Rosacea treatment with mussel adhesive protein delivered via microneedling: In vivo and clinical studies

Rosacea is a prevalent chronic dermatological condition marked by facial inflammation and erythema, significantly compromising the quality of life for affected individuals. Current treatment methods for rosacea are not considered ideal because of the complex etiology of the disease. Mussel adhesive protein (MAP) is a glycoprotein derived from the foot gland of mussels. The protein exhibits anti‐inflammatory properties, relieves skin itching, and promotes wound healing.


| INTRODUC TI ON
Rosacea is a chronic inflammatory skin disorder that causes distinct changes in the central part of the face.It is characterized by recurrent facial flushing, pustules, papules, erythema, and edema.These symptoms can be combined and may be associated with telangiectasia with disease progression. 1 Owing to the involvement of facial skin, patients often experience severe psychosocial pressure, which consequently affects their social interaction, and work and significantly reduces their quality of life. 2 The key pathological characteristics of rosacea lesions are hair follicle and perivascular immune cell infiltration, vascular and lymphatic dilatation, sebaceous gland hyperplasia, and skin fibrosis. 3Rosacea can be classified into four types based on its clinical manifestations, among which erythematotelangiectatic rosacea (ETR) is the most prevalent and challenging to treat. 4 Its pathogenesis has not been fully elucidated, and recent reports indicate that genetic susceptibility, local immune disorders of the facial skin, and neurovascular dysfunction contribute to the disease. 5Exposure to microorganisms and environmental factors can also induce the disease.
Mussel adhesive protein (MAP), is a type of mucin belonging to the glycoprotein family.It is produced and stored in the foot glands of mussels.It has three functions: biological bonding, protective film formation, and promoting cell adhesion and crawling.

It exerts anti-inflammatory effects and has been demonstrated
to enhance wound healing in clinical settings. 6Microneedling, which helps form temporary skin microchannels, can significantly increase the absorption of drugs.Concurrently, it can induce collagen regeneration and promote skin tissue remodeling.In this study, we explored the anti-inflammatory effect of MAP and its ability to repair the skin barrier in rosacea treatment.We established a rosacea-like mouse model using LL-37, 7 assessed the improvement of skin lesions and histological changes in response to the treatment, and analyzed the therapeutic mechanism of MAP.
Building upon the substantial efficacy observed in the animal experiments, we implemented this treatment in clinical settings.
Here, we present our findings from a preliminary investigation on the use of MAP in rosacea treatment to provide a reference for clinical treatment.

| Ethics statement
The study received approval from the Ethics Committee of Yanbian University Hospital (experimental license number for animal production: SCXK (X) 2018-0007, experimental unit use license number: SYXK (X) 2020-0009, ethics committee approval number: no.2021250), following the guidelines of the Helsinki Declaration.Patients provided voluntary signed informed consent.LL-37, synthesized by Gill Biochemical Co., Ltd.(Shanghai, China), was injected intradermally into the backs of mice using a microneedle roller with a length of 0.5 mm.The mice's back hair was shaved 24 h prior to LL-37 injection.The peptide, dissolved in sterile injectable water (320 μM), was administered in 40 μL doses, creating a dermal bubble.Subsequently, injections were given every 12 h for a total of 4 consecutive doses.The mice in the blank control group were solely administered subcutaneous injections of sterilized water.After the model construction was completed, images were captured using a digital camera and dermatoscope at the 12 h mark.Subsequently, treatment initiation commenced, with therapy administered at 36-h intervals.The treatment regimen comprised two sessions in total.

| Rosacea-like mouse model
Images were recorded 12 h after the treatment.The evaluation of rosacea-like lesions severity was based on the assessment of erythema and the condition of the capillaries.The degree of disease was measured on a scale of 1-5, with 5 indicating the highest level of severity.Twelve hours after completing the treatment, mice were euthanized with an intraperitoneal injection of pentobarbital sodium (150-200 mg/kg) under deep anesthesia.The mice were promptly dissected, and samples were collected for hematoxylin and eosin (HE) staining and immunohistochemical analysis.

| Histological examination
Skin samples were fixed in a 4% formaldehyde solution, embedded in paraffin, sectioned (5 μm), stained with HE, and then observed under a light microscope.The histopathological scoring analysis involved counting infiltrating cells in the dermis, with six randomly selected areas from three slices per specimen.

| Patient samples
(5) patients who had strong sun exposure in the preceding 1 month; (6) patients with scar diathesis.

| Instruments
The skin was assessed using the ISEMECO skin imaging analyzer from Shanghai Meice, China, and a dermatoscope from Dr. Camscope, Korea.MAP Wound Recovery Dressing from Jiangyin USUN Biochemical, China, was employed for delivering MAP.The microneedle tool employed for the procedure was 1 mm in length and manufactured by Suzhou CYNOUR, China.

| Methods
After the patient cleaned their face, lidocaine cream was applied externally for 30 min, followed by iodine disinfection.Transdermal administration involved using a 1.0 mm rolling needle across the entire face, rolling three to five times outward from the periphery to the center and from top to bottom, forming a "well" pattern with a steady rolling needle motion.Prior to each procedure, MAP was generously applied, and after the rolling needle procedure, the remaining medication was used.When the facial skin showed uniform redness and punctate bleeding, the treatment was considered completed.After the procedure, patients were guided on routine care requirements and strict sunscreen application.Patients were treated every other month, three times during each treatment.The course of treatment depended on the recovery of each patient.During each visit, the patients were asked about adverse reactions and underwent follow-up for 3 months posttreatment.

| Efficacy evaluations
During the posttreatment follow-up, clinical images were captured utilizing the ISEMECO skin analyzer and a dermatoscope for comprehensive evaluation.The degree of facial redness and flushing was evaluated through clinician erythema assessment (CEA) 9 and the global flushing symptom score (GFSS), 10

| Statistical analysis
Statistical analysis and graph preparation were conducted using SPSS version 22 and GraphPad Prism version 7 software.Continuous variables with a normal distribution are presented as mean ± standard deviation (SD).Paired t-tests were employed to compare paired samples within groups, while one-way ANOVA was used to compare multiple groups.

| Therapeutic effect of MAP administered via microneedle delivery on rosacea-like mice
The intradermal administration of LL-37 in mice resulted in the development of characteristic rosacea-like lesions, characterized by erythema, telangiectasia, and cutaneous inflammation.However, the application of MAP treatment via microneedling led to a significant improvement in these symptoms (Figure 1A).After the treatment, the capillary density of the lesions decreased and the area of erythema decreased significantly, as observed under the dermatoscope (Figure 1B).We scored the capillary density and erythema in the dermoscopy images after treatment to assess the severity of the lesions.The capillary density and erythema score were significantly reduced in the group treated with MAP via microneedle delivery compared to the LL-37 group (95% CI: 1.14-1.97,p = 0.001, Figure 1C; 95% CI: 1.73-2.37,<0.001, Figure 1D).The histological analysis using HE staining (Figure 2A) revealed that treatment with LL-37 resulted in the proliferation and dilation of blood vessels in mice, accompanied by extensive infiltration of inflammatory cells, which closely resembled the histological characteristics observed in patients with rosacea.Importantly, the histological manifestations showed significant improvement upon microneedle delivery of MAP (95% CI: 352.91-573.76,p < 0.001, Figure 2B).

| Analysis of the therapeutic effect of MAP microneedle delivery
The demographic and baseline characteristics of the 27 patients who completed the follow-up after receiving treatment are summarized in clearly demonstrate the improvement in the patient's condition following the treatment.In addition, images acquired using the dermatoscope showed that the facial capillary density decreased significantly, and a part of the dilated capillaries shrunk and ruptured in response to MAP treatment (Figure 5).

| Safety and patient satisfaction
During the treatment, the patients experienced slight pain, and some patients showed transient erythema, which disappeared within 2-3 days.The patient satisfaction score was assessed at various time points: immediately after treatment, and at 1, 3, 6, and 12 months posttreatment.A satisfaction score scale was used to evaluate individual subjective satisfaction with the treatment effect, incorporating responses such as "very satisfied," "satisfied," "average," "dissatisfied," and "very dissatisfied."This scale ranged from 1 to 5. The satisfaction rate was calculated using the formula: satisfaction rate = (number of very satisfied cases + satisfied cases)/ total cases × 100%.Throughout the follow-up period, there were no instances of complications, including issues like uneven pigmentation or the formation of scars.Additionally, the patients exhibited a notable level of satisfaction, as indicated by the data presented in Table 2.

| DISCUSS ION
ETR is a persistent inflammatory dermatological condition primarily characterized by alterations in the central facial region.This disorder significantly impacts patients' physical appearance and exerts a profound influence on their psychological well-being and overall quality of life. 11The pathogenesis of this disease has not been fully elucidated and may be related to innate immunity, inflammatory cell infiltration, and abnormal neurovascular regulation. 12In patients with rosacea, various external stimuli affect the skin and damage its barrier function, which leads to the activation of TLR-2 on the surface of keratinocytes and the upregulation of KLK-5 expression induced by an increase in TLR-2 expression in the skin.KLK5, as a key enzyme, converts the antimicrobial peptide hCAP18 translated by CAMP protein into active LL-37, which emerges as a pivotal determinant in the pathogenesis of rosacea.Although rosacea can be treated in various ways, the efficacy and safety of such methods remain limited.In this study, MAP delivered by microneedling considerably improved the transdermal absorption rate of the drug in rosacea.
MAP is a high-purity protein that is easily absorbed by the human body.It possesses the ability to create a protective barrier, enhance wound healing, and bolster the skin's resilience against diverse external stimuli. 13However, most high-molecular-weight drugs only act superficially on the skin and do not penetrate the skin layers.Therefore, it is usually necessary to expose the epidermal channel and deliver the drug to the deeper layers of the skin to achieve the desired effect.Microneedle therapy, alternatively referred to as microneedle percutaneous collagen induction therapy, represents an emerging cosmetic dermatology treatment modality. 14In this treatment, skin permeability is increased by mechanically piercing the epidermal barrier with a microneedle.Upon the skin's puncture by the microneedle, it incurs micro-damage that triggers the self-repair mechanism, stimulating the synthesis of growth factors, and collagen, and facilitating wound healing. 15Dong et al. show that MAP delivered via a microneedle can be used to effectively treat sensitive skin. 16 the current research, multiple methods have been reported for constructing an animal model of rosacea, among which LL-37 is the most commonly used agent in the construction of animal models of rosacea. 16,17Therefore, we selected the mouse model of rosacea induced by the intradermal injection of LL-37 to explore the histopathological characteristics and molecular mechanism of action of MAP in rosacea treatment.The mice exhibited conspicuous scratching behavior subsequent to the administration of LL-37.Erythema and inflammation were observed in the dorsal area of injection, which led to the formation of a skin lesion similar to that observed in rosacea.Inflammatory cell infiltration and telangiectasia were  The microneedle delivery of MAP significantly downregulates the expression of TLR2, KLK5, and CAMP. 21In addition, upon identification of external triggers, TLR2 can orchestrate immune responses and unleash inflammatory mediators, including IL-1β and TNFα. 22markably, we observed a downregulation of IL-1β and TNFα expression in rosacea lesions following the microneedle delivery of MAP.To sum up, our findings show that the microneedle delivery of MAP can be used to treat rosacea by modulating the immune response.
Rosacea is a skin disease associated with neurovascular dysfunction, characterized by increased nerve fiber density and vasodilation. 23Individuals with rosacea experience a multitude of interactions between the neuroimmune and neurovascular systems within their skin. 24Furthermore, external triggers can activate ion channels and heighten the body's sensitivity to environmental stimuli.TRPV1, TRPV2, and TRPV4, which belong to the TRPV superfamily, play a role in regulating blood vessels, contribute to neurovascular disorders, and trigger early symptoms of rosacea, such as temporary flushing. 25Additionally, rosacea tissue exhibits abnormally elevated levels of VEGF expression, which contributes to the development of rosacea by promoting angiogenesis and inflammation. 7To confirm the inhibitory effect of MAP microneedle delivery on neurological function and blood vessels, we conducted immunohistochemical staining for TRPV1, TRPV2, TRPV4, and VEGF.
The MAP microneedle delivery group exhibited significantly lower expression levels of TRPV1, TRPV2, TRPV4, and VEGF compared to the LL-37 group.This suggests that MAP microneedle therapy can regulate abnormalities in vascular and nerve fiber function.
Expanding upon the favorable results observed in animal models, we introduced MAP microneedle therapy into clinical practice.
By employing a skin image analyzer, clinical images demonstrated a substantial enhancement in the patient's condition following MAP microneedle therapy compared to their pre-treatment state.This improvement was evident in the form of reduced red values, CEA levels, and GFSS scores, thereby offering additional validation of the efficacy of MAP microneedle therapy for treating rosacea.
However, this study also had certain limitations and shortcomings, such as a small sample size in clinical studies, the absence of a control group, and a short follow-up time.We acknowledge these limitations and plan to address them in future studies to enhance the quality and robustness of our research.

| CON CLUS ION
Our findings indicate that the microneedle delivery of MAP can ameliorate the symptoms of ETR by reducing inflammation and modulating immunity and neurovascular abnormalities.This approach may be regarded as a novel, safe, and efficacious therapeutic method for the treatment of ETR.

FU N D I N G I N FO R M ATI O N
Funding not received for the study.

The 7 -
week-old BALB/c mice were purchased from the Animal Experimental Center at Yanbian University in Jilin, China.At the time of purchase, each mouse weighed approximately 20 g.Before the experiments, the animals underwent a one-week acclimatization period.All animal experiments were conducted under specific pathogen-free conditions.The mice were randomly allocated into the following four groups: blank control, LL-37 (rosacea-like mouse), microneedle (LL-37 + MN), and MAP microneedle delivery groups (LL-37 + MN + MAP) (n = 6 in each group).

A
total of 27 patients diagnosed with ETR were enrolled in the Department of Dermatology at Yanbian University Hospital and Suzhou Mylike Cosmetic Hospital.The diagnosis of rosacea is primarily based on international standards.Here are the exclusion criteria 8 : (1) patients with severe organic or autoimmune diseases; (2) women who were pregnant or lactating; (3) patients who took photosensitive drugs or exhibited a photosensitive reaction in the past; respectively, by a dermatologist who was not directly involved in the clinical study.The scoring criteria used in the study were as follows: The CEA score was assigned on a scale of 0-4, where 0 indicated clear skin without any signs of erythema, 1 represented almost clear skin with slight redness, 2 denoted mild erythema with definite redness, 3 indicated moderate erythema with marked redness, and 4 represented severe erythema with extreme redness.For the GFSS, patients were evaluated based on the presence and intensity of symptoms and signs associated with skin flushing, including skin redness, warmth, tingling, and/or itching.The GFSS score categories were defined as follows: 0-3 indicated none or mild symptoms, 4-6 represented moderate symptoms, 7-9 denoted severe symptoms, and 10 indicated extreme symptoms.These scoring systems were applied by an independent dermatologist who was not directly involved in the clinical study.

Table 1 .
Different durations of treatment were selected according to the severity and recovery of the patient (3-6 times, mean 4.67 ± 1.42 times).The representative images depicting the patient's condition before and after treatment are presented in Figure 4.The images F I G U R E 1 Compared with that in the LL-37 group, the microneedle delivery of MAP improved erythema and capillary abnormalities in mice.(A) Comparison of skin lesions in each group after treatment.(B) Comparison of skin lesions under a dermatoscope (dermoscopy ×10, polarized light).(C) Capillary density score.(D) Erythema score.n = 6, **p < 0.01.F I G U R E 2 The infiltration of inflammatory cells was significantly lesser in the MAP microneedle delivery group compared with that in the LL-37 group, and vascular dysfunction was significantly better in the former group.(A) HE staining images recorded under a microscope; scale bar = 100 μm.(B) The statistical map shows the average total number of inflammatory cells per HPF.Data represent the means ± SEM from three independent experiments.n = 6, Magnification: 20×, **p < 0.01.F I G U R E 3 Microneedle delivery of MAP inhibited the expression of core molecules (TLR2, KLK5, CAMP, IL-1β, IL-6, TNFα, VEGF, TRPV1, TRPV2, and TRPV4) in rosacea.Data represent the means ± SEM from three independent experiments.Scale bar = 100 μm, n = 6, Magnification: 20×, **p < 0.01.

TA B L E 1
Demographic and baseline characteristics.

FitzpatrickF I G U R E 5
in the lesion area.In addition, key factors (TLR2, KLK5, and CAMP) and inflammatory factors (IL-1β, IL-6, and TNFα) involved in the pathogenesis of rosacea were upregulated.These findings are consistent with the modeling results reported previously,18 and the inflammatory pattern is consistent with the pathological phenotype of clinical rosacea.Immune imbalance plays a pivotal role in the pathogenesis of rosacea.19TLR2, an essential immune molecule, is intricately involvedF I G U R E4 Improvement of facial skin before and after treatment (ISEMECO skin image analyzer), baseline (A, D), 1 month after the first course of treatment (B, E), and 1 month after the second course of treatment (C, F).The improvement of facial skin before and 1 month after the second course of treatment (dermatoscopy ×10).(A) Baseline (natural light).(B) After treatment (natural light).(C) Baseline (polarized light).(D) After treatment (polarized light).in the progression of rosacea and exhibits elevated expression levels in the skin of individuals affected by this condition. 20TLR2 exerts its influence by promoting the enzymatic activity of KLK5, inducing the synthesis of KLK5, and amplifying the production of CAMP.

F I G U R E 6
Changes in the red value, CEA, and GFSS score before and after the microneedle delivery of MAP.n = 27, *p < 0.05 and **p < 0.01.TA B L E 2 Subject satisfaction (n = 27).