Abstract
Psoriasis is a chronic, immune‐mediated disorder characterized by
immune regulation disorders and abnormal keratinocyte proliferation.
Deucravacitinib (Deu), a selective oral Tyrosine Kinase 2 (TYK2)
inhibitor, shows promise in treating psoriasis but may cause systemic
side effects and fail to address persistent localized thickened
lesions. Herein, a self‐locking microneedle (MN) patch with a polyvinyl
alcohol (PVA) inner ring loaded with Deu is developed, designed to
penetrate the transdermal barriers and dissolve rapidly, downregulating
the IL‐23/IL‐17 pathway and serve as the first line of defense against
the spread of skin‐originated inflammation. Additionally, Calcipotriol
(Cal), a vitamin D derivative, is incorporated into a methacrylated
hyaluronic acid (HAMA) backing layer and outer ring that mimics
occlusive administration, maintaining localized skin surface retention
for prolonged anti‐proliferative therapy. The Deu@Cal MN demonstrates
satisfactory adhesiveness due to swelling‐mediated mechanical
interlocking via the outer ring, ensuring targeted drug release at
lesion site. Besides its effectiveness in alleviating both skin
inflammation and proliferation, it inhibits the differentiation of Th17
cells in the spleen, suggesting potential to reduce systemic
inflammation. These findings offer a new therapeutic approach for
treating psoriasis and other autoimmune and inflammatory conditions.
Keywords: antiproliferation, deucravacitinib, immunology, microneedle,
psoriasis
__________________________________________________________________
A self‐locking MN patch for psoriasis delivers TYK2 inhibitor to target
skin‐originated inflammation and modulate local immune
microenvironment, while simultaneously releases Cal for sustained
anti‐proliferative effects on persistent hyperproliferative lesions.
This dual‐action MN therapy effectively reduces both local and systemic
inflammation, presenting a novel therapeutic strategy for autoimmune
and inflammatory conditions.
graphic file with name ADVS-11-2409359-g007.jpg
1. Introduction
Skin and subcutaneous diseases are prominent contributors to the global
burden of nonfatal illnesses. Approximately 0.09%–11.43% of the global
population suffers from psoriasis, making it one of the most prevalent
chronic skin diseases worldwide.^[ [36]^1 ^] Psoriasis is characterized
by widespread erythematous, scaly plaques, and itching, and is
associated with significant psychosocial comorbidities, including
anxiety and depression, suicidal ideation, and substance misuse.^[
[37]^2 ^] Pathogenesis involves factors such as heredity, abnormal
keratinocyte proliferation and differentiation, and immune
dysregulation. Psoriasis is characterized by intricate interactions
among infiltrating leukocytes, resident skin cells, and various
pro‐inflammatory cytokines produced within the skin, all governed by
the cellular immune system. The inflammatory processes that originate
in the skin can subsequently lead to systemic inflammation. Thus,
effective management of skin inflammation is essential for the
treatment of psoriasis and for mitigating its systemic consequences.^[
[38]^3 , [39]^4 ^] Current oral treatments for moderate to severe
psoriasis, such as methotrexate, acitretin, and cyclosporine, target
widespread intracellular pathways, potentially leading to broad‐ranging
effects and various side effects (e.g., bone marrow suppression, liver
fibrosis, teratogenicity).^[ [40]^5 ^]
Deucravacitinib (Deu), a first‐in‐class oral Tyrosine kinase 2 (TYK2)
inhibitor, has demonstrated favorable therapeutic outcomes in
real‐world clinical practice for the treatment of psoriasis compared to
many oral therapies such as methotrexate and apremilast.^[ [41]^6 ,
[42]^7 ^] Deu stabilizes the TYK2 pseudokinase (JH2) domain through
allosteric inhibition, disrupting the interaction between TYK2's
regulatory and catalytic domains. This blockade inhibits downstream
signal transduction and signal transducers and activators of
transcription (STAT)‐dependent gene transcription.^[ [43]^8 ^] The
Janus kinase (JAK)‐STAT pathway is engaged at various points within the
interleukin‐23 (IL‐23)/T helper 17 (Th17) signaling pathway, which is
considered central to the pathogenesis of psoriasis.^[ [44]^9 ^] Deu
may provide an improved therapeutic index and reduce the toxicities
associated with pan‐JAK inhibitors, as it demonstrates ≈100‐ to
200‐fold greater selectivity for JAK1/JAK3 and over 3000‐fold greater
selectivity for JAK2.^[ [45]^10 , [46]^11 ^]
Despite its effectiveness in treating psoriasis, Deu can cause adverse
effects such as upper respiratory infections and herpes zoster, and is
contraindicated in patients with severe hepatic impairment or latent
tuberculosis infection.^[ [47]^7 , [48]^10 , [49]^12 , [50]^13 ^]
Therefore, developing transdermal formulations of TYK2 inhibitors is
essential for psoriasis treatment with reduced systemic exposure, yet
such formulations for Deu are currently unavailable.
Although oral TYK2 inhibitor can improve overall clinical
manifestations through systemic immune regulation, some patients still
experience persistence of localized thickened lesions.^[ [51]^6 ,
[52]^7 ^] Calcipotriol (Cal), a vitamin D derivative, induces a
dose‐dependent decrease in keratinocyte proliferation and promotes
terminal differentiation.^[ [53]^14 ^] Cal can be locally used to
address the limitations of systemic medications in managing localized,
stubborn hyperproliferative lesions. Occlusion therapy with Cal
effectively reduced the scaliness and thickness of psoriatic lesions by
enhancing therapeutic response and drug penetration, facilitated by
increased temperature and humidity that improve stratum corneum
permeability.^[ [54]^15 ^] However, the poor adhesiveness of occlusive
materials limits their convenience of use.^[ [55]^15 , [56]^16 ^]
Based on these considerations, we developed a dual‐release microneedle
patch. The polyvinyl alcohol (PVA) needles fully penetrate the
transdermal barriers to rapidly release the TYK2 inhibitor in a
single‐dose manner, effectively mimicking “localized oral
administration.” This approach modulates the local immune
microenvironment of the lesions, serving as a primary defense against
the propagation of skin‐originated systemic inflammation. Concurrently,
Cal is stationed in the outer ring of the microneedle patch and backing
layer for sustained anti‐proliferative therapy on the surface of
psoriatic skin (Figure [57] 1 ). The occlusion‐like Cal administration
could in turn promote Deu penetration through scaly and thickened
lesions. To enhance packaging adhesion and facilitate clinical
application, we developed swelling‐mediated mechanical interlocking via
the outer ring material to ensure targeted drug release at the lesion
site. In vivo experiments conducted in our study demonstrated that
Deu@Cal MN effectively downregulated psoriasis‐associated IL‐23/IL‐17
pathways and alleviated epidermal hyperplasia, thereby achieving a
combined synergistic effect of immunomodulation and antiproliferation
on the local lesions. Additionally, Deu@Cal MN inhibits the
differentiation of Th17 cells in the spleen, suggesting its potential
to mitigate systemic inflammation. Our findings present a novel
therapeutic strategy for treating psoriasis and other autoimmune and
inflammatory conditions.
Figure 1.
Figure 1
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Schematic illustration of design and mechanism of self‐locking Deu@Cal
MN‐mediated antiproliferative and immunomodulatory effects for
psoriasis therapy.
2. Results and Discussion
2.1. Synthesis and Characteristics of Deu@Cal MNs
Using a template vacuum filling method, solutions containing different
drug matrix materials were filled into distinct regions of the mold to
fabricate microneedle patches with dual drug release capabilities
(Figure [59] 2a). These patches improved3 adhesion through
swelling‐mediated mechanical interlocking via the outer ring material.
Methacrylated hyaluronic acid (HAMA) was selected as the outer ring
material due to its superior biocompatibility, swelling behavior, and
photopolymerization properties, which are ideal for Deu@Cal MN
fabrication. To confirm the synthesis of HAMA, its molecular structure
was analyzed using Fourier‐transform infrared (FTIR) spectroscopy and
hydrogen nuclear magnetic resonance (^1HNMR) spectroscopy. The FTIR
spectrum of hyaluronic acid (HA) exhibited an ─OH stretching vibration
peak at 3220 cm⁻¹ and a bending vibration peak at 1400 cm⁻¹. Following
the reaction, the product exhibited strong peaks at 1610 and 1220 cm⁻¹,
corresponding to C═O stretching and C─O─C stretching vibrations,
respectively, indicating ester bond formation and successful grafting
of methacrylic anhydride (MA) onto the HA molecular chain
(Figure [60]2b). The ^1HNMR spectrum revealed two distinct proton peaks
at chemical shifts of 5.66 and 6.09, characteristic of the double bonds
in the grafted methacrylate (Figure [61]2c). Upon photopolymerization
and soaking in phosphate‐buffered saline (PBS), HAMA rapidly absorbed
water and swelled, achieving a swelling ratio exceeding 650% within 2 h
(Figure [62]2d). After freeze‐drying, the material exhibited a
microporous structure with pore sizes in the hundreds of micrometers
(Figure [63]S1, Supporting Information), validating its superior
swelling performance to ensure rapid mechanical interlocking and
improved adhesion and encapsulation of Cal upon insertion.
Figure 2.
Figure 2
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Preparation and characteristics of Deu@Cal MNs. a) Schematic diagram of
the microneedle preparation process. b) FTIR, c) 1H NMR, and d)
dissolution property evaluation of HAMA. e) Deu@Cal MNs in microscope
bright field, fluorescence field and SEM topography (Scale bar: 5 mm,
1 mm, 100 µm). Evaluation of mechanical properties of MNs, f)
mechanical displacement curves, g) sealing film piercing performance,
and h) pig skin piercing test (Scale bar: 3 mm).
Microscopic examination revealed that the Deu@Cal MN array maintained
its structural integrity. The array consisted of conical needles
measuring 600 µm in height and 300 µm in base diameter, arranged in a
circular pattern. Rhodamine B (red) representing Deu was primarily
distributed within the inner ring PVA needles, while calcein (green)
representing Cal was found in the HAMA backing layer and needles (outer
ring) (Figure [65]2e). Scanning Electron Microscopy (SEM) analysis
demonstrated that the needle surfaces were smooth, with no evident drug
precipitation, and had sharp tips, ensuring effective skin penetration.
The mechanical displacement curve and in vitro insertion performance
tests confirmed the Deu@Cal MN's ability to penetrate the stratum
corneum, thereby enhancing drug absorption and delivery efficiency. The
material composition significantly influenced mechanical performance;
although HAMA exhibited lower mechanical strength compared to PVA, it
still generated sufficient force (0.06 N per needle at 0.32 mm
displacement) to meet the minimum skin insertion requirement
(Figure [66]2f). Parafilm M film insertion tests demonstrated that all
microneedle types could penetrate three layers of film (each 125 µm
thick) (Figure [67]2g). Fluorescent model drugs were utilized to
evaluate the penetration efficiency of the microneedles into porcine
skin. As expected, the Deu@Cal MN arrays successfully penetrated the
skin, forming distinct fluorescent insertion points (Figure [68]2h).
Deu@Cal MNs demonstrated exceptional biocompatibility, as indicated by
the negligible hemolysis observed during co‐incubation with blood. The
hemolysis rate was consistently below 2%, with red blood cells
remaining intact and unruptured (Figure [69]S2, Supporting
Information). Furthermore, when fibroblasts were co‐incubated with
varying concentrations of Deu@Cal MNs for 24 h, the cells exhibited
high viability. AM‐PI staining confirmed the absence of significant
cell death (Figure [70]S3, Supporting Information), underscoring the
superior biocompatibility of Deu@Cal MNs.
2.2. Characterization of Mechanical Embedding and Encapsulation Properties of
Deu@Cal MNs
The dual‐release behavior of the microneedle patches is characterized
by the rapid dissolution of the PVA needles in the inner ring, which
are loaded with Deu. Upon subcutaneous insertion, PVA needles quickly
dissolved, releasing Deu to inhibit the regulatory domains of TYK2.
Concurrently, the HAMA needles in the outer ring swell upon insertion,
forming a mechanical interlocking structure that enhances patch
adhesion and enables the sustained release of Cal. To visualize the
structural characteristics of the Deu@Cal MNs, they were placed in a
95% humidity environment, where the inner ring PVA dissolved rapidly
within 60 s, while the outer ring HAMA gradually swelled as it absorbed
water (Figure [71] 3a). In vivo behavior of the microneedles in mice
confirmed these observations. Optical coherence tomography (OCT)
imaging can serve as a non‐invasive alternative to histopathology,
providing insights into the longitudinal structure of the skin. The OCT
images showed that the needle tips of both the PVA inner circle and
HAMA outer circle of the Deu@Cal MNs penetrate beneath the basal layer.
However, post‐insertion images revealed that the PVA component of the
inner ring dissolved nearly completely within 5 min, resulting in
closure of the channels. Simultaneously, the HAMA component swelled,
forming a spherical interlocking structure under the skin, thereby
enhancing mechanical adhesion (Figure [72]3b). The micro‐holes created
by Deu@Cal MNs were largely healed within 2 h after removal without
inducing local allergic reactions such as erythema (Figure [73]3c).
Tensile testing to measure the minimum separation force between the
microneedle patch and the skin showed that the cross‐linked HAMA
increased the separation force from 1.84 ± 0.35 to 5.11 ± 0.78 N
following subcutaneous insertion, with further increases over time
(Figure [74]3d,e). The improved adhesion was attributed to the
subcutaneous mechanical interlocking structure formed by the swollen
HAMA, which enhanced adhesion as its volume expanded over time.
Figure 3.
Figure 3
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Characterization of mechanical embedding and encapsulation properties
of Deu@Cal MNs. a) In vitro microneedle morphological and structural
changes in high humidity environment (Scale bar: 1 mm). b) OCT imaging
of the structural changes of microneedle puncture into the subcutaneous
skin of mice (Scale bar: 2mm, 500 µm). c) Changes in the recovery of
Deu@Cal MNs insertion sites on the skin surface of mice. d) Schematic
illustration of mechanical embedding and e) evaluation of adhesion
force of Deu@Cal MNs. f) Change in skin surface moisture before and
after MNs treatment. g) Representative in vivo fluorescence images of
mice at different time points after administration of the respective
simulated fluorescent drugs. h) Relationship between mean square
displacement and time for Cy3 (substitute for Deu) and Cy5.5
(substitute for Cal) (n = 3).
The transepidermal water loss (TEWL) exhibited a very slow decline in
cross‐linked MNs (Figure [76]3f), indicating their capacity to maintain
microchannels in the stratum corneum during the drug delivery phase,
thereby facilitating prolonged delivery of Cal and achieving extended
drug release. Furthermore, due to the packaging effect of MNs on the
skin surface, the increased humidity within the MN system further
enhanced effective drug penetration and therapeutic outcomes.^[ [77]^15
^] To further validate the drug release behavior mediated by the
Deu@Cal MNs and to infer drug absorption kinetics and duration, thereby
guiding subsequent dosing protocols, Cyanin 3 (Cy3) was employed as a
surrogate for Deu and Cy5.5 for Cal. The changes in corresponding
fluorescence signals were monitored using a small animal in vivo
imaging system. The fluorescence signal from the inner ring
representing Deu exhibited a gradual decrease as the PVA dissolved and
the drug was metabolized and absorbed, with the signal diminishing to
≈40% of its initial intensity within 24 h. In contrast, the
encapsulated Cal released through the swelling channels of HAMA and
backing layer showed a sustained and slow absorption profile,
maintaining a relatively stable fluorescence signal over time. This
indicated a prolonged release of Cal on the skin surface, supporting
long‐term therapeutic effects on the lesions, similar to localized
occlusive therapy. (Figure [78]3g,h; Figure [79]S4, Supporting
Information).
2.3. Therapeutic Effect of Deu@Cal MNs in Skin Lesion of Psoriatic Mouse
Model
The in vivo therapeutic efficacy of MNs was assessed using an imiquimod
(IMQ)‐induced psoriatic mouse model.^[ [80]^17 ^] IMQ was applied to
the mouse skin for 8 consecutive days from day 0, and MN patches were
applied every day from Day 3 (Figure [81] 4a). The IMQ‐induced
psoriasis‐like mice were randomly divided into four treatment groups
(n = 6): Blank MNs, Cal MNs, Deu MNs, and Deu combined with Cal MNs
(Deu@Cal MNs). These groups were compared with a normal group and a
model group (untreated IMQ group).
Figure 4.
Figure 4
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Therapeutic effect of Deu@Cal MNs in mice psoriatic lesions. a)
Schematic illustration of Deu@Cal MNs for the treatment of IMQ‐induced
psoriatic mice. b) Representative images of mice dorsal skins in
different groups from days 0 to 8 (n = 6 for each group). c) Heatmap of
PASI score (total) of each individual. d) The PASI scores including
desquamation, erythema, thickness and total score of different
treatment were recorded from days 0 to 8, each score ranged from 0 to
4, and the total score from 0 to 12. (n = 6, ** p < 0.01, ***
p < 0.001, and **** p < 0.0001 versus IMQ group, ^# p < 0.05 and ^##
p < 0.01. Data are presented as mean ± SD).
The model group exhibited typical physiological characteristics of
psoriasis, including white scales, erythema, and thickened skin. After
5 days of treatment, both the Deu MNs and Cal MNs groups showed
significant therapeutic effects on psoriasis, with the Deu@Cal MNs
group demonstrating the most pronounced relief of symptoms
(Figure [83]4b). The Psoriasis Area and Severity Index (PASI), which
assesses erythema, scaling, and skin thickening as indicators of skin
inflammation, was used to evaluate psoriasis severity throughout the
experiment period.^[ [84]^18 ^] Mice treated with Deu@Cal MNs showed
significant alleviation in erythema by day 4 and day 5, improved skin
thickness by day 5, and reduced desquamation by day 6, while the
untreated psoriasis group began returning to normal around day 8. Blank
MNs exhibited minimal therapeutic effects compared to the untreated
group, suggesting that MNs alone have limited ability to reverse the
disease. PASI scores and heatmaps were consistent with the symptoms
observed in images, indicating that Deu@Cal MNs proved most effective
in alleviating typical psoriatic symptoms, particularly in erythema and
desquamation scores. (Figure [85]4c,d).
Furthermore, the biosafety of Deu@Cal MN in vivo was evaluated through
monitoring body weight and examining organ histology. Throughout the
experimental period, mice in all treatment groups showed no significant
weight loss. (Figure [86]S5, Supporting Information). The histological
analysis of heart, liver, spleen, lung, and kidney did not show obvious
tissue injuries (Figure [87]S6, Supporting Information). The excellent
biosafety profile of Deu@Cal MNs highlights their promising clinical
potential.
2.4. In Vivo Antiproliferation Effect of Deu@Cal MNs
Psoriasis‐like mice and those treated with blank MNs exhibited typical
histopathological features of psoriasis, including hyperkeratosis,
parakeratosis, psoriasiform epidermal hyperplasia, and inflammatory
cell infiltration.^[ [88]^19 ^] Treatment with Cal MNs markedly reduced
epidermal thickness, however, obvious acanthosis and slightly elongated
rete ridges were still observed in Deu MNs group. Notably, Deu@Cal MNs
restored epidermal structure to nearly normal levels, with regular
epidermal layers. (Figure [89] 5a).
Figure 5.
Figure 5
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In vivo antiproliferation effect of Deu@Cal MNs. a) Hematoxylin and
eosin staining of the mice dorsal skin after different treatments on
day 8. b) Histopathologic illustration of ETmin and ETmax in psoriatic
lesions. c) ETmin with different treatments on day 8. d) ETmax with
different treatments on day 8^th. e) Immunofluorescence staining of the
Ki67 (Scale bar: 100 µm). f) Mean fluorescence intensity of Ki67 of
different groups. (* p < 0.05, ** p < 0.01, and *** p < 0.001 ****
p < 0.0001 versus IMQ group, ^## p < 0.01, ^### p < 0.001, and ^####
p < 0.0001. Data are presented as mean ± SD. ns, not significant).
Due to the undulated structure of the dermoepidermal junction (DEJ) in
psoriasis, we used the parameters Epidermal Thickness Minimum (ETmin)
and Epidermal Thickness Maximum (ETmax) to measure thickness from the
top to the bottom of the dermal papillae, respectively
(Figure [91]5b).^[ [92]^20 ^] ETmin and ETmax in Deu@Cal MN‐treated
group were significantly decreased compared to untreated group (55.8 µm
vs 123.8 µm, and 113.8 µm vs 290.6 µm respectively, p < 0.0001),
indicating substantial alleviation of acanthosis and psoriasiform
hyperplasia in psoriatic lesions. Remarkably, Cal MNs exhibited a more
pronounced effect on acanthosis compared to Deu MNs, with ETmin values
of 79.9 ± 2.6 µm versus 90.6 ± 3.4 µm (p < 0.01) (Figure [93]5c,d).
Although both Cal MNs and Deu MNs alleviated epidermal thickening, the
mitigation effects of Deu@Cal MNs on hyperplasia were significantly
superior, consistent with measurements of dorsal skin thickness in mice
(Figure [94]S7, Supporting Information).
Ki67, a nuclear proliferation marker, is indicative of keratinocyte
proliferation and reflects the severity of psoriasis.^[ [95]^21 ^]
Immunofluorescence staining demonstrated that the Cal MN group
inhibited Ki67 expression more effectively compared to the Deu MN group
(p < 0.001), and the Deu@Cal MN group exhibited the lowest expression
of Ki67 (Figure [96]5e,f). These findings were consistent with
histopathological observations and in vivo measurements of epidermal
thickness.
These results indicated that Cal was more effective than Deu in
alleviating epidermal hyperplasia, and this phenomenon agreed well with
the prior and our study suggesting Cal decreased proliferation of
keratinocytes in vitro (Figure [97]S8, Supporting Information).
Occlusion‐like application of Cal in vivo could amplify this effect,
demonstrating significant inhibition of hyperplasia when it was
combined with Deu.
2.5. In Vivo Immunomodulatory Effect in Psoriatic Skin Lesions
The inflammatory environment in psoriatic lesions is characterized by
immune cell infiltration and the secretion of various cytokines.
Notably, the tumor necrosis factor‐alpha TNF‐α and IL‐23/IL‐17 axis are
considered crucial mediators for regulating the functions of dendritic
cells and Th17 cells, thereby promoting the development of psoriasis.^[
[98]^22 ^] IL‐6 is elevated in the serum and skin lesions of psoriatic
patients and may serve as an indicator of inflammatory activity in
psoriasis.^[ [99]^23 ^]
The immunohistochemistry (IHC) staining results for TNF‐α, IL‐23,
IL‐17, and IL‐6 in the skin tissue are shown in Figure [100] 6a. These
cytokines exhibited significantly elevated expressions during psoriasis
progression and noticeable declines following Deu@Cal MN treatment.
Additionally, the mRNA expression levels of these cytokines were
measured using quantitative reverse transcription polymerase chain
reaction (qRT‐PCR) (Figure [101]6b). As expected, consistent with the
IHC results, the upregulated IL‐17 expression induced by IMQ was
significantly more decreased in the Deu MN group compared to the Cal MN
group (p < 0.01), with the combined Deu@Cal MN therapy showing the most
pronounced reduction. The Cal MN group showed no significant difference
in TNF‐α levels compared to psoriatic mice, whereas Deu@Cal MN therapy
markedly decreased TNF‐α expression (p < 0.01). Deu@Cal MN therapy
decreased the levels of IL‐23 and IL‐6, the efficacy was significantly
higher than that of the Cal MNs (p < 0.05). Taken together, Deu@Cal MNs
exhibited a satisfactory immunomodulatory effect, with Deu playing a
leading role in downregulating the IL‐23/IL‐17 axis, thereby regulating
immune responses and inflammation in local lesions.
Figure 6.
Figure 6
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In vivo immunomodulatory effect in psoriatic skin lesions. a)
Immunohistochemical staining of IL‐6, IL‐17, IL‐23, and TNF‐α (Scale
bar: 100 µm). b) mRNA expression of IL‐6, IL‐17, IL‐23, and TNF‐α in
mice dorsal skin measured by qRT‐PCR. (* p < 0.05, ** p < 0.01, and ***
p < 0.001 **** p < 0.0001 versus IMQ group, ^## p < 0.01, ^###
p < 0.001, and ^#### p < 0.0001. Data are presented as mean ± SD. ns,
not significant).
2.6. Evaluation of Spleen Inflammation and Th17 Cell Differentiation
Given the key role of the spleen in the immune system, it may
contribute to psoriasis‐like inflammation by modulating immune
responses.^[ [103]^24 ^] Previous studies have reported that topical
application of IMQ could cause significant splenomegaly, typically
regarded as an indication of elevated systemic inflammation.^[ [104]^25
^] We observed that the markedly increased spleen volumes in
IMQ‐induced mice were dramatically reduced following Deu@Cal MNs
treatment (Figure [105] 7a; Figure [106]S9, Supporting Information),
demonstrating the systemic immune adjustment effect of Deu@Cal MNs.
Moreover, Deu@Cal MNs significantly decreased the spleen index, whereas
the Cal MNs alone had a limited impact on relieving splenomegaly (0.87
vs 0.64, p < 0.05) (Figure [107]7b).
Figure 7.
Figure 7
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Analysis of splenomegaly and Th17 cells in mice spleen. a)
Representative images of mice spleen after different treatments on day
8. b) Spleen/body weight of mice in different groups. c) Representative
flow cytometry charts of CD4+IL‐17A+ cells in mice spleen. d) The ratio
of CD4+IL‐17A+ cells in spleen analyzed by flow cytometry. (* p < 0.05,
** p < 0.01, and *** p < 0.001 **** p < 0.0001 versus IMQ group, ^##
p < 0.01, ^### p < 0.001 and ^#### p < 0.0001. Data are presented as
mean ± SD. ns, not significant).
Th17 cells play an essentially immunoregulative role in the
pathogenesis of psoriasis by secreting various cytokines, including
IL‐17A, IL‐17F, IL‐22, and TNF‐α.^[ [109]^26 ^] The Th17 subset,
identified as a lineage of CD4+ T cells, primarily produces IL‐17A. In
our study, we analyzed the proportion of IL‐17A+ CD4+ T cells in the
spleen by flow cytometry and found that the proportion was
significantly lower in the Deu@Cal MN group compared to other groups
(Figure [110]7c). Deu@Cal MN treatment achieved substantial inhibition
of Th17 cells compared to the IMQ group (0.48% vs 4.17%, p < 0.0001).
Specifically, the proportion of Th17 cells in the spleen was much lower
in the Deu MN group than in the Cal MN group (1.29% vs 2.24%,
p < 0.0001) (Figure [111]7d), indicating that Deu exerts a potent
immunoregulatory effect at both local and systemic levels. These
results suggested that Deu@Cal MN can inhibit spleen inflammation,
thereby attenuating systemic psoriasis‐like inflammation.
2.7. Therapeutic Mechanisms Based on Transcriptomics
To further elucidate the therapeutic mechanisms underlying the
immunoregulatory effects of Deu@Cal MNs in the treatment of psoriasis,
the bulk RNA sequencing was conducted to uncover biological functions
and molecular mechanisms within psoriatic lesions.
A total of 5120 differentially expressed genes (DEGs) were identified
in skin treated with Deu@Cal MN compared to the untreated group, with
2692 up‐regulated and 2428 down‐regulated genes (Figure [112] 8a) using
1.2 as the fold change threshold. Venn diagram showed the shared 3147
DEGs between Deu MN versus IMQ and Deu@Cal MN versus IMQ group
(Figure [113]8b). Furthermore, the significantly downregulated genes
were highly enriched in type I IFN, IL‐22, and IL‐17 signaling pathways
after Deu@Cal MN treatment, which were distinct inflammatory and
keratinocyte‐response pathways involved in psoriasis. The mean mRNA
expression levels of type I IFN‐associated genes (such as Ifnr1, Ifnr2,
Ifnr1, and Ifnr2, etc.), IL17‐associated genes (Il17a, Il17f, and
Il17ra, etc.) and IL 22 associated genes (IL22, Il22ra1, Stat1, and
Stat3, etc.) were significantly decreased in Deu@Cal MN group compared
with IMQ group (p < 0.01), and the level of IL‐22 in Deu@Cal MN group
was significantly lower than Deu MN treatment alone (p < 0.05)
(Figure [114]8c,d).
Figure 8.
Figure 8
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Transcriptome profiling to elucidate genes significantly affected in
IMQ, Deu MN, and Deu@Cal MN treatment group by RNA‐Seq. a) Volcano
plot showed differentially expressed genes (DEGs) of the IMQ, Deu MN,
and Deu@Cal MN treatment group. b) Venn diagram showed the
co‐expression of DEGs by comparative analysis of Deu MN versus IMQ,
Deu@Cal MN versus IMQ, Deu@Cal MN versus Deu MN. c) Mean mRNA
expression associated with type I IFN, IL‐22, and IL‐17 signaling
pathways of each group. d) Clustered heat map showed genes that belong
to the type I IFN, IL‐22, and IL‐17 signaling pathways that were
differentially expressed between Deu@Cal MN, Deu MN, and IMQ group. (*
p < 0.05, ** p < 0.01, and *** p < 0.001. Data are presented as
mean ± SD. ns, not significant).
Gene Ontology (GO) functional annotation and enrichment analyses were
conducted to elucidate the biological processes associated with DEGs.
Deu@Cal MN treatment had significant effects on down‐regulating
production of molecular mediators of immune response, negative
regulation of immune system process, and somatic recombination of
immune receptors from immunoglobulin in biological processes (BP); and
extracellular organelle, coated vessels, and apical plasma membrane in
cell components (CC) (Figure [116] 9a). While regulation of cell cycle
phase transition, adenosine triphosphate (ATP)‐dependent activity
acting on DNA and protein phosphatase inhibitor activity were
up‐regulated after Deu@Cal MN therapy (Figure [117]9b; and Table
[118]S1, Supporting Information). Furthermore, Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analysis revealed that
following Deu MN and Deu@Cal MN treatment, psoriasis‐associated
pathways such as the IL‐17, JAK‐STAT, and TNF signaling pathways were
significantly down‐regulated due to a higher number of downregulated
DEGs compared to upregulated DEGs in each pathway. (Figure [119]9c).
Notably, STAT1, STAT3, and STAT6 expression were substantially
inhibited after Deu@Cal MN therapy (Figure [120]9d), which served as
downstream targets of TYK2, previously shown to provide protection
against multiple autoimmune and chronic inflammatory disorders.^[
[121]^27 ^]
Figure 9.
Figure 9
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Transcriptome profiling to elucidate pathologic pathways and immune
infiltrates in IMQ, Deu MN, and Deu@Cal MN treatment group. a) Gene
ontology (GO) enrichment analysis of the down‐regulated DEGs. b) GO
enrichment analysis of the up‐regulated DEGs. c) KEGG enrichment
analysis of up‐regulated and down‐regulated genes in each comparison.
The color from red to purple represents the significant size of the
enrichment. d) Mean mRNA expression of Stat1, Stat3 Stat 6 of each
group. e) MCP‐counter was used to show the immune composition of each
sample. f) The quantification analysis of immune infiltrates. (*
p < 0.05, ** p < 0.01, and *** p < 0.001. Data are presented as
mean ± SD. ns, not significant).
Additionally, we calculated immune infiltration scores using
microenvironment cell populations counter (MCP‐counter)
(Figure [123]9e), and the quantification analysis showed that
neutrophils and monocytic lineage were significantly decreased when
treated with Deu@Cal MNs compared with the untreated group (p < 0.01)
(Figure [124]9f). Neutrophils, a regulator between the innate and
adaptive immune systems, appear early in new psoriatic lesions and
contribute to sustained inflammation in psoriasis.^[ [125]^28 ^]
Moreover, neutrophils are associated with the IL‐17‐producing Th17
subset of CD4 T cells.^[ [126]^29 ^] Therefore, Deu@Cal MN therapy
effectively downregulated the expression of key genes and pathogenic
pathways (e.g., JAK‐STAT signaling pathway) involved in psoriasis, and
also reduced associated immune cell infiltration.
3. Conclusion
Psoriasis is characterized by epidermal hyperproliferation, abnormal
differentiation of epidermal keratinocytes, and inflammation with
immunologic alterations in the skin. Thus, treatment targeting the
immune microenvironment in local lesions can prevent the spread of skin
lesions and systemic complications. In this study, we introduced a
self‐locking Deu@Cal MN with satisfactory adhesiveness to ensure
targeted drug release at the lesion site. Deu released rapidly to
regulate local immune‐mediated cytokine‐initiated inflammation and
minimize systemic exposure. Cal showed a sustained release and slow
absorption profile, indicatig that Cal provided prolonged
anti‐proliferative effect on the the surface of persistent
hyperproliferative lesions. Our study demonstrated that Deu@Cal MNs
exhibited excellent therapeutic efficacy with a combined synergistic
effect of immunomodulation and antiproliferation when treating
psoriasis.
IL‐23‐driven Th17 pathways played a crucial role in chronic
inflammation in psoriasis and in numerous other inflammatory
conditions. Deu significantly downregulated IL‐17 expression in
lesions, and inhibited the differentiation of Th17 cells in spleen,
indicating the immunoregulation effect on both local and systemic
levels. Additionally, in vitro and in vivo studies demonstrated that
Cal significantly reduced keratinocyte proliferation, inhibited Ki67
expression, and substantially alleviated acanthosis and psoriasiform
hyperplasia in psoriatic lesions. The combination of Cal with Deu
results in a more pronounced reduction in lesion thickness, as Deu can
indirectly inhibit keratinocyte proliferation by blocking inflammatory
cascade responses within the lesions. Notably, the occlusion‐like Cal
administration improved humidity and permeability of the stratum
corneum, which reduced surface scaling and, in turn, improved Deu
penetration and amplifying both therapeutic effects.
Our study demonstrated that Deu@Cal MN treatment effectively targets
both adaptive immunity (mediated by IL‐23) and innate immunity
(mediated by type I IFN). The downregulated type I IFN‐associated gene
expression suggested that Deu@Cal MN therapy may prevent autoimmune
cell activation, and treat autoimmune diseases with a predominant role
for type I IFN, such as cutaneous lupus.^[ [127]^30 ^] The reduced
neutrophil infiltration in the lesion suggested that Deu@Cal MNs could
inhibit the formation of new lesions, such as those seen in the Koebner
phenomenon.^[ [128]^31 ^] Moreover, Deu@Cal MN therapy could also
regulate the TYK2‐STAT cascade, indicating its protection from multiple
autoimmune and inflammatory disorders. In conclusion, Deu@Cal MN hold
significant clinical potential for treating autoimmune diseases with
skin involvement.
4. Experimental Section
Materials
Polydimethylsiloxane (PDMS, Sylgard 184) was obtained from Dow Corning
(Midland, USA). MA was purchased from Bidepharm (Shanghai China). D2O,
HA (400 kDa), and PVA (9‐10 kDa) were purchased from Sigma‐Aldrich (MO,
USA). Cy3, Cy5.5‐NHS was purchased from Duofluor Inc. (Wuhan China).
PBS, Dulbecco's Modified Eagle Medium (DMEM), penicillin‐streptomycin
antibiotics, and 0.25% trypsin‐EDTA solution were purchased from Gibco
(New York USA). Certified fetal bovine serum (FBS) was purchased from
VivaCell (Shanghai China). Cell counting kits (CCK‐8) were purchased
from Beijing Solarbio Science & Technology Co., Ltd (Beijing China).
Calcein‐AM/PI Double Stain Kit were purchased from Beyotime (Shanghai
China). Photoinitiator 2959, Ninhydrin were purchased from Aladdin
(Shanghai China). Antibodies involved in immunohistochemistry and
immunofluorescence were purchased from Wuhan Servicebio Co., Ltd (Wuhan
China).
Preparation and Characteristics of HAMA
To achieve a composite microneedle structure, HAMA was selected as the
base material, following previously reported methods. Specifically, a
precise amount of HA was dissolved in PBS and stirred at 600 rpm at
45 °C until fully dissolved. The HA solution was then cooled in an
ice‐water bath at 4 °C for 30 min. Subsequently, 2 mL MA was added
dropwise at 300 rpm under ice bath conditions, and NaOH was used to
adjust the pH to 8.0. The reaction was allowed to proceed for 24 h,
yielding the initial product of HAMA. The next day, the reaction
mixture was centrifuged to remove any precipitates. The supernatant was
transferred to a 12 kDA dialysis bag and dialyzed at room temperature
for one week, with the deionized water changed twice daily. After
dialysis, the purified product was filtered through a 0.44 µm aqueous
microfiltration membrane and lyophilized to obtain HAMA.
To verify the modification, 10.0 mg of HA and HAMA were dissolved in
1.0 mL ultrapure water, vacuum‐dried to form transparent films, and
analyzed using a Thermo Fisher FTIR to measure transmittance in the
range of 500–4000 cm^−1. The molecular structure of the products and
the degree of substitution of HAMA were determined by dissolving 5.0 mg
of HA and HAMA in 500 µL D2O and analyzing with an AV‐600 1H‐NMR.
Additionally, to study the swelling properties of HAMA, a 4% (w/v) HAMA
solution containing 0.5% (w/v) Irgacure 2959 was prepared. This
solution was poured into a mold (8 mm × 8 mm × 1 mm) to create HAMA
patches. After 30 min, the patches were cross‐linked under UV light for
10 s. The patches were then fully dried and weighed, followed by
immersion in PBS solution at 37 °C. At various time points, the patches
were removed, blotted to remove surface moisture, weighed, and returned
to the PBS solution. After 48 h, the patches were freeze‐dried for one
day and weighed again. Freeze‐dried samples were also sectioned and the
pore morphology observed via SEM. The swelling ratio was calculated
using the following equation:
[MATH: SwellingDegrees(%)=ms−mdms×100 :MATH]
(1)
where m[s] was the mass of swollen HAMA patches and m[d] was the mass
of freeze‐dried HAMA patches.
Preparation and Characteristics of Deu@Cal MNs
To achieve the swelling‐induced mechanical interlocking of
microneedles, a custom PDMS mold with an inner and outer ring structure
was designed. The inner ring consisted of 19 needles arranged in 1, 6,
and 12 needle arrays, while the outer ring comprised 81 needles in 18,
27, and 36 needle arrays. A custom mask was applied to the PDMS mold,
and a 25% PVA solution containing Deu was added to the inner ring. The
setup was vacuumed for 10 min to remove excess solution, followed by
vacuum drying for 30 min. Subsequently, a 4% HAMA solution containing
Cal was added to the outer ring, vacuumed for 30 min, and UV
cross‐linked for stabilization, followed by an additional 60 min of
vacuuming. The mold was dried overnight and demolded using a
custom‐made baseplate, then stored in a vacuum desiccator for further
characterization and use. Rhodamine B and calcein were used as model
drugs to observe drug distribution within the microneedles using
brightfield and fluorescence microscopy, with SEM used to examine the
integrity and tip morphology of the microneedle arrays.
To ensure the microneedle arrays possessed sufficient mechanical
strength for skin insertion, an advanced digital dynamometer (ESM301,
Mark‐10, Force Gauge Model, USA) was employed to determine their
mechanical and insertion properties. The microneedles were affixed to a
test platform, and the force gauge probe descended at a rate of
10 mm min^−1 to record the force‐displacement curve. Additionally, the
microneedles were inserted into Parafilm M film or fresh excised
porcine skin with a fixed force of 10N using the force measurement
device to observe the insertion performance.
Characterization of Mechanical Eembedding and Encapsulation Properties of
Deu@Cal MNs
The prepared microneedles were placed in an environment with 95%
humidity, and changes in the structure of the inner and outer ring
needles were observed and recorded at different time points using a
handheld microscope. Additionally, the microneedles were inserted into
the dorsal skin of mice, and their subcutaneous morphological changes
were observed using OCT. After removing the microneedles, the recovery
of the needle holes on the mouse skin surface was documented.
To assess the impact of swelling‐mediated mechanical interlocking on
adhesion, cross‐linked and non‐cross‐linked microneedles were inserted
into porcine skin, and the force required for separation was measured.
The water content at the corresponding skin sites was determined using
a skin moisture analyzer to compare the drug encapsulation effects of
different microneedle patches.
For in vivo drug release analysis, Cy3 was used as a substitute for
Deu, and Cy5.5 was used as a substitute for Cal, loading them into the
inner and outer rings of the microneedles, respectively. The in vitro
transdermal delivery efficiency was evaluated using Franz diffusion
cells, with mouse abdominal skin serving as the model. After inserting
the Deu@Cal MNs, receptor fluid samples were collected at various time
points and analyzed for drug concentration using a fluorescence
spectrophotometer, allowing to characterize the drug release profile.
In vivo release behavior was assessed using a small animal in vivo
imaging system.
Animals and Psoriasis‐Like Inflammation Model
The female BALB/c mice (6–8 weeks old, 18–22 g) were obtained from SPF
(Beijing) Biotechnology Co., Ltd. All the mice were anesthetized with
Isoflurane (RWD, China), and a 2.5 × 3 cm area was selected on the back
of the mouse and shaved carefully. Each mouse was applied with 62.5 mg
IMQ ointment on the prepared back skin once daily in the morning, and
MNs treatment at night, except for the control group. Skin thickness
was measured once a day, and all mice were killed on the 8th day for
follow‐up detection. All animal experiments were approved by the Animal
Ethics Committee of China‐Japan Friendship Hospital (CRDWLL 230 155)
and were carried out following the National Guidelines for the Care and
Use of Laboratory Animals.
Evaluation of the Severity of Skin Inflammation and Spleen Index
PASI score involved skin erythema, scales, and thickness. Erythema,
scales, and thickness were scored independently from 0 to 4, where 0
represents “none,” 1 represents “slight,” 2 represents “moderate,” 3
represents “marked,” and 4 represents “severe.” The total severity of
skin inflammation and lesion score was calculated as the sum of the
three indexes (0–12). All mice in each group were scored daily for 8
consecutive days from day 0. The body mass and spleen mass of all mice
were measured on the seven days. This was used in the calculation of
the spleen index using the formula:
[MATH: spleenindex=spleenmass(mg)/bodymass(g)
:MATH]
(2)
Histological, Immunofluorescence Staining, and Immunohistochemistry Analysis
Dorsal skin samples of all mice were collected and fixed with 4%
paraformaldehyde for 24 h, embedded in paraffin, and then sliced into
4 µm thick sections. For histological analysis, the aforementioned
thick sections were stained with H&E, and Image‐pro Plus 6.0 was used
for the calculation of the epithelial thickness of the skin. The
expression of Ki67, IL‐6, IL‐17, IL‐23, and TNF‐α was evaluated by
immunofluorescence staining and immunohistochemical staining. The
sections were visualized by panoramic scanner (WISLEAP‐10, Jiangsu,
China).
Real‐Time Quantitative PCR
Total mRNA was extracted from biopsies of the dorsal skin isolated
after sacrificing the mice using RNAiso Plus (TaKaRa, Japan) according
to the manufacturer's instruction, and reverse transcription of mRNA
was performed using 4×Hifair III SuperMix plus (Yeasen, Shanghai,
China). Through the application of SYBR Green reagent (Yeasen,
Shanghai, China), IL‐6, IL‐23, IL‐17A, TNF, and GAPDH mRNA levels were
detected by the Gentier 32R Real‐Time PCR System. Sequences for the PCR
primers were seen in Table [129]S2 (Supporting information). Gene
expression levels in all samples were normalized using the 2^−ΔΔCt
method with GAPDH as internal controls for comparison.
Flow Cytometric Assays
The spleen was grinded and filtered using a 40 µm cell strainer to
obtain single cell suspension, then was incubated for 5 h in a 37 °C,
5% CO2 incubator. One microliter of Brefeldin A Solution (1000×)
(BioLegend, 420 601) was added to each 1 mL of cell suspension to
stimulate cells. APC/Cyanine7 anti‐mouse CD45 Antibody
(BioLegend,157 203), Pacific Blue anti‐mouse CD3 Antibody (BioLegend,
100 213), and APC anti‐mouse CD4 Antibody (BioLegend, 116 013) were
added to lymphocytes for cell staining. For IL‐17A intracellular
staining, cells were fixed and permeabilized by Intracellular
Fixation/Permeabilization Buffer Kit (Elabscience Biotechnology
Co.,Ltd) and FITC anti‐mouse IL‐17A (BioLegend, 506 907) was added.
Flow cytometric analysis was performed on the BD LSRFortessa SORP. The
data were analyzed by Flowjo software.
Transcriptome Profiling
For further investigation of therapeutic mechanism, RNA sequencing
(RNA‐seq) was performed on lesion skin tissues from IMQ, Deu MNs, and
Deu@Cal MNs treatment groups by Novogene Co., Ltd. Data analysis was
conducted using DESeq to identify DEGs with a p‐value < 0.05 and
Fold‐change ≥ 1.2 considered significant. Further analysis included GO
enrichment, KEGG pathway enrichment, and MCP‐counter.
Statistical Analysis
In this study, data were analyzed using GraphPad Prism 8.0 software and
presented as means ± standard deviation (M ± SD). One‐way analysis of
variance (ANOVA) was then used to analyze the levels of variance within
the groups at a significance threshold of p < 0.05.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
Z.Y.W. and Z.Q.Z. contributed equally to this work as co‐first authors.
Z.Y.W. and Z.Q.Z. conceived the idea, performed the experiment and
drafted the paper. K.J.C. assisted with experiment conduction. Y.J.S.
and B.Z.C. designed the overall experimental planning. Z.Y.W. and
Z.Q.Z. contributed to the data interpretation. Y.C. and X.D.G. directed
the research and provided financial support.
Supporting information
Supporting Information
[130]ADVS-11-2409359-s001.doc^ (17.7MB, doc)
Acknowledgements