Sele-targeted siRNA liposome nanoparticles inhibit pathological scars formation via blocking the cross-talk between monocyte and endothelial cells: a preclinical study based on a novel mice scar model

Abstract

Background

Pathological scars (PS) are one of the most common complications in patients with trauma and burns, leading to functional impairments and aesthetic concerns. Mechanical tension at injury sites is a crucial factor in PS formation. However, the precise mechanisms remain unclear due to the lack of reliable animal models.

Results

We developed a novel mouse model, the Retroflex Scar Model (RSM), which induces PS by applying controlled tension to wounds in vivo. RNA sequencing identified significant transcriptome changes in RSM-induced scars. Elevated expression of E-Selectin (Sele) was observed in endothelial cells from both the RSM model and human PS (Keloid) samples. In vitro studies demonstrated that cyclic mechanical stretching (CMS) increased Sele expression, promoting monocyte adhesion and the release of pro-inflammatory factors. Single-cell sequencing analysis from the GEO database, complemented by Western blotting, immunofluorescence, and co-immunoprecipitation, confirmed the role of Sele-mediated monocyte adhesion in PS formation. Additionally, we developed Sele-targeted siRNA liposome nanoparticles (LNPs) to inhibit monocyte adhesion. Intradermal administration of these LNPs effectively reduced PS formation in both in vivo and in vitro studies.

Conclusions

This study successfully established a reliable mouse model for PS, highlighting the significant roles of mechanical tension and chronic inflammation in PS formation. We identified Sele as a key therapeutic target and developed Sele-targeted siRNA LNPs, which demonstrated potential as a preventive strategy for PS. These findings provide valuable insights into PS pathogenesis and open new avenues for developing effective treatments for pathological scars.

Graphical Abstract

Introduction

Scars, the final outcome of tissue repair following skin damage, manifest in various forms, including superficial scars, hypertrophic scars, and keloids. The latter two types are often grouped as pathological scars (PS) [1]. PS is a common disease, which can occur after surgery, trauma, burn, acne, or other cases. Epidemiological data has reported that PS affected 35% of individuals with surgery wounds [2] and 70% of individuals with burns wounds [3,4,5]. PS not only causes cosmetic concerns but also leads to severe symptoms such as pain, itching, contractures, and functional impairments, which can detrimentally affect a patient’s mental health and quality of life [1, 6], imposing substantial social and economic burdens, contributing greatly to disability-adjusted life-years (DALYs) lost and morbidity [7]. Despite the enormous demand for patient health and medical society, there has been little progress in treating patients with PS. Current treatments for PS, including surgical interventions [8], laser therapy [9, 10], fat transplantation [11], medications [12], and physical therapy [13], often yield unsatisfactory results. To improve the therapeutic effect of PS, standardized and innovative therapeutic strategies are needed.

To investigate PS formation and identify potential therapeutic targets, it is crucial to establish stable and easy-conducted scar model. Recent studies have focused on developing mice models to study hypertrophic scars under controlled mechanical tension. For example, S Aarabi et al. employed a tension device to apply mechanical stress on mouse dorsal wounds [14], simulating hypertrophic scar formation. Similarly, silicone plates were used to limit wound contraction and apply surface tension [15], successfully inducing hypertrophic scars in mice. Qingfeng Li et al. used a silica gel splint to delay the healing of the incision and applied a special tension device to apply tension to wound site to promote scar formation [16]. However, there is still no unified modeling method, which greatly hindered the research on the pathogenesis of PS. Therefore, an easy-conducted, more reliable, and stable PS model is urgently needed.

Although the precise pathogenesis of PS remains elusive, it is well-known that persistent high-tension stimulation at the wound site plays a critical role [17,18,19]. Hypertrophic scars typically appear in high-tension anatomical areas (anterior to the sternum) or throughout the entire joint (potentially contracted areas) [18, 20], and keloid lesions can occur in high mechanical stress areas after any form of deep dermal injury [21]. Tension in human wound skin is approximately double that of normal skin [22,23,24]. Repeated tension stimuli can prolong inflammatory responses, leading to the recruitment of effector cells and the secretion of cytokines and fibrotic factors, thereby promoting PS formation [25, 26]. Studies have shown that high skin tension could impair endothelial function, increasing the infiltration of inflammatory cells and the levels of inflammatory mediators at the wound site [27,28,29]. Inflammation is an important factor in the occurrence and development of PS [30]. Evidence suggested that sustained inflammation in susceptible individuals can lead to persistent changes in the extracellular matrix, resulting in a fibrotic phenotype [1]. Some studies have shown that the expression of proinflammatory cytokines in PS increases, including interleukin-6 (IL-6), IL8, IL18, and IL-17 when compared with normal skin and normal scar [31], which indicated that a proinflammatory immune ecology existed in PS tissues [32, 33]. Previous studies have found differences in the immune infiltration between PS tissues and adjacent normal tissues via single-cell RNA sequencing, especially the increase of monocyte-macrophages [34].

The recruitment of immune cells by endothelial cells is a pivotal event in the inflammatory response [35, 36]. Selectins are an evolutionarily ancient family of 3 cell adhesion molecules with known roles in mediating immune cells rolling and homing to tissues [37]. E-(endothelial) selectin (Sele) and P (platelet and endothelial) selectin (Selp) are expressed on activated or inflamed endothelium, which are usually expressed together with integrin ligands [38]. Selectins and their ligands facilitate immune cell recruitment by recognizing specific carbohydrates on cell surfaces, which are crucial in various physiological and pathological processes, including inflammation [39], infection, cancer [40], and immune surveillance [41].

Since the approval of Patisiran in 2018 for treating hereditary amyloidosis-related peripheral neuropathy underscores the therapeutic potential of small interfering RNA (siRNA) [42], siRNA-mediated gene silencing offers a promising approach for disease treatment by effectively inhibiting the expression of disease-related genes at the mRNA level [43]. Numerous siRNA-based therapies are currently in advanced clinical trials, with some already approved for use [44,45,46]. Lipid nanoparticles (LNPs) represent a clinically validated delivery system for siRNA, offering high encapsulation efficiency, protection against enzymatic degradation, and enhanced cellular uptake [47, 48]. These features make LNPs suitable for the safe and effective delivery of siRNA to target tissues. Local siRNA injections have demonstrated efficacy in targeting organs such as skin, the central nervous system, eyes, and lungs [49,50,51], where direct injection into wounds can enhance retention and cellular uptake. Recently, the delivery of siRNA to skin tissues has also made breakthroughs. Studies have successfully targeted the pathogenic genes of psoriasis through RNA interference (RNAi) therapy [52], thereby inhibiting the excessive proliferation of keratinocytes in psoriatic skin tissue and enhancing immune activation. These research advances and technologies inspired us to propose gene therapies to inhibit PS formation.

In this study, we established a tension-based mice scar model named Retroflex scar model (RSM). According to the analysis of the molecular biology characteristics of mice RSM and human PS tissues, we found that a high-tension environment could promote the expression of Sele in endothelial cells, thereby promoting the homing of inflammatory cells in blood vessels during wound healing and affecting the immune ecology of the wound. Based on this, we designed and validated an LNP-mediated RNAi method targeting Sele to suppress high-tension-induced inflammation and inhibit PS formation in RSM. We have developed LNPs using cationic lipid materials (DOPE, DOTAP) to encapsulate siRNA targeting Sele, forming stable LNPs that protect siRNA from nuclease degradation and promote its delivery. This construction is named siSele@LNPs. siSele@LNPs were delivered to the wound tissue via intradermal injection (ID), it was found that siSele effectively downregulated the mRNA expression level of Sele in endothelial cells, reduced recruitment of monocyte at the wound site, reduced collagen deposition, and decreased expression of actin, significantly improving scar features such as surface area, cross-sectional area, and collagen deposition rate. This study firstly demonstrated that ID of LNPs loaded with siRNA could effectively block Sele-mediated cross-talk between monocyte and endothelial cells in the wound site, providing a promising treatment method for PS inhibition.

Materials and methods

Experimental design

The purpose of this study is to utilize tension to induce and establish a stable and reproducible mouse scar model, which can be used to conduct in-depth research on the pathogenesis of tension-induced PS In this study, we identified and validated the key role of Sele in the formation of tension-induced PS. Based on this discovery, we designed feasible siRNA lipid nanoparticles (LNPs) targeting Sele to specifically silence Sele expression in endothelial cells, and verified the therapeutic effect of this strategy in scar model mice. The physicochemical properties of the targeted siSele LNP were characterized by measuring size, morphology, zeta potential, encapsulation efficiency (EE), and stability. Primary vascular endothelial cells will be extracted from the skin to assess the safety, uptake ability, and ability to target and silence genes in LNP. According to the protocols approved by the Institutional Animal Care and Use Committee of the Ninth People’s Hospital affiliated with Shanghai Jiao Tong University School of Medicine, the in vivo targeting ability and therapeutic effect will be evaluated in C57/BL6 mice. Mice will be randomly assigned to each study group. All experiments in this study will be conducted using at least five independent replicates.

Ethics statement

Six to eight weeks old Male C57/BL6 were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. The study was carried out in compliance with ethical guidelines and was approved by the ethics committee of Shanghai Ninth People's Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, under the approval number SH9H-2020-A314-1. Efforts were made to minimize the number of animals used and alleviate any discomfort they might experience. PS (keloid) tissue and normal skin tissue were both taken from the Department of Plastic Surgery and Dermatology of Shanghai Ninth People's Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, including 5 cases of keloid tissue (Table 1) and 5 cases of normal skin tissue as a control group. The clinical study was approved by the ethical review board of, under the approval number SH9 H-2024-TK400-1.

Table 1 Clinical characteristics of keloid patients

Retroflex scar model (RSM) in mice

In our study, a linear incision with a length of 1.5 cm was made on the dorsal side of mice, and the wound was sutured with 5–0 suture. Remove the suture on the third day to ensure that the linear wound does not rupture. After removing the stitches, retroflex the skin 1 cm away from both sides of the linear wound along the curved tensile device, and use 3–0 suture at the top of the tensile device to fix the skin on both sides together through small holes, forming a cylinder composed of the skin on both sides curled up to keep the skin below the wound tight. The design drawing of the tensile device manufacturer is shown in Fig. 1B. The mold manufacturer is a 300-degree cylindrical structure made of Polyethylene (PE) material, using 3D printing technology, with small holes at the top for fixing the skin on both sides. The tension of the skin fixed on the cylinder is adjusted according to the width of the insert in the middle of the cylinder. Measure the distance of skin extension at the wound site on days 0, 3, 7, and 14 after modeling, and ensure that the skin at the wound site is always kept under high tension by timely inserting spacers of different widths, the detailed calculation formulas are provided in supplementary Text. Mice in static group underwent the same linear incision and suture as the RSM model. After suture removal, the wounds were allowed to heal naturally without any device intervention.

Fig. 1

Construction and characterization of a novel stretch-mediated mice RSM. A Side and front views of the mice Retroflex scar model (RSM). B Schematic of the RSM tensile device, highlighting the dimensions of cylindrical body and insert. C The procedure of molding. D Gross morphology of scars in control (Static) and RSM groups on days 14 and 60 post-incision. Histological assessments at day 14: E H&E staining, F Masson’s trichrome, and G CD31 immunostaining scale bar = 200 μm. H–J Semi-quantitative analysis of histology for control and RSM groups on day 14. K–L Western blot analysis and semi-quantification of Fibronectin, Col1a1, and Acta2 at day 14 (n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Extraction of primary human dermal vascular endothelial cells and cell culture

Fresh human skin tissue was sourced from plastic surgery, informed consent was obtained from the patients or their legal representatives. Skin tissue was cut into small pieces (approximately 1–2 mm³). The tissue pieces are then digested using a mixture of collagenase I (biosharp, BS163), hyaluronidase (20426ES60) and trypsin–EDTA (Gibco, 25200056). The digested tissue is then gently triturated to release cells, and the cell suspension is filtered through a 70 μm cell strainer (corning, CLS431751) to remove undigested tissue fragments. The cell suspension is centrifuged at 300g for 10 min, the supernatant is discarded, and the cell pellet is resuspended in endothelial cell growth medium EGM-2 (Clonetics, CC-3162). Endothelial cell line HMEC-1 was obtained from National Collection of Authenticated Cell Cultures, and cultured in endothelial cell growth medium EGM-2 (Clonetics, CC-3162). Monocyte cell-line THP-1 was cultured in RPMI-1640 medium (SH30809.01, HyClone, USA) containing 10% FBS and 1% penicillin/streptomycin/fungizone (15240-062, Gibco, USA).

Monocyte-endothelial cell adhesion assay

Primary endothelial cells were digested from a static CMS device or a stretched CMS device and cultured in confocal dishes (FCFC020-10pcs, Beyotime). THP-1 cells were stained with CellTrace (40717ES50, Yeasen) for 20 min before coincubation with the endothelial cells for 30 min. Observation and counting of adherent THP-1 cells using a microscope (Zeiss, Germany) and ImageJ software.

CRISPR/Cas9 mediated Sele knockout

The deletion of the Sele gene in HMEC-1 cell lines was performed. (Cas9X, China). In brief, the gene sequence of Sele was retrieved from the NCBI database, and appropriate sgRNA was designed and used to construct plasmids. Using CRISPR/Cas9 gene editing technology, the Cas9 and sgRNA plasmids were electroporated into the cells. CRISPR/Cas9-mediated Sele endothelial cells were then screened using PCR and confirmed to be deleted.

Histology and immunohistochemistry

Cut off the scar tissue and quickly immerse it in 4% paraformaldehyde for 24 h. Dehydrate the scar tissue and embed it in avidin, then slice it and use H&E, and Masson’s tri-cochrome staining. For immunohistochemistry analysis, tissue sections were treated with appropriate primary antibodies followed by incubation with HRP-conjugated secondary antibodies. The 3,3′-diaminobenzidine (DAB) substrate was used for direct visualization of the distribution of target proteins.

Western blot and co-immunoprecipitation (CO-IP)

Protein samples from cells and tissue were extracted using the Radio Immunoprecipitation Assay (PC101, Epizyme) and quantified using the BCA Protein Assay (ZJ102, Epizyme). After denaturing and separating on 7.5% or 12.5% Sodium Dodecyl Sulfate–Polyacrylamide Gels (SDS-PAGE), the protein on SDS-gels were transferred to a PVDF membrane (IPVH00010, Milipore), and incubating with the primary antibodies as follows: Sele (1:1000, Santa Cruz, sc-137054), Atpb (1:1000, P06576, Abmart), Gapdh (1:50,000, 60,004-1-Ig, Proteintech), Col1a1(1:5000, 67288-1-Ig, Proteintech), Acta2(1:20,000, 67735-1-Ig, Proteintech), PSGL-1 (1:1000, T58338, Abmart), CD44(1:1000, T55122, Abmart), CD43(1:1000, PU202528, Abmart). Secondary antibodies were further used as follows: HRP-labeled Goat Anti-Rabbit IgG(H+L) (1:1000, A0208, Beyotime), HRP-labeled Goat Anti-Mouse IgG(H+L) (1:1000, A0216, Beyotime). For co-IP, protein sample was lysed by lysis buffer for IP (P0013, Beyotime). Before the co-IP, pre-incubated lysate with Protein A/G Magnetic Beads (HY-K0202, MedChemExpress) with rotator at 4 °C for 2 h for removing non-specific proteins bound to the beads. Subsequently, add diluted antibody (Ab) to the lysate above into a 1.5 mL tube for overnight at 4 °C, Mouse IgG (1ug for IP, B900620, Proteintech) were used as a negative control. Add the Protein A/G Magnetic Beads into antigen–antibody (Ag-Ab) complex and rotate tube for 2 h at 4 °C. Collect the protein–protein complexes, they were later subjected to Western blot for the further experiment.

RT-qPCR

Trizol reagent (Invitrogen, United States) was employed for total RNA extraction. The extracted RNA samples were then reverse-transcribed into cDNA using the PrimeScriptTM RT reagent Kit (TAKARA, Japan). RT-qPCR was carried out on a Light Cycler thermal cycler system (Bio-Rad, United States) using SYBR® Premix Ex Taq™ II (TAKARA, Japan). For mRNA normalization, GPADH was used as the internal control. The relative expression was compared to the control group. The sequences of the primers used in the present study are shown in Table 2.

Table 2 Primers for mRNA real-time polymerase chain reaction

Transcriptome sequencing

Second-generation sequencing was performed on the PS tissue from the RSM group and normal scar tissue from the control group. Sequencing was performed using the Illumina NovaSeq™ 6000 platform with paired-end (PE) 150 bp sequencing mode. The results were analyzed via R (v4.3.1) software.

Immunofluorescence

After administering anesthesia to the mice, the scar tissue was extracted with 4% paraformaldehyde. The scar tissue was dehydrated using sucrose and embedded in OCT compound (Sakura, Japan) before rapidly frozen at − 20 °C. Subsequently, the scars were coronally sliced into sections with a thickness of 30 μm. The scar sections underwent a triple wash with PBST and were incubated overnight at 4 °C with specific antibodies CD31(1:100, 11265-1-AP, Proteintech), CD11b (1:100, sc-52600, santa Cruz) and Sele (1:100, TD6914, Abmart). Following another round of triple washes with PBST, the sections were exposed to different secondary antibodies (Invitrogen, United States) for 1 h at room temperature. After a final triple wash with PBST, the skin sections were mounted using a DAPI-containing mounting medium (southenbiotech, United States). Digital pathology syestem was used to observe and capture images of sections.

Single-cell RNA sequence data analysis

The single-cell RNA-seq data for this study were obtained from GSE181316 and GSE156326, including human normal scar, human Keloid and human HS. Quality control and cell annotation were performed according to a prior study. R package Seurat (v4.1.3) was used for data integration, cell filtration, normalization, clustering, and Uniform Manifold Approximation and Projection (UMAP) dimensional reduction. Cell progression genes were defined based on DEGs among Seurat clusters. Inference and analysis of cell–cell communication were used via R package CellChat to v2.

Mechanical stretch devices

Using FX-5000T ™ The Flexcell Tension Plus system (Flexcell International Corporation, Hillsborough, NC, USA) applies cyclic mechanical stretching (CMS) to endothelial cells. Endothelial cells were cultured at a rate of 2 * 105 cells/mL on a 6-well BioFlex culture (Flexcell International Corporation, Hillsborough, NC, USA) with a flexible silicon membrane bottom. CMS is applied to the stretching group in a sinusoidal mode with an amplitude of 0%–20% at 0.5 Hz. Static cells cultured in the same type of plate but kept stationary were incubated in the same incubator.

Inflammation array and ELISA array

Quantibody® Human Inflammation Array 1 by RayBiotech (Norcross, GA, USA) was utilized to quantitatively detect the supernatant of static or stretched Endothelial-Monocytes co-culture system. It employs specific antibody pairs designed for this purpose, the experimental protocol and data analysis procedures recommended by Ray Biotech were strictly followed. Cytokines displaying statistical significance (p < 0.05) across different experimental groups were considered statistically significant. To facilitate data visualization and interpretation, a volcano plot was generated using the R statistical software version 4.3.1, allowing for the identification of trends and patterns in the data. To investigate the specific secretion pro-inflammation cytokines, ELISA were employed. After various experimental interventions, culture medium was harvested, followed by centrifugation at 400×g for 5 min at 4 °C. The resulting supernatants were meticulously collected and subjected to quantification using a Human IL-1α ELISA kit (PI565, Beyotime), Human IL-6 ELISA kit (ab178013, Abcam), and Human TNF-alpha ELISA Kit (ab108908, Abcam).

Preparation of siRNA@LNPs

LNPs are prepared by thin filming-rehydration method from cationic lipids DOTAP (MCE, USA), DOPE (MCE, USA), cholesterol (McLean, USA), and DSPE-PEG2000 (MCE, USA). Dissolve DOTAP, DOPE, cholesterol, and DSPE-PEG2000 (in order of 5, 5, 0.5, and 0.11 mg) in chloroform (3 mL), and transfer the lipid solution to a round bottom flask after ultrasonic treatment to fully dissolve it. Steam under vacuum conditions for 30 min (75 r/min, room temperature) to allow the organic solvent to completely evaporate and obtain a homogeneous lipid film. Hydrate with 3 mL of enzyme free water for 10 min (100 r/min) and then homogenize with ultrasound. Then, the siRNA solution was mixed with LNPs by electrostatic interaction at a weight ratio of 1:30, and concentrated by centrifugation in a Macrosep Advance filtration device (MAP001C37) (3000g/min, 4 °C). The molecular weight was cut off to 10 kDa to remove the unsealed drug.

siRNA@LNPs stability assessment and physicochemical characterization

To test the stability of naked siRNA and siRNA@LNPs, incubating naked siRNA or siRNA@LNPs with RNase (10 μg/mL) at 37 °C for 0, 15, 30, 60, 120, or 240 min. Then collect LNPs by centrifuging at 12,000 rpm for 10 min. Then extracted siRNA in 0.5 M NaCl containing 0.1% SDS (58). Electrophoresis was performed on 3% agarose gel injected by GelRed, and then imaging was performed under ultraviolet (UV) light. The particle size of LNPs was measured before and after stretching using DLS (Malvern, UK). Transmission electron microscope (TEM) (Thermo Fisher Scientific, USA) was used to characterize the morphology of LNPs before and after stretching. The size and zeta potential of LNPs were measured using DLS.

Determination of encapsulation efficiency

Measure the encapsulation efficiency (EE) according to the instructions of Quant-iT RiboGreen reagent. Prepare LNP loaded with siRNA according to the above method. Mix 3 μ l volume of LNP solution with 117 μL of 1 × TE (Tris EDTA) buffer or 2% Triton X-100, and vortex for 2 min. Dilute the free siRNA standard with a series of concentrations. Incubate NP samples and siRNA standards with an equal volume of 1:200 diluted RiboGreen reagent for 5 min. Measure fluorescence intensity using an enzyme-linked immunosorbent assay (excitation/emission, 480/520 nm; Tecan, Switzerland). Calculate the encapsulation efficiency (EE%) using the following formula:

$$ {\text{EE}}\left( \% \right) = \left( {{\text{fluorescence}}\;{\text{of}}\;{\text{B}} - {\text{fluorescence}}\;{\text{of}}\;{\text{A}}} \right)/\left( {{\text{fluorescence}}\;{\text{of}}\;{\text{B}}} \right) \times {1}00\% $$

Biodistribution study

Twenty-four hours after local injection of the cy5-siSele@LNPs at the wound site, mice were subjected to live imaging on the IVIS system (Perkin Elmer, USA). Immediately after imaging, the mice were sacrificed and their hearts, liver, spleen, and lungs were removed for imaging on the IVIS system. To determine cy5-siSele@LNPs Cell targeted collection, shredding, and digestion of scar tissue in PBS containing collagenase I (201.3U/ml; Yeason), 0.92 M Hepes (Beyotime), and DNase I (50.3U/ml; Sigma Aldrich) at 37 °C for 1 h. Filter the obtained digests using a 70 μm cell filter and treat with red blood cell lysis buffer for 5 min. Then centrifuge the sample at 400g, resuspend it in PBS containing 0.5% BSA, and filter it through a 40 μm cell filter. Subsequently, the sample was incubated with antibodies targeting epithelial cells (EpCAM Alexa Fluor 647), immune cells (CD45 phycoerythrin), and endothelial cells (CD31 Bright Purple 421) markers at 4 °C for 30 min. Using flow cytometry (BD Biosciences, USA) to determine various cell subtypes cy5-siSele@LNPs level.

Statistical analysis

All experimental data were expressed as the mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 9.0 software (La Jolla, CA, USA). A t-test was used for comparisons between two groups. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s multiple comparison test was conducted. All tests were two-tailed, with p < 0.05 indicating statistical significance.

Results

Construction and characterization of a novel stretch-mediated mice RSM

To construct a stable and reliable scar model, RSM was designed to induce PS on the back of mice by exerting controllable tension. Figure 1A showed the photograph of the side and front view of the mice RSM. The video in the supplementary movie S1 showed that the tensile device could load on the back of the mice stably, and did not affect the mobility of the mice. Figure 1B provided a detailed demonstration of the structure of the tensile device, and the detailed molding procedure is shown in Fig. 1C. As shown in Table S1, we measured the distance between the retroflex skin on both sides at different time points and derived a calculation program (Supplementary Text) to calculate to the required width of the insert, we used inserts with widths of 5.4 mm, 5.8 mm, 6.3 mm, and 6.4 mm on days 0, 3, 7, and 14, respectively, to maintain high tension on the skin at the wound site. To evaluate the effect of the tensile device, we evaluated the wound healing status at 14 and 60 days after loading the tensile device, respectively. As it shown in Fig. 1D, compared to the scars of mice in the static group, the scars of mice in the RSM group were more obvious on 14 days after injury, and there was no atrophy of the scars on day 60, with no hair growth on the surface. Further evaluation of the cross-sectional area of the scar was conducted using HE staining (Fig. 1E), the cross-sectional area of scar tissue in the RSM group was five-fold to that in the static group (Fig. 1H). The results of Masson staining indicated that the loaded mold marker significantly promoted collagen deposition, the proportion of collagen fiber in the RSM group was about twice or more than that in the static group (Fig. 1F and I). Additionally, Masson staining revealed that the collagen protein in the scar tissue was disorderly arranged in a vortex shape in the RSM group, which is one of the important characteristics of PS in humans (Fig. 1F). Furthermore, we evaluated the number and distribution of blood vessels in these scar tissues, IHC of CD31 showed that the scar tissue of RSM group distributed with more vascular than that in static group (Fig. 1G and J). Western blotting showed a significantly higher protein level of muscle fibroblast biomarkers Acta2,

ECM protein Fibronectin, and Col1a1 of the scar tissue in RSM mice compared to that in the control group (Fig. 1K and L).

Sele expression was increased in human and mice RSM PS tissues

To explore the molecular mechanism of PS formation caused by high-tension, we performed second-transcriptome sequencing on the scar tissues in mice from the static and RSM groups. Enrichment analysis on differentially expressed genes (DEGs) was conducted (Fig. 2A), and it was found that a large number of biological processes related to collagen or extracellular matrix synthesis were activated in the scar tissues from the RSM group, which was consistent with the histological phenotype in mice RSM PS tissues. Additionally, we also found that genes related to the CAMs process were significantly upregulated in the RSM group, including Mpzl1, Sele, Jam3, and Igsf11 (Fig. 2C). Furthermore, the expression levels of these 4 genes in human normal skin and PS (keloid) tissues were tested. The results found that Sele mRNA was significantly upregulated in human PS (Fig. 2G), while the expression levels of Isgf11 (Fig. 2D), Jam3 (Fig. 2E), and Mpzl1 (Fig. 2F) showed no difference between normal skin and keloid tissues in human. Consistently, western blotting also confirmed that the protein expression level of Sele was upregulated in human and mice RSM PS tissues (Fig. 2H). Since Sele is mainly expressed on the activated endothelial cells. The flow cytometry results of scar tissue in mice showed that the proportion of endothelial cells (CD31+) within the PS tissue of the RSM group was higher than that in the normal scar (NS) tissue within the static group, and the proportion of Sele+ endothelial cells was also significantly increased, increasing by about 6 times (Fig. 2I). IF detection also indicated that compared with the normal scar in the static group, the number of endothelial cells and the expression level of Sele within PS tissue in the RSM group were significantly increased, and Sele protein was mainly localized in endothelial cells (Fig. 2J). To further investigate the relationship of Sele, endothelial cells, and monocytes in high-tension-induced PS formation, we analyzed the single-cell lineages of NS and PS (keloid) tissues in human (Fig. 2K). As shown in Fig. 2L, M, a significant increase of cluster 0_endothelial cells were found in keloid tissues. After enriching and analyzing the functions of cluster 0_endothelial cells, we found these cells mainly involved in the differentiation and activation of immune cells, and CAMs were highly activated in the cluster 0_endothelial cells (Fig. 2N). Furthermore, we found Sele was also upregulated in (keloid)_endothelial cells, and its expression mainly concentrated on cluster 0_endothelial cells (Fig. 2O). Therefore, in this study, we named cluster 0_endothelial cells as Sele_high endothelial cells, while other endothelial cells were referred to as Sele_low endothelial cells. Human PS was classified into two subtypes, including keloids and HS. Therefore, to further confirm the upregulation of Sele_high endothelial cells in PS tissues, we also compared scRNA-seq data from human NS and HS tissues. The results also confirmed that Sele_high endothelial cells are significantly more abundant in HS tissue (Fig. S2).

Fig. 2

Sele expression was increased in human and mice RSM PS tissues. A Enrichment analysis of differentially expressed genes highly expressed in RSM-derived pathological scar (PS) tissues. B Schematic diagram of tissue sequencing and validation of PS tissue in mice and humans. C Cell Adhesion Molecules (CAMs)-related genes upregulated in RSM-derived PS tissues. Relative mRNA expression levels of Igsfl1 (D), Jam3 (E), Mpzl1 (F), and Sele (G) in normal skin and human PS (keloid) tissue. H Western blot analysis of Sele protein in mice-derived normal scar and PS, and human-derived normal skin and keloid tissue. I Flow cytometry analysis of PS tissue in mice. J Immunofluorescence images of normal scar in control (Static) and PS in RSM groups. Scale bar = 10 μm. K UMAP image of clustering from single-cell RNA sequencing (scRNA-seq) dataset GSE181316 comparing normal scar and keloid tissue in humans. L, M Cluster and number proportion bar charts of endothelial cells in various clusters of human normal scar and keloid tissue from dataset GSE181316. (N) Functional enrichment analysis of cluster_0 endothelial cells. O Expression distribution map of Sele on endothelial cells clustering map (n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

CMS stimulated Sele overexpression and Sele-dependent monocyte adhension to endothelial cells

To explore the role and mechanism of increased Sele expression in PS formation under high-tension. A CMS model was implemented in vitro to mimic the high-tension during PS formation, and the protein expression of Sele was measured in primary human dermal vascular endothelial cells exposed to different tensile strengths. Notably, it was found that Sele expression was upregulated as strength increased from 0 to 15%, while no significant change in Sele expression as strength increased from 15 to 20% (Fig. 3A, B). Previous studies have confirmed that Sele could mediate cell adhesion to the inner wall of blood vessels, leading to the accumulation of white blood cells in the inflammatory site [53, 54]. Therefore, we further tested the adhesion ability of endothelial cells exposed to different tensile strengths. Consistent with the changes in Sele expression levels, the adhesion of endothelial cells to monocytes significantly increased as the tensile strength increased from 0 to 15%. However, there was no significant change in adhesion when the tensile strength increased further from 15 to 20% (Fig. 3C, D). Therefore, 15% tensile strength was selected as the tensile strength in subsequent experiments. IF assay further confirmed that endothelial cells in the RSM group showed higher expression of Sele with more monocyte adhesion compared to that in the static group (Fig. 3E). A linear regression model was established based on the quantitative analysis of Sele expression level and the counts of adherent monocytes, which indicated that the ability of monocytes adhesion were positively correlated with the expression level of Sele, with a calculated correlation coefficient of 0.9420 (Fig. 3F). In addition, the supernatants from co-culture systems of endothelial cells and monocytes under different tension environments were collected and inflammatory array was conducted (Fig. 3G). The results showed that IL-1a, TNF-a, and IL-6 were significantly upregulated after CMS treatment (Fig. 3H). To further verify the role of Sele in promoting monocyte adhesion and inflammatory response induced by high-tension, we built the Sele knockout (KO)-HMEC-1 cell line. As shown in Fig. 3I, monocyte adhesion in wild-type (WT) HMEC-1 was increased in the stretched group compared to that in the static group, while the KO-treatment reversed this increase (Fig. 3I, J). Meanwhile, the ELISA test verified that high-tension significantly promoted the release of inflammatory factors, including IL-1a (Fig. 3K), TNF-a (Fig. 3L), and IL-6 (Fig. 3M). However, knocking out the Sele gene could effectively inhibit the release of pro-inflammatory factors in high-tension status (Fig. 3K–M). These findings suggested that Sele played a key role in stimulating the monocyte adhesion to endothelial cells and increasing inflammatory infiltration. Additionally, they imply that inhibiting Sele could be an effective strategy to prevent excessive and persistent inflammatory responses, and consequently, inhibit the formation of PS.

Fig. 3

CMS stimulated Sele overexpression and Sele-dependent monocyte adhension to Endothelial cells. A Protein expression of Sele in primary human dermal vascular endothelial cells assessed in a tensile strength-dependent manner. B Quantitative analysis of Sele expression levels. C, D Representative fluorescence images and quantitative analysis of the monocyte adhesion to endothelial cells in a tensile strength-dependent manner. Scale bar = 200 μm. E High-resolution confocal microscopy image of co-cultured endothelial cells and monocyte. Scale bar = 50 μm. F Correlation analysis between Sele expression levels and monocyte adhesion numbers. G, H Volcano plot of the detection results of the multi-inflammation chip for the supernatant from static and stretched groups. I, J Representative fluorescence images and quantitative analysis of monocyte adhesion to HMEC-1 cells in control, stretched, and stretched with Sele knocking out groups. Scale bar = 200 μm. Release of IL-1a (K), TNF-a (L), and IL-6 (M) in co-cultures of HMEC-1 cells and monocytes (n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Endothelial cells recruited monocytes via Sele-CD44 interaction upon high-tension

Previous studies had reported various selectins and selectin ligands were involved in the transport of monocyte to target-organs during autoimmune diseases. These selectin ligand-receptor pairs included Sele-CD44, Sele-CD43, and Sele-PSGL-1 [37]. To investigate the molecular mechanism of Sele-mediated monocyte adhesion in endothelial cells, the co-culture system of endothelial cells HEMC-1 and monocyte THP-1 was established and CO-IP array was conducted. Cells were collected after co-culturing for 24 h, and the expression levels of CD44, CD43, and PSGL-1 were detected. CMS significantly increased the expression level of CD44 and CD43, and the expression of protein PSGL-1 was not detected. Notably, Sele-KO effectively reversed the high expression of CD44 and CD43 after CMS stimulation (Fig. 4A, D). CD43 protein was not detected in the pull-down protein using the IP antibody of Sele (Fig. 4B), which indicated that Sele did not interact with CD43. CD44 protein was detected in the pull-down proteins of the stretched group, while almost no expression of CD44 was detected in the static group. Remarkably, Sele-KO treatment significantly reduced the pulldown level of CD44 under CMS stimulation (Fig. 4C). These results suggested that the interaction between Sele-CD44 might be the main reason for monocytes’ recruitment to endothelial cells. Furthermore, we validated the role of Sele-CD44 in single-cell lineages of human normal scar and keloid tissue. It was found that the interaction between Sele_high endothelial cells and monocytes was significantly stronger than that between Sele_low endothelia and monocytes (Fig. 4E, F). Further analysis of intercellular communication between Sele_high endothelial cells and monocytes revealed that Sele-CD44 is the strongest mode of interaction among them (Fig. 4G, H), which was consistent with the findings in above vitro studies.

Fig. 4

Endothelial cells recruited monocytes via Sele-CD44 interaction upon high-tension. A, D Western blot results and quantitative analysis of Sele, CD44, CD43, and PSGL-1 in endothelial cell-monocyte co-cultures. Co-Immunoprecipitation (CO-IP) of Sele (IP protein) with CD43 (B) and CD44 (C). E, F Analysis of cell communication numbers and interaction weights in human normal scar (normal scar) and keloid tissue using single-cell RNA sequencing (scRNA-seq) dataset GSE181316. G, H Cell communication relationships and proportions in human normal scar and keloid tissue from scRNA-seq dataset GSE181316 (n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Preparation and characterization of Sele-targeted siRNA LNPs

LNPs were synthesized using cationic lipids DOPE and DOTAP via the thin film hydration method, and siRNA was encapsulated within LNPs through electrostatic interactions. As shown in Fig. 5A, LNPs effectively condensed siRNA at a weight ratio of 1:20 or higher, with electrophoresis confirming no siRNA leaching and thus complete complexation by the cationic liposomes. For subsequent experiments, a weight ratio of 1:30 was chosen to prepare LNPs. To evaluate the protective effect of LNPs on siRNA, naked Si-Sele or Si-Sele encapsulated within LNPs (Si-Sele@LNPs) was incubated with ribonuclease (RNase) for varying durations (0, 15, 30, 60, 120, and 240 min). Naked siRNA degraded rapidly, while siRNA from LNPs retained its structural integrity for up to 4 h (Fig. 5B, C). The Rio-Green assay confirmed that the encapsulation efficiency of siRNA in Si-Sele@LNPs exceeded 94.2%. Compared to unlodeed LNPs, the zeta potential of Si-Sele@LNPs decreased (Fig. 5D) as the encapsulation of si-Sele reduced the relative counts of cations in LNPs. Stability tests revealed that no significant changes happened in the particle size or polydispersity index (PDI) of Si-Sele@LNPs when they were incubated in phosphate-buffered saline (PBS) at pH 6.8 for 1 week (Fig. 5E), suggesting that Si-Sele@LNPs can maintain their structure in the slightly acidic environment of skin scar tissues. The impact of tensile forces generated during the modeling procedure on LNP structure was examined, and DLS results showed that no significant change happened in the average size of Si-Sele@LNPs after CMS stimulation (Fig. 5F). TEM imaging also confirmed that Si-Sele@LNPs retained their spherical morphology after CMS stimulation (Fig. 5G, H). Cellular uptake studies demonstrated a significantly increased uptake of si-Sele when they were encapsulated in LNPs compared to that of naked si-Sele (Fig. 5I, J). Biocompatibility tests in endothelial cells showed that Si-Sele@LNPs could be incubated without causing cytotoxicity, even at the highest concentration of 150 μg/mL (Fig. 5K). Furthermore, RT-qPCR analysis indicated that treatment with Si-Sele@LNPs resulted in a nearly 70% reduction of Sele mRNA expression in endothelial cells (Fig. 5L). Therefore, Si-Sele@LNPs were subsequently applied in vitro and in vivo validation and intervention experiments.

Fig. 5

Preparation and characterization of siRNA@LNPs targeting Sele. A Study of the interaction between lipid nanoparticles (LNPs) and siRNA. B, C Stability of naked siRNA and LNP-encapsulated siRNA against RNase. D Zeta potential of empty LNPs versus siRNA-loaded LNPs. E Changes in polydispersity index (PDI) and particle size of siRNA-loaded LNPs in PBS at pH 6.8 over time. F Particle size changes of siRNA-loaded LNPs before and after CMS stretching. G, H Transmission electron microscopy images of siRNA-loaded LNPs before and after CMS stretching. I, J Uptake of Cy5-labeled naked siRNA and LNP-encapsulated Cy5-labeled siRNA by endothelial cells. K Viability of endothelial cells treated with LNPs at different concentrations. L Comparison of Sele mRNA silencing efficiency between naked siRNA and LNP-encapsulated siRNA (n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Transfection efficiency and biodistribution of Cy5-labeled LNPs on scar in vivo

To evaluate the delivery efficiency and biodistribution of si-Sele-loaded LNPs (si-Sele@LNPs) in scar tissue, Cy5-labeled si-Sele was encapsulated in LNPs (si-Sele@Cy5_LNPs) and locally injected intradermally into the scar tissue of mice. Figure 6A depicted the schematic diagram of the treatment protocols for the mice in the RSM group. As shown in Fig. 6B, fluorescence imaging using the IVIS Spectrum system revealed a significantly stronger fluorescence signal in the dorsal scar tissue from the si-Sele@Cy5_LNPs-treated group compared to that from the saline-treated group. This finding indicated that the LNPs enabled uniform penetration and distribution of the si-Sele therapeutic agent within the scar tissue. Then the mice were sacrificed, and their organs including the heart, liver, spleen, and lungs, were collected for analysis. No fluorescence signal was detected in these organs (Fig. 6C, D), demonstrating that si-Sele@Cy5_LNPs remained localized in the scar tissue and did not spread to other organs. Furthermore, to identify the primary cell types that internalized si-Sele@Cy5_LNPs, the scar tissues were digested, and the cells were analyzed via flow cytometry using markers for epithelial cells (EpCAM), endothelial cells (CD31), and immune cells (CD45). The results showed that skin endothelial cells were the predominant cell type that si-Sele@Cy5_LNPs entered, with 20.70% of the total endothelial cell population being Cy5 positive. In comparison, 6.43% of epithelial cells and 4.48% of immune cells were Cy5 positive (Fig. 6E, F).

Fig. 6

Transfection efficiency and biodistribution of Cy5-labeled LNPs on scar in vivo. A Schematic of different therapeutic treatments injected into mice RSM. B–D In vivo imaging of mice 24 h post intradermal (ID) injection, including IVIS imaging of organs (heart, liver, spleen, and lungs), with mice receiving saline treatment imaged as controls. E, F Cellular localization and quantitative analysis of siSele@LNPs in scar tissues of mice under various treatments (n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Sele-targeted siRNA LNPs inhibit stretched-induced PS formation in vivo

To evaluate the potential clinical effects of si-Sele@LNPs in reducing scar formation in the RSM model, we performed a series of intradermal (ID) injection treatments. Mice with RSM were randomly assigned to 2 groups: one group received si-Sele@LNPs injections, while the control group received saline injections. These treatments were administered every 3 days until the mold was removed after 14 days (Fig. 7A). On the 14th day, the mice were sacrificed, and scar tissues from static mice, saline-treated RSM mice, and si-Sele@LNPs-treated RSM mice were collected for analysis. As shown in Fig. 7B, the si-Sele@LNPs treatment significantly improved the appearance of the scars, reducing the scar surface area to approximately one-third of that in the saline group (p < 0.01, Fig. 7D), and making it comparable to the static group. HE staining indicated that the si-Sele@LNPs treatment markedly decreased the formation of new granulation tissue compared to saline treatment (p < 0.001, Fig. 7C, E). Similarly, Masson staining showed that si-Sele@LNPs significantly inhibited excessive collagen deposition under high-tension (p < 0.001, Fig. 7C, F). Further analysis with IF revealed that si-Sele@LNPs treatment significantly reduced the expression of Sele in endothelial cells within the scar tissue and notably decreased the recruitment of CD11b+ monocytes compared to the saline-treated group (Fig. 7G), si-Sele@LNPs treatment also significantly reduced inflammation cytokines like IL-1β (p < 0.001, Fig. S1A), IL-6 (p < 0.01, Fig. S1B), and TNF-α (p < 0.001, Fig. S1C) compared to the saline-treated group. Western blot analysis corroborated these results, showing that ID injection of si-Sele@LNPs significantly decreased the levels of muscle fibroblast marker Acta2, extracellular matrix protein Fibronectin, and Col1a1 (Fig. 7H, I). Additionally, the expression of the endothelial cell activation marker Vegfa was significantly lower in the si-Sele@LNPs treatment group compared to the saline group (Fig. 7H, I). Overall, si-Sele@LNPs treatment effectively reduced scar formation and improved the histological and molecular characteristics of scar tissues in the RSM model, highlighting its potential for clinical application in PS reduction.

Fig. 7

Sele-targeted siRNA LNPs inhibit stretched-induced PS formation in vivo. A Experimental design of the in-vivo study, including intradermal injection (ID). B, D Images and surface area measurements of scars on mice 14 days post full-thickness longitudinal skin incisions under various treatments. Scale bar = 1 cm. C H&E (scale bar = 200 μm) and Masson’s trichrome staining (scale bar = 50 μm) images of mice scars 14 days post-incision under different treatments. E–F Cross-sectional area and collagen fiber proportion statistics in scar tissues 14 days post-incision under various treatments. G Representative immunofluorescence images of scar tissues 14 days post-incision under different treatments. Scale bar = 50 μm, 10 μm (partial enlargement). H, I Western blot results and semi-quantitative analysis of Fibronectin, Col1a1, Acta2, Sele, CD44, and Vegfa in scar tissues 14 days post-incision under various treatments (n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant)

Discussion

In our study, the mice scar model was successfully constructed based on RSM. Several studies have previously reported mouse scar models based on tension [16, 55, 56], but our model demonstrates unique advantages in several aspects. First, previous devices were mostly placed on open wounds [56], while we use sutured wounds, which better mimic the scar formation process in humans. Secondly, we constructed the device using lightweight polyethylene (0.9–1.3 g/cm³) to minimize the burden on mice and ensure their normal movement, which meets animal welfare standards. Thirdly, the “retroflex scar model” design ensures secure attachment to the mouse, preventing detachment and enhancing stability and success rates. Finally, tension is easily adjusted using fixed-width spacers, ensuring high reproducibility without requiring operator expertise. These features make our model more reliable, scalable, and accessible for studying scar formation.

To explore the mechanism of high-tension-induced PS formation. We conducted transcriptome sequencing of mice normal scar tissue and PS tissue, and discovered that the cell adhesion process was activated in PS tissue. Cell adhesion molecules (CAMs), a large class of transmembrane proteins, are involved in cell-to-cell or cell-to-extracellular matrix adhesion, playing roles in cell proliferation, differentiation, migration, transport, apoptosis, and tissue structure [57]. To further investigate the role of CAMs in PS formation, we collected PS tissue in high-tension anatomical areas from PS patients and normal skin tissue in healthy individuals. We found that Sele mRNA and protein levels were significantly higher in both scar tissue from mouse RSM and human PS. Sele, a member of the selectin family, is primarily expressed in endothelial cells of the skin, mediating the adhesion of circulating immune cells to the endothelium [54, 58, 59]. We observed increased monocyte infiltration and elevated Sele expression in endothelial cells within the PS tissue of the RSM group. To explore the relationship between tension, Sele, and monocyte infiltration, we established an in vitro CMS system for endothelial cells. We found that with increased stretching intensity, Sele protein levels of endothelial cells and monocyte adhesion rates consistently increased, showing a positive correlation between Sele protein levels and monocyte adhesion rates. Knocking out Sele in vitro can reverse the increase in monocyte adhesion caused by stretching and the evaluated expression of pro-inflammatory factors. These findings suggested that mechanical stretching exacerbated inflammation and promotes PS formation through Sele-mediated monocyte adhesion in the endothelium.

Previous studies have shown that Sele mediated the adhesion of circulating immune cells to the endothelium by binding to its ligands [60], which are glycosylated molecules with sLex and sLea determinants expressed on leukocytes [61], including PSGL-1, CD43, and CD44 [62]. Research has indicated that Sele regulated leukocyte adhesion to brain vessels in multiple sclerosis (MS) by interacting with CD44 or PSGL-1, affecting the susceptibility to relapsing–remitting multiple sclerosis [63,64,65]. Sele also plays a significant role in inflammatory skin diseases, with high Sele expression in blood vessels co-locating with inflammation zones, and skin-infiltrating leukocytes preferentially expressing CD43 and CLA antigens, facilitating leukocyte transport to the skin during psoriasis inflammation [66]. Our study is the first to discover that endothelial cells under stretching promote monocyte adhesion to moncyte through the Sele-CD44 interaction. scRNA-seq analysis defined a subset of endothelial cells with high Sele expression in human keloid and HS tissues, constructing their cell–cell interaction network. Compared to endothelial cells with low Sele expression, those with high Sele expression had closer and stronger interactions with monocyte, with CD44-Sele being the most robust receptor-ligand pair, which is consistent with our in vitro CMS model observations. These findings suggestd that targeting Sele could be a promising approach to inhibiting scar formation by blocking monocyte infiltration mediated by Sele-CD44, potentially opening new avenues for scar treatment.

RNAi therapy is a novel gene therapy that achieves restrictive selection by base pairing to directly target specific genes with high specificity [43]. Compared to antibody and small molecule drugs, siRNA offers a rich variety of targets, ease of synthesis, short preclinical development cycles, and controllable adverse reactions [67, 68]. However, its structure makes it immunogenic and susceptible to nuclease degradation [69], limiting its in vivo application. Thus, safe and efficient modification and delivery systems are needed to enhance siRNA stability and bioavailability. Skin, as a relatively enclosed and accessible tissue [70], can enhance siRNA retention and ensure cellular uptake through local injection. Thus, the delivery of LNP-encapsulated siRNA locally to the skin is a promising application strategy. We developed a unique LNP-siRNA system, siSele@LNPs, delivering siSele@LNPs treatment directly to the new granulation tissue in wounds through local ID. LNPs consist of neutral lipids, ionizable cationic lipids, cholesterol, and PEG, forming a nucleic acid drug delivery system to neutralize the negative charge of siRNA. LNPs can prevent siRNA degradation by blood nucleases, and promote endosomal escape. In vitro, siSele@LNPs were demonstrated to be good stability and biocompatibility. In vivo, local injection of siSele@LNPs mainly deposited siSele in the wound area without spreading to other tissues, showing good tissue targeting. Flow cytometry analysis indicated that siSele primarily accumulated in endothelial cells. Previous studies thought that endothelial cells form the lining of blood vessels and have more opportunities for contact with external substances [71], studies also have shown that siRNA delivered via liposomes can preferentially target endothelial cells in organs [72], partly due to the unique uptake properties of endothelial cells as the first barrier of the blood vessel wall.

In PS tissues from mice RSM, we observed significant scar formation and collagen deposition, with disorganized collagen fibers consistent with human PS in appearance and histology. Knocking down the level of Sele through siSele@LNPs significantly reduced the scar size and decreased the expression of myofibroblast marker and ECM-related protein, which were recognized as the characteristics of PS. Histological analysis of scar tissues showed that siSele@LNPs treatment alleviated collagen deposition and improved collagen arrangement in new granulation tissue.

In summary, we propose a mouse scar model and a targeted siRNA@LNP therapeutic system, investigating the therapeutic effect of local injection delivery of siSele@LNPs. Local injection of siSele@LNPs offers repeatable operations, stable local effects, low systemic toxicity, and high targeting specificity. This work shows promising translational potential for siRNA-based treatment for PS.

Conclusion

Our study successfully constructed a novel stretch-mediated mouse Retroflex Scar Model (RSM) to investigate high-tension-induced PS formation. Elevated expression of Sele was observed in both RSM and human PS tissues. This upregulation of Sele under CMS conditions promoted monocyte adhesion to endothelial cells and increased pro-inflammatory factor release. Intradermal administration of Sele-targeted siRNA liposome nanoparticles effectively inhibited monocyte adhesion and PS formation in vivo and in vitro. These findings highlight the critical role of Sele in PS pathogenesis and suggest that targeting Sele with siRNA-loaded nanoparticles could be a promising therapeutic strategy for PS prevention and treatment, offering new insights into the development of effective interventions for pathological scars.