Lactobacillus-derived extracellular vesicles provide multi-target acne treatment by enriched proteins and skin microbiota protection

Abstract

Numerous studies have highlighted the therapeutic potential of probiotics in acne. Given the complex and multifactorial nature of the disease, probiotic-based multi-target therapies may be promising. Additionally, due to their non-replicative and nanosized characteristics, probiotic-derived extracellular vesicles might provide a safer and more efficient alternative. Here, we selected three anti-acne targets—Propionibacterium acnes (P. acnes) inhibition, anti-inflammatory effects, and sebum suppression—for high-throughput screening, identifying Lactobacillus plantarum 5b4m2 with multi-target anti-acne potential. Next, we isolated 5b4m2-derived EVs (LP-EVs) and discovered that they retain the anti-acne potential of the parental bacteria while exhibiting superior skin permeability. Further studies in P. acnes-induced acne mice demonstrated that LP-EVs significantly alleviated inflammatory symptoms such as redness and swelling and effectively inhibited the colonization of P. acnes, leading to enhanced therapeutic efficacy. Then, proteomic analysis identified nine enriched proteins in LP-EVs directly associated with acne improvement, including mucus-binding protein, cell wall hydrolase, lipase, and thioredoxin. Correspondingly, pathway changes in the host were revealed by transcriptomic analysis, such as the chemokine signaling pathway, lipid metabolism, and tissue tight junctions. Moreover, LP-EVs effectively maintain the skin microbiota balance, which may also contribute to acne improvement. Collectively, our study offers a new perspective on probiotic-based multi-target strategies, which may facilitate the treatment of acne and other skin diseases in the future.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04291-8.

Introduction

Acne is a common chronic inflammatory skin disorder, characterized by comedones, pustules, excoriations and hypertrophic scars on the face or torso [1]. In addition to significant physical suffering, acne can also cause psychological trauma and may even lead to suicidal thoughts and attempts [2]. It was reported that 16.5% of patients have engaged in self-harming behaviors and 22.3% have contemplated suicide due to acne [3]. According to epidemiological analysis, acne currently affects up to 20.5% of the global population, with its incidence rates continuing to increase rapidly [4, 5].

As a typical multifactorial disease, acne primarily results from excessive sebum production, abnormal follicular keratinization, colonization by P. acnes, and inflammatory mediators, with diet and environmental factors also implicated in the process [6]. The complex pathophysiology of acne poses significant challenges for effective treatment. Current therapies primarily focus on topical medication (benzoyl peroxide, etc.), systemic administration (isotretinoin, etc.), and physical therapy (photodynamic therapy, etc.) [1, 7]. However, these conventional treatments have disadvantages, such as poor effect, high recurrence rate, or serious adverse effects (skin irritation, teratogenicity, antibiotic resistance, etc.), which make them fail to achieve satisfactory outcomes [1, 8, 9]. Thus, there is an urgent need for novel, highly effective, and safer therapeutic strategies.

Recent studies have highlighted the potential of probiotics in managing various skin disorders including acne. Specifically, Lactobacillus plantarum has been shown to reduce sebum production by modulating the signaling pathways of insulin and insulin-like growth factor-1 [10, 11]. Deidda et al.. reported that Lactobacillus salivarius can inhibit the growth of P. acnes and the release of P. acnes-induced interleukin-8 (IL-8) through bacteriocins, thereby reducing skin inflammation and improving acne [12]. Additionally, according to the study of Jung et al.., Lactobacillus rhamnosus could enhance the expression of tight junction proteins and improve skin barrier function [13]. In addition to the probiotics, probiotics-derived extracellular vesicles (EVs) have emerged as a promising therapeutic strategy in recent years for their capacity to improve skin homeostasis and alleviate pathology [14]. For instance, EVs from L. plantarum, L. rhamnosus, and Lactobacillus druckerii have been shown to exert multiple skin health benefits, such as anti-inflammatory effects, microbiome homeostasis restoration, etc [15–18]. Particularly, EVs possess unique advantages over their parent bacteria. Their nanosized structure, non-replicative nature, low immunogenicity, and exceptional biocompatibility enable efficient skin penetration and biological activity with fewer safety concerns [19]. These attributes highlight that probiotic-derived EVs hold promise as a novel strategy to replace parent bacteria in combating acne.

Nowadays, multi-target therapeutic strategies have garnered increasing attention due to their synergistic effects and reduced side effects [20–22]. Given the complicated pathogenesis of acne, we propose that probiotic-based multi-target therapies hold significant potential for effective acne treatment. Here, we targeted three key aspects of acne pathogenesis: inflammation, excessive sebum secretion, and P. acnes colonization. Through in vitro high-throughput screening, we identified a probiotic strain L. plantarum 5b4m2 with multi-target anti-acne potential. Subsequently, we isolated and characterized L. plantarum-derived EVs (LP-EVs), and validated their effects in a P. acnes-induced acne mouse model. We then employed proteomic and transcriptomic techniques to identify key proteins and potential pathways implicated in acne improvement. We found the therapeutic effects of LP-EVs may be attributed to their anti-inflammatory, antibacterial, lipid-regulator proteins, etc., which further regulate related pathways within the host. Additionally, skin microbiome analysis revealed that LP-EVs intervention could maintain the balance of skin microbiota, which may also contribute to the therapeutic outcomes. Taken together, our findings suggest that LP-EVs may serve as a promising multi-target therapy for acne.

Materials and methods

Bacteria and cell culture

All 71 probiotics used in this study were preserved in the laboratory (Table S1), and P. acnes type IA (ATCC 6919) was purchased from the American Type Culture Collection. All the isolates were identified by sequencing their full-length 16 S rRNA genes and comparing them with sequences obtained from GenBank using BLAST analysis. Probiotics and P. acnes were cultured in MRS and BHI liquid medium, respectively, supplemented with 0.05% L-cysteine and grown anaerobically under an atmosphere of 10% CO₂, 10% H₂ and 80% N₂ at 37 °C. The immortalized human sebaceous gland cells (SZ95) [23] were provided by Professor Christos C. Zouboulis. THP-1 human monocytic cell lines and HaCaT cells were purchased from BNCC (China, Henan). SZ95 and THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, while HaCaT cells were cultured in DMEM medium with the same supplements. Cells were cultured in a 37 °C incubator with 5% CO₂ in air and saturated humidity.

P. acnes inhibition assay

The fresh suspension of P. acnes was adjusted to OD_600nm = 0.3 using BHI medium. In a 96-well plate, 180 µL of the prepared P. acnes solution was added to each well. The blank control group, positive control group, and experimental groups were supplemented with 20 µL of MRS medium, clindamycin at the final concentration of 20 µg/mL [24], various probiotic solutions (OD_600nm = 1), and LP-EVs at the final concentration of 200 µg/mL, respectively. The OD_600nm was measured before and after 20 h of incubation to calculate the antibacterial rate of the probiotics. The inhibition rate (%) was determined using the following formula: (OD_600nm before incubation - OD_600nm of the experimental group) / (OD_600nm before incubation - OD_600nm of the blank control group) × 100%. Correspondingly, the growth curves of the experimental and the control group were compared to evaluate the inhibitory effect of LP-EVs on the growth of P. acnes.

Sebum secretion inhibition assay

SZ95 sebocytes were transferred into the medium containing 200 µM oleic acid and cultured for 24 h to establish a sebaceous gland cell model of oil hypersecretion [25, 26]. Subsequently, the cell suspension was co-incubated with probiotic solution at a proportion of 10% (20µL, OD_600nm=1) or LP-EVs at the final concentration of 200 µg/mL for 24 h. A total of 0.1% DMSO was set as the control. Lipogenesis was detected using the Oil Red O staining method as described previously by Green et al. [26]. Briefly, cells were washed with PBS and fixed with 10% formaldehyde for 20 min. Then, they were stained with 0.5% Oil Red O staining solution prepared by dissolving the powder in the isopropanol-distilled water mix (v/v = 3/2) for 30 min. After staining, the cells were washed with PBS and observed under a microscope. To quantify lipid content, the cells were incubated with 100% isopropanol for 5 min and the OD_510nm was measured.

Cell inflammatory factor inhibition assay

In a 96-well plate, 1 × 10⁴ HaCaT cells were inoculated per well and cultured for 24 h. Correspondingly, THP-1 cells were inoculated in a 24-well plate at a concentration of 1 × 10⁶ cells/mL, and phorbol 12-myristate 13–acetate (PMA) at the final concentration of 200 nmol/L was added to the medium to induce the cells for 24 h to promote differentiation into macrophages. Then, 20 µL of heat-killed P. acnes at a concentration of 1 × 10⁶ CFU/mL was added to each well, and the cells were co-incubated in a 37 °C incubator with 5% CO₂ for 24 h to establish the inflammatory cell model [27, 28]. The HaCaT and THP-1 inflammatory cell was co-incubated with 20µL probiotic bacterial liquid (OD_600nm =1) or LP-EVs at the final concentration of 200 µg/mL for 24 h. The supernatant after treating the cells was collected, and the contents of the inflammatory factors IL-8, IL-1β, and TNF-α secreted by cells were detected according to the instructions of the human IL-8 ELISA KIT (Solarbio, China), human IL-1β ELISA KIT (Solarbio, China), and human TNF-α ELISA KIT (Solarbio, China), respectively.

MTT colorimetric assay

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Mosmann, 1983) [29]. SZ95, THP-1 and HaCaT cells were seeded in a 96-well plate at a density of 1 × 10⁵ cells/well and allowed to reach 90% confluence. Following this, the cells were treated with 50 µg/mL, 100 µg/mL and 200 µg/mL LP-EVs, respectively. After 24 h of incubation, the cells were incubated with 5 mg/mL MTT reagent (Sigma Aldrich, Germany) for 4 h at 37 ℃. Formazan crystals formed within the cells were dissolved by adding 100 µL of dimethyl sulfoxide (Sigma Aldrich, Germany) to each well. Absorbance was measured at 570 nm using an EnSpire 2300 microplate reader (Perkin Elmer, USA). Viability was expressed as a percentage of live cells compared to untreated control cells (100%).

Preparation of LP -EVs

For the extraction and purification of vesicles, we made slight modifications to the method proposed by Li et al. [30]. The specific operation is as follows: L. plantarum 5b4m2 was cultured in MRS medium. After reaching the logarithmic growth phase, it was transferred to fresh MRS medium and incubated at 37 °C for 24 h. The culture was centrifuged at 4 °C and 12,000 g for 20 min twice to obtain the supernatant, which was further concentrated to 1/100 of its initial volume using a 100 KDa tangential flow membrane cassette. Ultracentrifugation was performed at 4 °C and 200,000 g for 1.5 h using an ultra-high-speed refrigerated centrifuge. The pellet was resuspended in Deionized water to obtain LP-EVs, which were filtered through a 0.22 μm filter and stored at – 80 °C until use. Total protein measurements were performed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

Characterization of the particle size, zeta potential, and protein concentration of LP-EVs

Sterile LP-EVs solutions were diluted appropriately, and 10 µL of the diluted solution was applied to a copper grid. After adsorption for 30 min, the excess solution was blotted with filter paper. The grid was then stained with 10 µL of 1% phosphotungstic acid (PTA) for 5 min, followed by blotting again with filter paper and allowing the grid to air-dry overnight. Morphological characteristics of LP-EVs were examined using transmission electron microscopy (TEM). Additionally, the size and Zeta potential of LP-EVs were measured using dynamic light scattering (DLS).

Cellular uptake of DiI-labeled LP-EVs in skin cells

For LP-EVs staining, 1 mL of LP-EVs sample was co-incubated with 1 µL of 1 mg/mL DiI dye at 37 ℃ for 30 min. The mixture was then centrifuged at 4500 g for 15 min using a 100 kDa ultrafiltration tube, and the sample was washed three times with PBS to obtain DiI-labeled LP-EVs. DiI-labeled LP-EVs were co-cultured with SZ95 cells for 2 and 6 h. Then, SZ95 cells were fixed and stained with 4’,6-diamidino-2-phenylindole (DAPI), and the stained images were observed under confocal laser scanning microscopy (CLSM).

Phylogenetic distribution and taxonomic relation analysis

Universal 16S rRNA bacterial primers (27F, 5’-AGAGTTTGATCCTGGCTCAG-3’; 1492R, 5’-GGTTACCTTGTTACGACTT-3’) were used to perform PCR amplification on the strains to obtain 16 S rRNA genes. Subsequently, we aligned these sequences and constructed the phylogenetic tree using the maximum likelihood method through MEGA 11.

Preparation of carbomer gel

1 g of carbomer powder was first dispersed in 99 mL of distilled water and soaked for 2 h to ensure complete swelling. This was followed by mechanical stirring at 330–380 r/min until homogeneous, after which the gel solution’s pH was adjusted to 7 using a low-concentration triethanolamine solution. Once the gel is prepared, it can be mixed with samples such as vesicles and applied to the ears of mice for intervention.

Experimental animals

Female 8-week-old ICR mice were purchased from the Hubei Province Center for Disease Control and Prevention (Wuhan, China). All mice were housed in a specific pathogen-free environment at a constant temperature (25 °C ± 1 °C) and 50% to 60% relative humidity. All mice were allowed to acclimatize for 1 week after arrival. During the experiments, all mice had free access to water and food and received humane care. On days 7 and 9, 60 µL of P. acnes suspension was injected into the inner side of the right ear, and the untreated left ear served as a control used for the 16 S rRNA sequencing analysis [31–33]. From the 11th day, 200µL 5b4m2 bacterial suspension with an OD_600nm of 1, Clindamycin at the final concentrations of 20 µg/mL [24], LP-EVs digested by 200 µg/mL protease K, protease K alone (200 µg/mL) and LP-EVs at the final concentrations of 200 µg/mL, all in carbomer gel, were applied to the ears of the corresponding groups of mice with the volume of 200µL twice daily for a week. The model group was treated with blank solvent. The mice were sacrificed at the end of the experiments, and the blood, skin, and skin microbial samples were collected respectively for follow-up testing. Levels of IL-6 and CXCL1 (The functional homolog of human interleukin-8 in mouse) in serum were quantified using mouse ELISA kits (Solarbio, China) according to the manufacturer’s instructions. Experimental methods for skin tissue and skin microbiota analysis are described in subsequent sections.

Observation of L. plantarum 5b4m2 and LP-EVs penetration in skin by CLSM

LP-EVs were stained following the same protocol as used in the “Cellular uptake of DiI-labeled LP-EVs in skin cells” assay. For staining of the parental L. plantarum 5b4m2, bacterial protoplasts were first prepared by incubating the cells in a protoplast buffer containing lysozyme (Sigma Aldrich, Germany) [34] stock solution. The resulting protoplasts were then stained using the same procedure as for LP-EVs, involving incubation with DiI dye followed by centrifugation and washing to obtain DiI-labeled 5b4m2. The mice were anesthetized, depilated, cleaned with saline, and monitored for 48 h to ensure complete recovery of the skin barrier. Subsequently, mice were randomly divided into three groups with an equal number. Then, the dorsal hairless skin was treated by applying DiI-labeled LP-EVs or DiI-labeled 5b4m2 solution 50 times using cotton swabs. After smearing, they were placed in a light-proof environment. Four hours after treatment, the mice in each group were euthanized. The skin surface was washed with PBS to remove unbound dye, and the stained skin was excised and rapidly frozen in liquid nitrogen. Skin samples were sliced at low temperature and imaged by CLSM to assess fluorescence penetration.

Real-time reverse transcription PCR (RT-qPCR)

Total RNA was extracted from HaCaT cells, SZ95 sebocytes, THP-1 cells and mouse skin tissues using the RNA Extraction Reagent Kit (Vazyme, China) according to the manufacturer’s protocol. Subsequently, total RNA was reverse-transcribed into cDNA using HiScript^®Ⅱ1st Strand cDNA Synthesis Kit (Vazyme, China) following the manufacturer’s instructions. qRT-PCR was performed with Universal SYBR Green Fast qPCR Mix (ABclonal, China). The amplification program consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Primer sequences are provided in Table S2. The specificity of amplification was evaluated by melting curve analysis, and RNA expression levels were normalized to those of the GAPDH housekeeping gene in each sample. All experiments were performed in triplicate, and the fold change in expression levels was analyzed using the 2^−ΔΔCt method.

Relative bacterial abundance in skin tissue was assessed through quantitative real-time PCR (qPCR)

The experimental procedures for intervention, sample collection, and extraction of microbial genomic DNA were performed as “Experimental animals” with minor modifications. Briefly, after topical intervention on P. acnes-induced acne mice, ear tissues were collected at the end of the experiment. Then, the microbial genomic DNA in the ear was extracted. qPCR was operated using Universal SYBR Green Fast qPCR Mix (ABclonal, China) according to the manufacturer’s cycling parameters. Bio-Rad CFX Maestro software was used to measure CT values, and a fold-change standardized to 16 S ribosomal DNA (16 S rDNA) was calculated in Microsoft Excel. The primers used for qPCR are listed in Table S2.

H&E staining

Tissue samples were fixed in 4% paraformaldehyde at room temperature overnight and then embedded in paraffin. Tissues were sectioned at 5 μm thickness and dipped in H&E following the standard protocols. All images were captured at 200× magnification using a Nikon Digital Imaging System.

RNA extraction, reverse transcription and sequencing

Total RNA of skin tissues was extracted using Trizol reagent (Thermo Fisher Scientific, USA). The integrity and purity of the RNA were determined using a nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and electrophoresis. Complementary DNA was obtained via a two-step reverse transcription kit (TaKaRa, Japan). RNA-seq was performed by Novogene.

Transcriptome data analysis

RNA-Seq reads from the FASTQ files were mapped to the Mus musculus (assembly GRCm39) using STAR (Spliced Transcripts Alignment to a Reference) [35]. The output files in BAM format were analyzed by featureCounts to quantify gene expression [36]. Raw counts were transformed to Counts per million (CPM) with the cpm method implemented in the edgeR package [37]. Then the sva seq function [38] implemented in R package sva [39] was used to remove the batch effect, followed by normalization with voom and differential expression analysis with limma [40] package. P-value was adjusted with BH method. Genes fulfilling the criteria adjusted p < 0.05 and fold-change > 0.5 were considered to be differentially expressed. All of the differentially expressed genes were utilized as the inputs for Gene Set Enrichment Analysis (GSEA) analysis with the R package ClusterProfiler, focusing on biological process, cellular component and molecular function. A p.adjust < 0.01 and the tags > 50% were considered statistically significant. Immune cell deconvolution analysis was performed using the cibersort R function [41]. The reference signature matrix for mouse immune cells was obtained from the study by Chen et al. [42]. Visualization of the results was generated using the online bioinformatics platform (https://www.bioinformatics.com.cn), a web-based tool for data analysis and visualization [43].

Label-free quantitative proteomics data analysis

The LP-EVs and 5b4m2 bacterial suspension samples were sent to SpecAlly company (Wuhan, China) for label-free quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) to identify differential proteins. Briefly, the data was collected with SWATH/DIA (Sequential Window Acquisition of all Theoretical Mass Spectra/Data-Independent Acquisition SWATH mode). In the first round of detection, the DDA model was used to collect data to build a spectral database; in the second round, the DIA model was used to collect quantitative data of various peptide segments and proteins for biostatistical and bioinformatics analysis. Missing values for each group are imputed using the nearest neighbour method after removing peptides missing in more than two. For the LP-EVs group, the peptides missing more than two were removed. For the control group, the peptides missing more than two were filled with the minimum value. The groups with one missing peptide were filled with an approximate value. The gene ontology enrichment analysis was conducted on DAVID [44], related biological process GO terms were classified and their p-value was calculated with the Hypergeometric test in R.

16 S rRNA sequencing

High-throughput sequencing was performed with the DNA of skin microbes from mice in each group. An amplification library was prepared with the primer pair 341 F/806R targeting 16 S rRNA gene V3-V4 and sequenced on an Illumina NovaSeq 6000 System (Novogene). The sequencing reads were analyzed using QIIME2 (version 2020.8). DADA2, wrapped in QIIME2, was used to filter the low-quality reads, errors, chimeras, obtain amplified sequence variants (ASVs) and generate a feature table. Bacterial features were classified using the Greengenes reference database classifier (version 13 − 8), with a sequence similarity threshold of 99%. The alpha and beta diversity analyses were also calculated in QIIME2. In addition, the vegan package was used to calculate the Bray-Curtis distance to perform the principal coordinate analysis (PCoA). Linear discriminant analysis effect size (LEfSe) analysis was used to do the Kruskal-Wallis test and the Wilcoxon rank-sum test, and both took p < 0.05 as statistical significance. The LDA score was set at 2.0 to determine the characteristics with rich differences between groups. Taxa with an average relative abundance above 0.0001 (0.01%) were saved in this study. Bars and heatmaps were visualized using the “ggplot2” and “pheatmap” software packages of the R software (version 4.0.5) (https://www.r-project.org/), respectively.

Statistical analysis

All data were presented as the mean ± SEM. Statistical analysis for multiple groups was performed by the Kruskal-Wallis test (the non-parametric equivalent of one-way ANOVA) in GraphPad Prism 7.0 software to ascertain the differences between the groups.

Results

In vitro high-throughput screening of probiotics with anti-acne targets

Probiotics can combat acne through multiple ways, including regulation of sebum secretion, inflammatory responses [45, 46], and the microecological balance on the skin surface [47, 48]. Here, we chose three acne-related targets to screen candidates from 71 probiotics isolated from healthy human intestine, including inhibiting excessive sebum secretion, inhibiting the growth of P. acnes and reducing inflammation (Fig. 1A). Then, we selected the top ten probiotics from the three targets as promising candidates and found that most strains address only one or two targets (Fig. 1B-C, Figure S1). Venn diagrams further showed that L. plantarum 5b4m2 was the only one harboring all three targets (Fig. 1B and C). It suggested that 5b4m2 may be the most promising probiotic for combating acne.

Fig. 1.

In vitro high-throughput screening of probiotics with anti-acne targets. (A) Schematic diagram showing the experimental design and screening process; (B) Scatter plot showing the effects of probiotics on P. acnes growth, sebum secretion, and IL-8 concentration. The indexes were represented by color, horizontal coordinate, and vertical coordinate, respectively. Red and black dashed lines indicate the top 10 strains in terms of sebum inhibition and anti-inflammatory activity. Green circles mark the top 10 strains inhibiting P. acnes; (C) Venn diagram illustrating the top 10 probiotic strains based on the three anti-acne targets: inhibit P. acnes growth (blue), inhibit sebum secretion (red), and suppress inflammation (green). The overlapping region shows that only 5b4m2 demonstrates inhibitory effects across all three targets; (D) Phylogenetic tree depicting multiple probiotic strains with different functions, including inhibition of P. acnes growth, sebum secretion, and suppression of inflammation. BF, Bifidobacterium faecale; BL, Bifidobacterium longum; BBL, Bifidobacterium bifidum; BB, Bifidobacterium breve; BA, Bifidobacterium animals; LS, Lactobacillus salivarius; LP, Lactobacillus plantarum; LPC, Lactobacillus paracasei; LR, Lactobacillus reuteri; LL, Lactococcus lactis; LAP, Lactobacillus acidophilus. Asterisks represent probiotics with multiple functions

To determine whether these targets were strain-specific, we analyzed their taxonomic relationships and phylogenetic distribution (Fig. 1D). Results showed that 4 and 5 Lactobacillus strains ranked in the top ten for inhibiting P. acnes growth and sebum secretion, respectively. And most strains with anti-inflammatory activity were Bifidobacterium, numbering 9. These results suggested a potential relationship between genus-level classification and beneficial characteristics. Furthermore, our results are consistent with previous studies, which support the idea that Lactobacillus and Bifidobacterium are promising probiotics for treating skin acne [49–51].

LP-EVs exhibited excellent multi-target anti-acne effects

Considering the unique advantages of EVs, such as excellent biocompatibility, non-replicative and nanosized features [52, 53], we collected EVs derived from 5b4m2 (LP-EVs) and investigated their anti-acne potential. Transmission electron microscopy (TEM) images showed that LP-EVs were intact, isolated, round-shaped nanoscale particles (Fig. 2A). And their diameters and Zeta potential detected by Dynamic light scattering (DLS) were 229.75 nm and − 28.91 mV, respectively (Fig. 2B and C).

Fig. 2.

LP-EVs exhibited excellent multi-target anti-acne effects. (A) TEM image of LP-EVs. Scale bar = 100 nm; (B) Size distribution of LP-EVs measured by DLS; (C) The Zeta potential of LP-EVs; (D) The absorbance values at 600 nm were recorded every 4 h up to 28 h using a microplate reader to obtain the growth curve of P. acnes treated with PBS (Control), LP-EVs and CLI, respectively; (E) Bar plot of the CFU/mL of P. acnes; (F-G) Oil red staining detected the effect of LP-EVs on lipid production in SZ95 sebocytes. Representative picture (F), quantitative analysis (G); (H-K) The mRNA levels of SREBP-1a, PAR-2, SREBP-1c and FAS in SZ95 sebocytes; (L-N) The mRNA levels of TNF-α, IL-1β, and IL-8 in THP-1 cells; (O-Q) The supernatant TNF-α, IL-8, and IL-1β levels in THP-1cells; (R-T) The mRNA levels of TNF-α, IL-1β, and IL-8 in HaCaT cells; (U-W) Cell viability of cells (HaCaT, SZ95 and THP-1 cells) incubated with different LP-EVs concentrations measured by MTT assay. PC: positive control(dexamethasone). CLI: clindamycin. P-values are marked in the Figure

Then, we examined the inhibitory effect of LP-EVs on the growth of P. acnes. As expected, LP-EVs could suppress P. acnes growth in a dose-dependent manner, with 200 µg/mL showing the greatest efficacy (Figure S2). This concentration was therefore used in subsequent in vitro experiments. We further verify the anti-P. acnes activity of LP-EVs through bacterial growth curves and viable P. acnes count at 28 h. Results showed that LP-EVs and clindamycin inhibited P. acnes growth significantly (Fig. 2D), with viable counts of 5.73 × 10⁴ CFU/mL and 1.17 × 10⁴ CFU/mL, respectively, both reduced compared to the control (Fig. 2E). These findings demonstrated the potential of LP-EVs for combating P. acnes, with an efficacy comparable to the antibiotic control.

Next, we evaluated the impact of LP-EVs on sebum secretion using the SZ95 sebaceous gland cell model. Treatment with LP-EVs could reduce lipid production in SZ95 cells compared to the model group, with an effect similar to that of the positive control dexamethasone (Fig. 2F and G). We further analyzed the expression of lipogenesis-related genes and observed that the expression of FAS, SREBP-1c and PAR-2 genes was significantly decreased following LP-EVs treatment, although SREBP-1a showed no significant change (Figs. 2H-K). These findings indicate the potential of LP-EVs to inhibit sebum secretion.

Moreover, we investigated the anti-inflammatory effect of LP-EVs using an in vitro THP-1 inflammatory cell model. Our results demonstrated that LP-EVs could downregulate the expression of inflammation-related genes, including IL-1β, IL-8, and TNF-α (Figs. 2L-N). Consistently, the levels of these inflammatory cytokines in the cell supernatants were also reduced to the normal level in the LP-EVs group (Figs. 2O-Q). To validate these results, we conducted additional experiments using a HaCaT cell inflammation model, which similarly showed the same reductions in the expression levels of TNF-α, IL-1β, and IL-8 (Fig. 2R-T). Furthermore, we performed a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity (MTT) assay on SZ95, THP-1 and HaCaT cells to assess the cytotoxicity of LP-EVs. The results confirmed that LP-EVs were nontoxic to all three cell types (Fig. 2U-W).

Finally, we assessed the uptake of LP-EVs by acne-relevant skin cells. As visualized by confocal microscopy, DiI-labeled LP-EVs were efficiently taken up by SZ95 sebaceous gland cells (Figure S3), highlighting their cellular accessibility to key skin cells involved in acne pathogenesis.

Taken together, our results indicate that LP-EVs exhibit excellent multi-target anti-acne potential by inhibiting P. acnes growth, reducing sebum secretion and mitigating inflammation responses.

LP-EVs mitigated lesion formation in P. acnes-induced acne mice

Given the promising in vitro experimental outcomes, we proceeded to evaluate the efficacy of LP-EVs in animal models (Fig. 3A). We first utilized fluorescent probes to investigate the ability of LP-EVs to penetrate the epithelial barriers in vivo. As shown in the CLSM images, LP-EVs exhibited greater penetration than parental bacteria, as indicated by an enhanced fluorescence signal observed in the deeper layers of the skin (Fig. 3B). And this suggests that LP-EVs possess superior skin permeability, which may potentially augment their therapeutic efficacy [54, 55].

Fig. 3.

LP-EVs mitigated lesion formation in P. acnes-induced acne-like mice. (A) Flowchart of animal experiment; (B) CLSM images of mouse skin after treatment with 1 mg/mL DiI in PBS, DiI-labeled 5b4m2 or DiI-labeled LP-EVs. Key skin structures including epidermis, dermis, and subcutaneous tissue are annotated. Arrows and boxes indicate regions of LP-EVs enrichment. Scale bar = 200 μm; (C) Overall appearance of mouse ears after treatment with PBS (control), CLI, 5b4m2 and LP-EVs for 7 days; (D) The weight difference between the left and right ear indicates the degree of ear swelling; (E) Relative copy number of P. acnes in mice ears; (F) H&E staining reveals the pathological changes in the mouse ear. The red box highlights an area with diffusely distributed blue spots, indicating inflammatory cell infiltration. Scale bar = 50 μm; (G-I) The mRNA levels of CXCL1, IL-1β, and IL-6 in ear tissue. P-values are marked in the Figure

Next, we validated the anti-acne effects of LP-EVs in a P. acnes-induced acne mouse model. As expected, mice in the model group exhibited notable inflammatory symptoms such as redness and swelling of the ears, which were effectively alleviated by LP-EVs treatment (Fig. 3C-D). Additionally, LP-EVs significantly inhibited the colonization of P. acnes in the ears, demonstrating a more pronounced effect than the parental 5b4m2 (Fig. 3E). Furthermore, the hematoxylin and eosin (H&E) staining of ear tissue revealed that LP-EVs reduced inflammatory cell infiltration and preserved skin structures (Fig. 3F). Consistent with the H&E staining results, qPCR analysis of the ear skin tissue showed that LP-EVs treatment significantly downregulated the local mRNA expression of key pro-inflammatory mediators (Cxcl1, IL-1β, and IL-6) compared to the model group (Fig. 3G, H, I), further confirming its potent anti-inflammatory action at the disease site.

Moreover, the body weight of each experimental group remained relatively stable (Figure S4), preliminarily indicating the biological safety of these treatments.

Collectively, our data indicate that LP-EVs markedly mitigate lesion formation in P. acnes-induced acne mice, outperforming both the positive control drug and parental bacteria in effectiveness. This demonstrates a promising potential for LP-EVs in the treatment of acne.

The anti-acne function of LP-EVs may be attributed to the enrichment of key proteins

Based on the superior therapeutic effects of LP-EVs compared to bacterial interventions, we hypothesize that, in addition to their nano-size features [56], the enriched components within vesicles may also play a role. To verify this, we performed a comparative proteomic analysis between purified LP-EVs and their parental bacteria. Our analysis identified a total of 584 proteins within LP-EVs, with 181 being significantly enriched relative to the parent strain 5b4m2 (Fig. 4A). Functional screening further identified 9 proteins directly implicated in acne improvement (Table S3). Notably, F9UKV7_LACPL (Lipoprotein) and F9USM7_LACPL (Mucus-binding protein) were associated with immune interactions and inflammation inhibition [57, 58]. Additionally, two significantly enriched proteins, F9UPB8_LACPL (Glycosyl hydrolase) and F9URD9_LACPL (Cell wall hydrolase/muramidase), were predicted to play roles in inhibiting P. acnes growth [59–62]. Besides, we also identified proteins associated with lipid metabolism [63–65], such as F9US95_LACPL (Lipase/esterase) and GLPF3_LACPL (Glycerol uptake facilitator). Further analysis also revealed proteins involved in oxidative stress regulation within LP-EVs [66–68], including F9ULY7_LACPL (Thioredoxin reductase), F9UN44_LACPL (Glutathione reductase), and F9UUB4_LACPL (Thioredoxin).

Fig. 4.

Key proteins and pathways involved in the alleviation of acne. (A) Volcano plot showing the proteins with significant quantitative differences in LP-EVs compared with those in 5b4m2. The up-regulated proteins are shown as red dots, and the down-regulated proteins are shown as blue dots. Among red dots, 9 proteins are directly associated with acne improvement. The green, purple, orange, and gray dots represent proteins involved in oxidative stress regulation, inhibition of P. acnes, regulation of lipid metabolism, and anti-inflammatory processes, respectively; (B) Overall appearance of mouse ears after treatment with LP-EVs, Protease K and LP-EVs digested by Protease K; (C) The weight difference between the left and right ear indicates the degree of ear swelling; (D-E) The mRNA levels of IL-6, and CXCL1 in ear tissue. (F) Bubble plot showing the top 25 enriched GSEA pathways for DEGs in LP-EVs treatment group; (G) Alluvial diagram illustrating potential interactions between proteins and host pathways. P-values are marked in the Figure

To verify whether proteins in LP-EVs are functionally essential for acne alleviation, we performed additional in vivo experiments in which LP-EVs were treated with proteinase K to degrade protein cargo. As expected, proteinase K treatment impaired protein integrity in vesicles, with most proteins existing as peptide fragments (Figure S5). Correspondingly, proteinase K digestion largely abrogated the efficacy of intact LP-EVs (Fig. 4B). Compared to the LP-EVs group, mice treated with digested vesicles exhibited aggravated ear swelling and elevated mRNA expression levels of inflammatory cytokines (IL-6 and CXCL1) (Fig. 4D-E). These findings suggest that the enriched proteins within vesicles are likely key contributors to acne alleviation.

Then, to elucidate the potential mechanisms within the host, we performed transcriptome analysis on ear skin tissues from the model and LP-EVs intervention groups, which identified nearly 30,000 differentially expressed genes (Figure S6). GSEA and KEGG enrichment analyses of these genes revealed that LP-EVs inhibited pathways associated with acne progression (Fig. 4F, Figure S7). Specifically, LP-EVs intervention led to the downregulation of multiple inflammation-related pathways, including chemokine signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway. Consistent with the observed downregulation of multiple inflammation-related pathways, immune cell deconvolution analysis using CIBERSORT revealed that LP-EVs treatment increased the relative abundance of M2 macrophages and Th17 cells while decreasing M1 macrophages (Figure S8), which suggests that the therapeutic potential of LP-EVs may stem from promoting an anti-inflammatory macrophage shift toward the M2 phenotype together with Th17-mediated tissue repair and barrier defense functions [69, 70]. Furthermore, pathways related to pathogenic infection and lipid accumulation were also downregulated, such as Staphylococcus aureus infection, lipid and atherosclerosis [71, 72]. These findings are consistent with our proteomic analysis conclusions which indicates that host responses may be influenced by the corresponding key proteins. Additionally, gene pathway analysis also revealed alterations related to the skin barrier. Although not statistically significant, LP-EVs promoted the upregulation of pathways associated with tight junctions and extracellular matrix (ECM) receptor interactions in skin tissues, changes that may also contribute to acne improvement [73, 74] (Figure S9).

In summary, our integrated analysis suggests that the therapeutic effects of LP-EVs may be attributed to their anti-inflammatory, antibacterial, lipid-regulatory and barrier-related proteins (Fig. 4G).

LP-EVs maintained the skin microbiota homeostasis in P. acnes-induced acne mice

Previous studies have indicated that acne development is associated with dysbiosis of the skin microbiota, while probiotic EVs have been shown to regulate skin microecology and inhibit disease progression [75–77]. Therefore, we investigate the impact of LP-EVs on skin microbiota homeostasis in the P. acnes-induced acne mice.

Alpha diversity analysis, utilizing the Chao1 and Shannon indices, revealed that LP-EVs treatment significantly increased skin microbiota diversity compared to the model group, while the model group showed an obvious reduction (Fig. 5A). Further beta diversity analysis using Principal Coordinates Analysis (PCoA) revealed that the microbiota composition in the LP-EVs group closely resembled that of the healthy control group, and was notably distinct from the model group (Fig. 5B). These findings indicate that LP-EVs protect against P. acnes-induced microbiota dysbiosis, helping to restore and maintain a healthier microbial balance. Next, we performed LEfSe analysis to identify differentially abundant bacterial taxa (Fig. 5C), and visualized their abundance via a heatmap to facilitate comparisons (Fig. 5D). Our results indicated that P. acnes significantly reduced the abundance of beneficial bacteria in model group, including Bacillus, Bacteroides, and Bifidobacterium [50, 78–80], while simultaneously increasing the abundance of pathogenic bacteria such as Stenotrophomonas and Delftia [81–83]. In contrast, LP-EVs treatment effectively decreased the levels of these pathogenic bacteria and restored normal skin microbiota. Notably, LP-EVs promoted the recovery of probiotic Staphylococcus species, which are widely recognized for their health benefits [84, 85]. Our data suggest that LP-EVs intervention can maintain the skin microbiota balance, which may also contribute to the improvement of acne.

Fig. 5.

LP-EVs maintained skin microbiome homeostasis in mice with acne. (A) Changes in the α-diversity of the Control, LP-EVs and Model group, determined using Chao 1 index and Shanon index; (B) β-diversity analysis was determined using PCoA; (C) Analysis of taxonomic abundances using LEfSe; (D) Heat map of the abundance of differential bacterial in Fig C. ns: Non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Discussion

In recent years, the global incidence of acne has continued to rise, significantly affecting both the physical and mental health of patients, while also imposing a substantial disease burden on society. Probiotic-based acne treatments have gained significant attention owing to their high safety and minimal side effects. However, most existing therapies focus on limited targets, often resulting in unsatisfactory outcomes [86–88]. In this study, we identified L. plantarum 5b4m2 as a multi-target probiotic and extracted its extracellular vesicles for effective anti-acne treatment.

We first screened 24 candidate probiotics with different anti-acne effects based on three primary targets: antibacterial activity, sebum reduction, and anti-inflammatory effects. These 24 probiotics belong to the genera Bifidobacterium and Lactobacilli, both of which have been widely reported to hold significant potential for the treatment of acne [89, 90]. Furthermore, we demonstrated that their beneficial characteristics were related to the genus/species level. Specifically, Lactobacillus strains showed superior efficacy in inhibiting P. acnes growth and reducing sebum secretion, while B. longum exhibited strong anti-inflammatory effects. These findings are consistent with previous studies. Xu et al.. reported that Lactobacillus suppresses P. acnes proliferation and regulates lipid metabolism through FAS/SREBP-1c pathway inhibition [91]. And Fang et al.. reported that B. longum can improve atopic dermatitis by regulating tryptophan and indole derivative metabolic pathways [92].

Based on the screening results, we found that most probiotics harbored only a single target, emphasizing the necessity of developing multi-target probiotic therapies. Further analysis revealed that L. plantarum 5b4m2 was the only strain that showed significant inhibitory effects on all three targets, which suggests its potential for multi-target synergistic action in acne treatment. However, 5b4m2 did not perform the best on all targets. Other strains, such as Lactobacillus salivarius cb13m2 and B. longum cb10y5, despite lacking multi-target synergy, also demonstrated significant therapeutic potential on specific targets. Therefore, we will attempt to combine these strains with 5b4m2 into a multi-target probiotic community, aiming to provide a more efficient multi-target strategy for acne treatment in the future research [20, 93].

Next, we isolated EVs from 5b4m2 which exhibited typical vesicular characteristics. It was shown that they were notably larger than expected, with sizes exceeding 200 nm, differing from the commonly reported range of 50–200 nm for L. plantarum-derived EVs [94, 95]. We hypothesize that this discrepancy may result from different mechanisms of EV formation, isolation methods, and culture conditions [96]. However, the size of EVs from L. plantarum reported in some studies is not universally confined to below 200 nm. Forsberg et al.., for instance, extracted vesicles averaging 214 nm in size and found that these larger vesicles might carry more functional proteins [97].

Then, we systematically evaluated the anti-acne potential of LP-EVs across the three targets. It was shown that LP-EVs significantly inhibited the growth of P. acnes and inflammation. This finding is consistent with the research of Li et al.., which discovered that Bacteroides fragilis outer membrane vesicles could attenuate renal inflammation through NF-κB pathway inhibition [98]. Additionally, a study by Zhou et al.. also demonstrated that Staphylococcus epidermidis extracellular vesicles (SE-EVs) could inhibit the growth of S. aureus and significantly reduce the expression of pro-inflammatory genes, which effectively alleviated atopic dermatitis in mice [99]. Furthermore, differing from previous studies, we discovered that LP-EVs also possess the ability to inhibit lipid accumulation in sebaceous cells. These findings demonstrate that LP-EVs exhibit anti-acne potential similar to that of the parent bacteria. Moreover, cellular uptake of EVs is regarded as one of the key steps that enable their biological activity [100]. Our assays showed that LP-EVs can be efficiently internalized by SZ95 sebocytes, a process likely foundational to their therapeutic effects.

Given the above promising results of LP-EVs, we further validated their anti-acne effect in a mouse model. Before animal experiments, we demonstrate that EVs exhibited superior skin permeability compared to live bacteria, likely due to their nano-sized properties. This finding was supported by a previous study reporting that EVs exhibit enhanced skin penetration and cellular uptake in the treatment of skin diseases, which facilitates their distribution in tissues [101]. These results suggest that EVs may be more effective than bacteria in the treatment of acne in mice, primarily owing to their superior skin permeability.

In the P. acnes-induced acne mouse model, we selected a topical dose of 80 µg LP-EVs per mouse per day for the intervention group. This dosage, which falls within the range reported in prior studies using bacterial EVs in mouse skin disease models [16, 102], demonstrated significant therapeutic effects with no observable toxicity. As reported, the proliferation of P. acnes can exacerbate acne by inducing inflammation and excessive sebum secretion [103–105], and most acne treatments rely on inhibiting inflammation induced by P. acnes [106, 107]. In our animal experiments, we found that LP-EVs reduced P. acnes colonization, significantly alleviated acne-like symptoms and associated inflammatory responses in skin tissues. Moreover, it also downregulated gene expression levels in lipid synthesis and accumulation pathways, as confirmed by transcriptomic analysis. These results are consistent with our in vitro experiments, suggesting that LP-EVs can exert a synergistic effect through the three targets, which resulted in an excellent anti-acne effect. Interestingly, as expected, we observed that LP-EVs were more effective than 5b4m2 in reducing inflammation and P. acnes colonization, which indicates that the unique skin permeability of vesicles may play a key role. Therefore, we believe that LP-EVs are a promising biologic with better efficacy in acne treatment.

Previous studies have found that the protein components of EVs are the key molecular basis for their anti-acne function [108–110]. To investigate this, we performed proteomic analysis, which identified the enrichment of 9 key proteins linked to acne improvement in LP-EVs. Among these, the Mucus-binding protein, which has been reported to regulate the release of immune factors, was notably more abundant than the other eight, thereby reducing inflammation caused by bacterial lipopolysaccharides [59–62]. Besides, lipoproteins located on the vesicle surface act as significant cell interaction receptors and may be associated with inflammation suppression [57, 58]. Additionally, antioxidant enzymes, such as thioredoxin and Glutathione reductase, were also significantly enriched. Natalia et al.. found that they can inhibit the release of pro-inflammatory factors and lower reactive oxygen species (ROS) levels, which may help to reduce cell damage and promote the recovery of damaged tissues [66–68]. Furthermore, the enrichment of Glycerol Uptake Facilitator Protein and Lipase suggests that LP-EVs may reduce lipid accumulation in host cells by regulating lipid metabolism [63–65]. Overall, our study revealed key proteins within LP-EVs linked to acne improvement, including antibacterial, anti-inflammatory, antioxidant, and lipid metabolism-regulating proteins. They may contribute to the excellent therapeutic effect greatly.

Consistent with the proteomic results, transcriptomic analysis revealed corresponding regulatory changes in the host. Specifically, the gene expression levels of key inflammatory pathways, such as the NF-κB, chemokine, and Toll-like receptor signaling pathways, were significantly downregulated. This finding was supported by the research of Huang et al. [103, 111, 112]., who demonstrated that P. acnes promotes acne by producing chemokines, and the downregulation of pro-inflammatory responses is directly associated with acne improvement. Additionally, we observed that LP-EVs intervention downregulated the pathways of S. aureus infection and Lipid and atherosclerosis. These results further support that LP-EVs may alleviate acne by inhibiting pathogen growth, suppressing inflammation, and regulating lipid metabolism, and the key proteins within LP-EVs may play a crucial role in these processes. Notably, impaired skin barrier function may contribute to microbial dysbiosis and exacerbate inflammation, potentially triggering or worsening acne [113, 114]. Transcriptomic data also indicated the upregulated pathways associated with tight junctions and extracellular matrix (ECM) receptor interactions in skin tissues after LP-EVs treatment, which suggest an improvement in skin barrier function, a change that is also evident in the mouse ear following LP-EVs treatment. Taken together, the proteomic and transcriptomic data suggest that the proteins enriched in LP-EVs exert multi-target anti-acne functions.

The colonization of P. acnes on the skin may induce dysbiosis, while a healthy skin microbiome can effectively regulate host metabolism and immune responses, contributing to the improvement of acne [115–117]. In this study, we found that P. acnes colonization led to the deterioration of the skin microbiome in mice, while LP-EVs intervention significantly improved the skin microbiome composition in acne mice, restoring it closely to a normal level. This finding was supported by a previous study reporting that Lactobacillus dregea extracellular vesicles can modulate skin microbiome composition toward a healthier state, further promoting wound healing and skin barrier restoration [18]. Additionally, Zhou et al.. isolated S. epidermidis outer membrane vesicles and demonstrated that they could reestablish skin microbiome homeostasis, thereby alleviating skin inflammation in atopic dermatitis mice [118].

Notably, we found that the skin microbiota of mice in the model group was primarily enriched with two pathogenic bacteria, Stenotrophomonas and Delftia, both of which have been widely reported to be associated with pyogenic otitis and pathogenesis in immunocompromised patients [119–122]. In contrast, LP-EVs-treated mice showed a higher abundance of S. epidermidis, a key skin probiotic. It benefits the host by metabolizing skin lipids to produce short-chain fatty acids [123] (SCFAs) and secreting proteases that degrade S. aureus adhesion proteins, thereby inhibiting harmful bacterial overgrowth and preventing skin inflammation [124]. Furthermore, S. epidermidis modulates immune responses by regulating pathogen-suppressing regulatory T cells and pathogen-eliminating effector T cells, which offers multiple protective effects [125]. Therefore, our study demonstrates that LP-EVs improve the skin microbiome composition and promote the colonization of beneficial skin probiotics, which may contribute to their anti-acne effects.

However, this study still has shortcomings. Firstly, as summarized by Tore Skotland et al.. in their review, the lipid components in EVs interact with the host and play a regulatory role, which may also be an important functional component of the vesicles. In the current study, we primarily focus on protein components, while lipids and nucleic acids may also serve as potential functional molecules. Additionally, although we have established a connection between the anti-acne effects of LP-EVs and their enriched proteins through proteomic analysis and proteinase inhibition assays, future investigations should employ targeted approaches, such as generating mutant bacteria lacking key proteins, exogenously expressing specific candidate proteins, and using small molecule inhibitors to precisely link individual proteins to downstream signaling pathways in the host identified by transcriptomic analysis above and their acne-related biological effects. Furthermore, despite consistent evidence from our in vitro and in vivo studies that LP-EVs suppress pro-inflammatory cytokines and inhibit inflammatory signaling pathways, which are consistent with the established notion in the literatures that EVs derived from probiotics generally exhibit immunomodulatory properties rather than strong immunogenicity [15, 126–129], it should be noted that we have not yet directly quantified the dynamic impact of LP-EVs on complex local immune cell subsets in the skin. To fully elucidate how LP-EVs reshape the inflammatory microenvironment in acne, future studies should employ in vitro co-culture systems with primary skin immune cells or leverage cutting-edge technologies including single-cell RNA sequencing, high-dimensional flow cytometry, and spatial transcriptomics. These approaches will enable tracking of immune cell state transitions and intercellular communication networks at single-cell resolution. Moreover, although the in vivo dose of LP-EVs in our study was chosen based on in vitro efficacy, experimental feasibility, and precedent from the literatures, the lack of comprehensive pharmacokinetic and biodistribution data following topical administration prevents a precise delineation of their spatiotemporal distribution, retention time, and clearance pathways in vivo, thereby limiting a deeper, quantitative understanding of the relationship between tissue exposure levels and therapeutic efficacy. Future investigations should employ techniques such as in vivo fluorescence imaging to dynamically monitor biodistribution, combined with longer-term, repeated-dose studies to comprehensively evaluate potential adverse effects on local skin, vital organ function, and the systemic immune response. Meanwhile, we propose that surface engineering strategies [130], particularly ligand-based modifications tailored to the acne microenvironment, could enhance the targeting specificity of LP-EVs and thereby further improve therapeutic safety. Lastly, our study lacks human data, and further clinical investigations are required to validate the safety and efficacy of LP-EVs.

Given the promising anti-acne effects and high safety of LP-EVs, our study still offers new insights into multi-target anti-acne treatment based on probiotic vesicles, which may contribute to achieving greater therapeutic efficacy in the future management of skin acne and other complex dermatological conditions.

Conclusion

We identified L. plantarum 5b4m2 based on three anti-acne targets and extracted its extracellular vesicles. We found that LP-EVs demonstrated excellent effects in antibacterial, sebum suppression, and anti-inflammatory activities, while also showing good safety in in vitro experiments. Animal studies indicated that LP-EVs effectively improved acne in mice, with better results than the parent bacteria. Further omics analysis and protease treatment experiment suggest that LP-EVs are enriched with key proteins that may exert multi-target anti-acne effects, treating acne by acting on pathways related to antibacterial, sebum suppression, and anti-inflammation. Moreover, LP-EVs effectively regulated the skin microbiota of acne mice towards a healthy state, further promoting acne improvement. Collectively, our findings confirm that multi-target vesicle intervention is an effective treatment approach and can serve as a reference for future promising probiotic therapies for acne.

Author contributions

X.L.Y., Z.L., and D.M.G. designed and directed the research; K.L., J.W., and Y.T.S managed the sampling and performed most of the experiments; K.L., J.W., and D.M.G. performed most of the analysis; Q.Q.Y., Y.T.S., Z.Q.Z., and X.Y.M. helped with the analysis. Q.Q.N., Z.Q.Z., X.Y.M., Z.H.M., H.Z., and C.C.Z. also helped with the sample collection and experiments; K.L., Z.Q.Z., X.Y.M., and D.M.G wrote the paper with results from all authors; X.L.Y., Z.L., and D.M.G. polished the manuscript through multiple iterations of discussions with all authors. All authors have read and approved the final manuscript.

Data availability

All data relevant to the study were included in the article or uploaded as supplementary information. The 16s raw sequencing data used in this study are available from the China National Center for Bioinformation (CNCB) - Genome Sequence Archive (GSA) under accession code PRJCA037243. The transcriptome raw sequencing data used in this study are available from the CNCB - GSA under accession code PRJCA037244. The proteomics raw sequencing data used in this study are available from the CNCB - Open Archive for Miscellaneous Data (OMIX) under accession code PRJCA037759.

Declarations

Ethics approval

All mice received the humane care and the experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Huazhong University of Science and Technology, as approved by the Animal Care Committee of Hubei Province, the assigned approval number of the laboratory is: [2020] IACUC Number: 2996.

Competing interests

The authors declare no competing interests.