2: tFNAs, 3: STs, 4: STTs.

Figure 4

STTs reduced TNF‐α‐induced inflammation in HaCaTs through inhibiting NF‐κB p65. (a) Statistical chart of CCK‐8 showing the cell viability of TNF‐α‐induced HaCaTs under the treatment of TA (i: 0, 12.5, 25, 50, 100, and 200 µg/mL), STs (ii: 0, 62.5, 125, and 250 nM), and other drugs (iii: 250 nM tFNAs, STs, STTs, 1000 nM siRNAs and 12.5 µg/mL TA) (n = 3). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. ii *: Blank group versus other groups; iii *: STT group versus other groups, #: ST group versus other groups. (b) The quantitative analysis of TNF‐α and IL‐6 in HaCaTs after different treatment for 24 h based on the immunofluorescence images (n = 3). (c) Immunofluorescence images of TNF‐α and IL‐6 in HaCaTs after different treatment for 24 h (cytoskeleton: green; nucleus: blue; TNF‐α and IL‐6: red; 3D thermal imaging: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of TNF‐α and IL‐6). Scale bars are 10 µm. (d) Immunofluorescence images of NF‐κB p65 and NF‐κB p‐p65 in HaCaTs after different treatment for 24 h. (cytoskeleton: green; nucleus: blue; NF‐κB p65 and NF‐κB p‐p65: red; 3D thermal imaging: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of NF‐κB p65 and NF‐κB p‐p65). Scale bars are 10 µm. (e) The quantitative analysis of NF‐κB p65, NF‐κB p‐p65 and IL‐1β in HaCaTs after different treatment for 24 h based on fluorescence microscopic images(n = 3). (f) Immunofluorescence images of IL‐1β in HaCaTs after different treatment for 24 h. (cytoskeleton: green; nucleus: blue; IL‐1β: red; 3D thermal imaging: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of IL‐1β). Scale bars are 10 µm. (g) WB analysis of the NF‐κB p65, NF‐κB p‐p65 and IL‐1β expression level. (h) The relative protein expression intensity NF‐κB p65, NF‐κB p‐p65 and IL‐1β in HaCaTs after different treatment for 24 h (n = 3). 1: Control, 2: TNF‐α, 3: TNF‐α+tFNAs, 4: TNF‐α+siRNAs, 5: TNF‐α+STs, 6: TNF‐α+TA, 7: TNF‐α+STTs; β‐actin was used as an internal control. Statistic differences are significant between the two groups (p < 0.05). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. *: STT group versus other groups, #: ST group versus other groups.

Figure 5

STTs reduced inflammation and maturation of DCs through inhibiting the expression of NF‐κB. (a) SEM images showing the morphological changes of DCs under different treatments. Scale bars are 10 µm and 20 µm. (b) ELISA detection of TNF‐α, IL‐6 and IL‐23 in DCs after different treatment for 24 h (n = 3). (c) and (d) Immunofluorescence images of TNF‐α, IL‐6, NF‐κB p65 and NF‐κB p‐p65 in DCs after different treatment for 24 h. (cytoskeleton: green; nucleus: blue; TNF‐α, IL‐6, NF‐κB p65 and NF‐κB p‐p65: red; 3D thermal imaging: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of TNF‐α, IL‐6, NF‐κB p65 and NF‐κB p‐p65). Scale bars are 10 µm. (e) Flow cytometry images and statistical analysis showed the changes of DCs labeled with CD80 (n = 3). (f) Flow cytometry images and statistical analysis showed the changes of DCs labeled with CD86 (n = 3). (g) WB analysis of the NF‐κB p65, NF‐κB p‐p65, TNF‐α, IL‐6, IL‐12 and IL‐1β expression level. (h) The relative protein expression intensity of NF‐κB p65, NF‐κB p‐p65, TNF‐α, IL‐6, IL‐12 and IL‐1β in DCs after different treatment for 24 h (n = 3). 1: Control, 2: LPS, 3: LPS+tFNAs, 4: LPS+siRNAs, 5: LPS+STs, 6: LPS+TA, 7: LPS+STTs; β‐actin was used as an internal control. Statistic differences are significant between the two groups (p < 0.05). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Therapeutic effect of STTs on IMQ‐induced psoriasis in mice. (a) Schematic diagram of IMQ‐induced psoriasis in mice. (b) General views of IMQ‐induced psoriasis in mice after treatment with Dex, siRNAs, tFNAs, STs, TA, and STTs for 6 days. (c)‐(f) The cumulative total score, erythema score, scales score, and thickness score change of IMQ‐induced psoriasis in mice after treatment with Dex, siRNAs, tFNAs, STs, TA, and STTs for 0, 1, 2, 3, 4, 5, and 6 days based on PASI rules. (n = 5). (g) Representative histopathological images of skin sections from normal and IMQ‐induced mice treated with Dex, siRNAs, tFNAs, STs, TA, and STTs on days 6 according to HE images (thickness of skin epidermis: green arrow). Scale bars are 50 µm. (h) Statistical analysis showing the thickness of the skin epidermis according to HE images (n = 5). (i) Representative IHC images of skin sections from normal and IMQ‐induced mice treated with Dex, siRNAs, tFNAs, STs, TA, and STTs on days 6 according to Ki67 staining. Scale bars are 100 µm. (j) Statistical analysis showing the positive area changes of Ki67 according to IHC images (n = 5). (k) Representative IHC of skin sections from normal and IMQ‐induced mice treated with Dex, siRNAs, tFNAs, STs, TA, and STTs on days 6 according to NF‐κB p65 staining. Scale bars are 100 µm and 10 µm. 1: Control, 2: IMQ, 3: IMQ+Dex, 4: IMQ+tFNAs, 5: IMQ+siRNAs, 6: IMQ+STs, 7: IMQ+TA, 8: IMQ+STTs; β‐actin was used as an internal control. Statistic differences are significant between the two groups (p < 0.05). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Anti‐inflammation and anti‐maturation of DCs by STTs on IMQ‐induced psoriasis. (a) Schematic diagram showing the mechanisms of inflammation induced by IMQ in mice (orange arrow) and the mechanisms of anti‐inflammation progression of STTs (blue arrow) in psoriasis. (b) and (c) Immunofluorescence images and its quantitative analysis of TNF‐α, IL‐1β and IL‐6 in skin tissues after different treatment for 6 days (cytoskeleton: green; nucleus: blue; TNF‐α, IL‐1β and IL‐6: red; 3D thermal imaging: 3D reconstruction of fluorescence microscopic images based on fluorescence intensity of TNF‐α, IL‐1β and IL‐6; n = 5). Scale bars are 100 µm. (d) and (e) Flow cytometry images and its statistical analysis showing the changes of DCs labeled with CD80 and CD86 (n = 4). (f) ELISA detection of TNF‐α, IL‐6, IL‐17A and IL‐23 in serum of psoriatic mice after different treatment for 6 days (n = 3). 1: Control, 2: IMQ, 3: IMQ+Dex, 4: IMQ+tFNAs, 5: IMQ+siRNAs, 6: IMQ+STs, 7: IMQ+TA, 8: IMQ+STTs; β‐actin was used as an internal control. Statistic differences are significant between the two groups (p < 0.05). Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.