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.


Materials.
The DC and HaCaT were obtained by ATCC.The single-strand DNAs (ssDNAs), siRNA, RNase H, RNase A and primer sequences designed for quantitative PCR were purchased from and purified by Sangon Biotech (Shanghai, China).TA and TNF-α were purchased from MCE (Shanghai, China).The lipopolysaccharides (LPS) was purchased from Sigma (Missouri, USA).

Isolation of BMDCs
After cervical dislocation, c57 mice (6-8 weeks old, male) were euthanized and soaked in 75% alcohol for 5 min.After fully exposing and isolating the leg bones, the cells in the bone marrow cavity were washed out with cold PBS by centrifuging for 5 min at 1500 rpm.Next, the cells were incubated with diluted red blood cell lysate and then washed with PBS.The final collected cells were resuspended in RPMI 1640 culture medium containing 20 ng/mL IL-4, GM-CSF and 10% FBS.We changed the medium on the 3rd and 5th days.On the seventh day, the cells suspended in the culture dish were removed for storage and were considered BMDCs.

Cell Culture
The DCs and HaCaTs, were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin.In addition, BMDCs extracted from mice were cultured in RPMI 1640 medium containing 20 ng/mL GM-CSF, IL-4 and 10% FBS.All cells were cultured in the incubator containing 5% CO 2 and 95% air at 37 °C.Next, an in vitro model of psoriasis was established using HaCaTs.According to the experimental requirements, we divided the cells into the following seven groups for the experiment: control group: no special treatment; TNFα group: HaCaTs were incubated in medium supplemented with 20 ng/mL TNF-α for 36 h; Other drug groups: HaCaTs were cultured in the medium containing 20 ng/mL TNF-α for 12 h and then continued for 24 h in medium supplemented with additional drugs (250 nM tFNAs, 1000 nM siRNAs, 250 nM STs, 12.5 μg/mL TA, STTs (250 nM STs, 12.5 μg/mL TA) ).
In addition, it is necessary to establish an inflammation model using DCs to verify the antimaturation effect of STTs on DCs.According to the experimental requirements, we divided the cells into the following seven groups for the experiment: control group: no special treatment; LPS group: DCs were cultured in a medium containing 100 ng/mL LPS; Other drug groups: DCs were precultured in the medium containing drugs (250 nM tFNAs, 1000 nM siRNAs, 250 nM STs, 12.5μg/mL TA, STTs (250 nM STs, 12.5 μg/mL TA) ) for 4 h, on this basis, DCs were continued to be cultivated for 24 h in the medium with additional 100 ng/mL LPS.

CCK-8 Assay
The HaCaTs were treated in groups: HaCaT cell was cultured in the medium with or without 20 ng/mL TNF-α for 12 h and then continued for 24h in medium supplemented with additional drugs.Later, at 37 ℃, the cells were cultured in 10% (v/v) CCK-8 solution.Finally, the absorbances were measured at 450 nm to determine the cell viability.

Migration Assay
Scratch experiments were used to investigate cell migration behavior.HaCaTs (2 × 10 5 ) were cultured in12 plates.After washing with PBS, a pipette tip was used to create a cross-shaped scratch in each well.The cells were rewashed three times and cultured with RPMI 1640 medium without FBS containing different drugs for 24 h.Images of the samples were taken after 0 h and 24 h of treatment.

Cell Apoptosis Assay
The KeyGEN's Annexin V-FITC Apoptosis Detection Kit (Jiangsu, China) was selected to detect apoptosis of the HaCaTs.Then, the outcomes were analyzed by flow cytometry.After drug and TNF-α treatment, the cells were cleaned, digested and harvested into the corresponding centrifugal tubes.After twice cleaning, the cells were then resuspended in binding buffer (500 μL/tube) at 4 °C.In the dark, dyestuffs were added in sequence.
Approximately 10−15 min later, the samples were tested by flow cytometry.

Quantitative PCR (qPCR)
The cells treated as previous were harvested in cellular lysis buffer (TRIzol, Invitrogen, Carlsbad, CA, USA).According to the manufacturer's instructions (RNA pure total RNA extraction kit, RP1202, BioTeke, Jiangsu, China), the RNA was extracted and purified.RNA was reverse transcribed into the cDNA based on PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan).Then, the cDNA was added to the Mix required by qPCR.The program set by qPCR was run to obtain the final RNA expression result.The primer sequences of TNFα, IL6, IL-1β, and NF-κB are provided in Table S3.

Cell Morphology Detection
In order to detect the mature state of DCs, we further analyzed the morphology of DCs through scanning electron microscopy (SEM).DCs were seeded into a 24 well plate with climbing plates and grouped according to the requirements mentioned earlier.After the cell culture is completed, the cells are fixed with cold 4% paraformaldehyde for 20 min.Then, the cell samples were subjected to dehydration treatment, specifically, 25% ethanol treatment for 15 min, 50% ethanol treatment for 15 min, 75% ethanol treatment for 15 min, followed by 100% ethanol treatment for 15 min.The samples were further dried and sprayed with gold, and finally images were obtained through SEM.

Determination of the Maturation Status of DCs
Flow cytometry was used to analyze the maturation status of DCs in spleen tissue and BMDCs extracted in vitro.The obtained spleen was immediately ground and passed through a 70 μm cell sieve to obtain a single-cell suspension.BMDCs were collected after grouping and culturing as mentioned earlier to obtain the single cells to be determined.After washing with PBS and performing lysis of red blood cells, these cells were resuspended in flow cytometry buffer and stained with 1*10 6 cells per tube.After being blocked by FCR, the cells were incubated with anti-mouse antibodies (MHC II-PE, CD11c-FITC, CD86-PE-Cy7, CD45-APC-Cy7, and CD80-APC) in the dark for 30 min at room temperature.Finally, DCs labeled with CD80 and CD86 were analyzed using flow cytometry (BD LSRFortessa, BD, USA).

Distribution of Cy5-labeled siRNA, tFNAs, ST, and STT In Vivo
To ensure that STT can enter and remain in the skin, adult BALB/c mice (6-8 weeks old, male) were selected.The skin of mice was subjected to hair removal and then coated with Cy5-labeled siRNAs, tFNAs, STs, and STTs for 24 h.The next day, we wiped the surface of the mouse skin clean to ensure that there were no residual drugs.Next, mice anesthetized with isoflurane were placed under an IVIS (Bio Real Quick View 3000, Austria) for fluorescence imaging of the skin.

Histological Staining
The obtained skin tissue was fixed in a 4% paraformaldehyde solution for 48 h, then dehydrated in a disposable tissue embedding box, soaked in wax, and embedded.Then the tissue wax blocks were cut wax into 5 μm sections.Then, after dewaxing and hydration, the slices were stained with hematoxylin solution and alcohol eosin staining solution for several minutes each.
IHC staining, on the other hand, involves sealing the section after hydration, repairing the antigen, and then staining with primary and secondary antibodies and DAPI.Finally, the stained sections were dehydrated with pure alcohol and then made the slices transparent by xylene for microscopic observation.Smooth skin without wrinkles 0

2.
Slight wrinkles appear on the skin at the edges of the area where the medication is applied 1

3.
The skin in the area where the medication is applied appears slightly wrinkled 2

4.
The skin wrinkles in the area where the medication is applied further deepen 3

5.
On the basis of a skin thickness score of 3, mice also experienced weight loss or poor condition 4

1.
The skin surface is smooth and free of scales 0

2.
Mild scaling of the skin in the area where the medication is applied 1

3.
The skin in the area where the medication is applied is completely covered with scales 2

4.
The skin in the area where the medication is applied is completely covered with scales, and the scales are further deepened 3

5.
On the basis of a skin scalling score of 3, mice also experienced weight loss or poor condition 4 C. Skin erythema

1.
The skin surface is smooth and free of erythema 0

2.
Mild erythemas of the skin in the area where the medication is applied 1

3.
The skin in the area where the medication is applied is completely covered with erythemas

4.
The skin in the area where the medication is applied is completely covered with scales, and the erythemas are further deepened 3

4.
On the basis of a skin erythema score of 3, mice also experienced weight loss or poor condition 4 Cumulative score: The sum of three scores 12 Table S3.Sequence of primers for q-PCR.

Figure. S2
Figure.S2 The encapsulation efficiency of TA in STTs.

Figure. S3
Figure.S3 The size and zeta potential of tFNAs and STTs.(a) and (b) Molecular size of STTs with different concentrations of TA measured by DLS.(c) and (d) Zeta potential of tFNAs, TA, STs and STTs measured by DLS.

Figure. S4
Figure.S4 The stability of siRNAs, STs and STTs.(a) Images of AGE showing Cy5 loaded free siRNAs, STs and STTs after incubation with 10% FBS for 0 h, 2 h, 6 h, 12 h, and 24h.(b) Images of AGE showing Cy5 loaded free siRNA and STs after incubation with 1 U/mL RNase A for 0 h, 1 h, 3 h, 6 h, 12 h, and 24h.

Figure. S9
Figure.S9 Circle gate strategy of flow cytometry showing the changes in the DCs labeled with CD80 and CD86.