RS-EVLP were extracted from 500 g of dried Rosa rugosa flowers. First, the flowers were crushed using a pre-cooled grinder to obtain a crude extract, which was filtered. The filtrate was then centrifuged at 10,000 × g for 30 min. The resulting supernatant was filtered using a magnetic stirrer to remove molecules with molecular weights < 100 kD. The concentrated liquid was then centrifuged at 10,000 × g for 60 min and filtered through a 0.22-μm membrane (Tianjin Jinteng Experiment Equipment Co., Ltd., Tianjin, China) to remove large fragments. The RS-EVLP were concentrated by centrifugation at 100,000 × g for 70 min at 4 °C using an ultracentrifuge (Sorvall^TM WX+ Ultracentrifuge Series; Thermo Fisher Scientific, Waltham, MA, USA), resuspended in phosphate-buffered saline (PBS) (Wuhan Procell Life Science & Technology Co., Ltd., Wuhan, China), and stored at -80 °C. Prior to use, the RS-EVLP were concentrated by centrifugation at 100,000 × g for 70 min at 4 °C.

The particle size and concentration of the RS-EVLP were first determined using a nanoflow cytometer (NanoFCM Co., Ltd., Xiamen, China). To evaluate lipid membrane integrity, the RS-EVLP were subsequently treated with Triton X-100 for 30 min to rupture the vesicle membranes, after which the particle size and concentration were re-measured under the same conditions. The morphology of the RS-EVLP was observed with a transmission electron microscope (JEOL-1400; JEOL Ltd., Tokyo, Japan). In brief, each sample (10 μL) was placed on a copper mesh in a 6-cm Petri dish, allowed to settle for 10-30 min, then aspirated, stained with 10 μL of phosphotungstic acid for 30 s, and aspirated again. Afterward, the copper mesh was transferred to filter paper and shade-dried for at least 2 h prior to electron microscopy.

The active components of R. rugosa (i.e., flavonoids, phenolic acids, coumarins, and flavonol derivatives^[34-38]) were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which revealed the presence of luteolin, ellagic acid, and proanthocyanidins. Based on the active ingredients reported in the literature and comparative analysis of rose hips by mass spectrometry, the key compounds of RS-EVLP were identified, and 47 core components were selected. Then, 372 potential targets were identified using the SwissTargetPrediction platform (http://www.swisstargetprediction.ch/). Based on the GeneCards database (https://www.genecards.org/) and the keyword “hyperpigmentation”, 1990 related genes were obtained. Finally, Cytoscape 3.9.1 bioinformatics software (https://cytoscape.org/release_notes_3_9_1.html) and the STRING database (https://string-db.org/) were used to construct multilevel interaction and protein-protein interaction networks. Additionally, Gene Ontology (GO) annotation pathway analysis of the common targets with reference to the Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed using the Metascape platform (https://metascape.org/).

The mass spectra of the VLNPs of Rosa moschata and Rosa sinensis were compared, and the substances quercetin, luteolin, apigenin, kaempferol, ellagic acid, and procyanidin B3 were selected for molecular docking. After searching the RCSB Protein Data Bank (https://www.rcsb.org), UniProt Protein Database (https://www.uniprot.org/), National Center for Biotechnology Information Protein Database (https://www.ncbi.nlm.nih.gov/protein/), and Protein Data Bank (www.wwpdb.org), among others, MITF and TRP-1 were selected for docking simulations, which were conducted using Molecular Operating Environment software (https://www.chemcomp.com/en/Products.htm) and visualized using the PyMOL molecular visualization system (https://github.com/schrodinger/pymol-open-source).

To explore the interactions between the proteins MITF and TRP-1 and the ligands procyanidins and luteolin, molecular dynamics simulations of the protein-ligand complexes were performed using GROMACS 2020 (https://manual.gromacs.org/). Based on the AMBER99SB-ILDN force field, we constructed the protein topology and set up a cubic water box with a buffer distance of 1.0 nm (SPC216 water model). We then optimized the system energy using the steepest descent method after charge neutralization with Cl^-/Na⁺ counterions. A 100-ps NVT (canonical ensemble, constant number of particles, volume, and temperature)/NPT (isothermal-isobaric ensemble, constant number of particles, pressure, and temperature) pre-equilibration (300 K, 1 bar) was then performed, followed by a production simulation for 30 ns (V-rescale temperature control, Parrinello-Rahman pressure control, and an integration step of 2 fs). Particle Mesh Ewald calculations were used to determine long-range electrostatic interactions (0.16-nm spacing), and the LINear Constraint Solver algorithm was used to calculate bond lengths. The trajectories were analyzed using VMD (Visual Molecular Dynamics) 1.9.3 (https://www.ks.uiuc.edu/Research/vmd/vmd-1.9.3/) and PyMOL 2.4.1 (https://pymol.org/), and the binding free energies were calculated using gmx_mmpbsa (https://valdes-tresanco-ms.github.io/gmx_MMPBSA/dev/). The SPR technique was used to study biomolecular interactions and analyze the affinity, kinetics, and other parameters of the binding of luteolin/procyanidin to MITF/TRP-1 by detecting changes in optical signals of biomolecules during binding and dissociation on the chip surface.

B16 cells (obtained from Shanghai Jisidenuo Biotechnology); RRID: (CVCL_F936); synonyms: (B16 sunline B78) in the logarithmic growth stage were inoculated into the wells of 96-well plates, which included blank controls (medium), negative controls (untreated cells), and the experimental group (RS-EVLP concentration gradient: 4.6 × 10^-4 to 4.6 × 10¹ µg/mL, “µg” refers to the protein content in the sample). Cells were cultured in Dulbecco’s modified Eagle’s medium [DMEM; (Thermo Fisher Scientific Inc, C11095500BT)] supplemented with 10% fetal bovine serum [FBS; (Zeta Life, Z7185FBS-500)] and 1% penicillin-streptomycin (Thermo Fisher Scientific Inc, 15140122) at 37 °C in a humidified atmosphere containing 5% CO₂. After incubation for 24, 48, or 72 h, CCK-8 reagent (10 μL) was added to each well, and the plates were incubated for a further 2 h at 37 °C in the dark. The optical density (OD) at 450 nm was then detected using an enzyme marker^[39]. Cell viability was calculated as (OD treated group - OD blank group)/(OD control group - OD blank group) × 100%.

DiI dye was mixed at a PBS:RS-EVLP:DiI ratio of 950:50:5 (v/v/v), protected from light, and incubated in a metal bath at 37 °C for 30 min. The mixture was then purified three times by ultracentrifugation (wX + ULTRA) at 100,000 × g for 70 min at 4 °C. B16 cells were inoculated into Corning CellBIND® dishes at a density of 5 × 10⁴ cells per dish. When cell fusion reached 50%, the cells were treated with 4.6 μg/mL DiI-labelled RS-EVLP for 2, 4, or 8 h. The nuclei and cytoskeleton were subsequently stained with fluorescein isothiocyanate and 4’,6-diamidino-2-phenylindole, respectively. Then, the cells were fixed with 4% paraformaldehyde for 15 min before quantitative analysis of the time-dependence of RS-EVLP, which was performed using three-channel imaging with a confocal microscope (Thunder DMi8; Leica Microsystems GmbH, Wetzlar, Germany)^[40,41].

B16 melanoma cells (1 × 10⁵) were inoculated into the wells of 6-well plates containing Roswell Park Memorial Institute 1640 complete medium. After 24 h, the culture medium was replaced with basic complete DMEM and the cells were incubated with RS-EVLP, along with 200 ng/mL of α-MSH (MedChemExpress, Monmouth Junction, NJ, USA) and 1,500 nM/mL of arbutin (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), at protein concentrations of 4.6 and 23 μg/mL, respectively. After 48 h, the cells were washed twice with PBS, examined under a microscope for melanin deposition phenotypes, and the solution was discarded. Then, 1 M NaOH (800 μL) was added to each well, and the solution was lysed at 80 °C for 1 h.

The lysates were transferred to the wells of a 96-well plate, and the relative melanin content was determined by measuring the absorbance at 405 nm using an enzyme marker^[42]. Equal numbers of treated cells were lysed with 1 mL of 1% Triton X-100, subjected to a freeze-thaw cycle at -80 °C (30 min → 10 min at room temperature), and then centrifuged at 12,000 × g for 15 min. The resulting supernatant was incubated with 2 mg/mL of L-3,4-dihydroxyphenylalanine (L-DOPA) substrate at 37 °C for 1 h. Afterward, the absorbance was measured at 475 nm to detect TYR activity.

Total RNA was extracted from B16 cells that had been treated for 48 h using TRIzol Reagent (Ambion®; Thermo Fisher Scientific) and reverse-transcribed into complementary DNA using 5× PrimeScript RT Master Mix (TaKaRa Bio, Inc., Shiga, Japan). The RT-qPCR assays were performed using 2× UltraSYBR® Green Mix (Beijing Kangwei Century Biotechnology Co., Ltd., Beijing, China) on a real-time fluorescence quantitative PCR analyzer (Thermo Fisher Scientific). β-actin was used as the internal reference gene. The transcriptional regulatory effects of MITF, TYR, and TRP-1/TRP-2 were quantitatively analyzed in control cells and groups treated with α-MSH (200 ng/mL), arbutin (1,500 nM/mL), and RS-EVLP (4.6/23 μg/mL)^[43] using the 2^-ΔΔCt method with the primer sequences listed in Table 1.

Drug-treated adherent cells were collected and washed with pre-cooled PBS before digestion with 0.25% trypsin containing ethylenediaminetetraacetic acid. The cell precipitate was collected by centrifugation. The freeze-thaw lysis method was then employed: the cell precipitates (1 × 10⁶ cells) were resuspended in 150 μL of PBS, centrifuged at 3,000 rpm for 5 min at 4 °C, and underwent three freeze-thaw cycles (-20 °C/30 min → 37 °C/10 min). The resulting supernatant was then extracted by centrifugation at 15,000 × g for 10 min at 4 °C. Key regulatory proteins of melanin synthesis were detected using a mouse MITF/TYR/TRP-1/TRP-2 ELISA kit (Beijing Dongge Boye Biotechnology, DG96647Q/DG96642Q/DG30784M/DG96648Q), and protein concentrations were calculated based on standard curves.

Experiments were performed using wild-type AB strain zebrafish embryos (Hangzhou Hunter Biotechnology Co., Ltd., Beijing, China). Embryos at 24 h post-fertilization (hpf) were inoculated into the wells of 6-well plates at a density of 15 embryos per well. The embryos were then grouped and treated as follows: a blank control group (standard zebrafish culture); a positive control group (1 mg/mL arbutin); and an experimental group (RS-EVLP gradient at concentrations of 0.046/0.23/0.46 mg/mL). The embryos in each group were exposed to the treatment for 24, 48, or 72 h at a constant temperature of 28.5 °C under a 14/10-h light/dark cycle. The culture medium containing the corresponding concentration of the treatment reagents was replaced every 24 h. Each experiment was performed with n = 15 zebrafish larvae per group. All animal experiments were approved by the Animal Ethics Committee of Ruiye Model Animal (Guangzhou) Biotechnology Co., Ltd. (ethical approval no. RYEth-20250627752).

MS-222 anesthetic (Hangzhou Jiuyan Biotechnology Co., Ltd., Hangzhou City, China) was diluted 100-fold in 6% methylcellulose (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) and used as the fixation matrix after vortexing. Zebrafish larvae at 24/48/72 hpf were treated, placed on concave slides pre-set with fixative, and imaged in vivo using an MZ62 somatic microscope (10× objective). The grey scale values of the pigmented region of the zebrafish heads were analyzed quantitatively using ImageJ software (https://imagej.net/ij/), and the survival rate of juvenile zebrafish in each group was calculated^[44].

Groups of zebrafish embryos at 24 hpf were exposed to basal culture medium, 1 mg/mL of arbutin, and gradient concentrations of RS-EVLP (0.046, 0.23, and 0.46 mg/mL) for 72 h. The embryos were then quickly frozen and ground in liquid nitrogen. The tissue homogenate was then centrifuged at 10,000 × g for 15 min at 4 °C, and the precipitate was lysed by adding 1 M NaOH containing 10% dimethyl sulfoxide (500 μL per sample) at 80 °C for 1 h. The lysate was transferred to the wells of 96-well plates, and the OD at 405 nm was detected using an enzyme marker. The relative melanin content of each group (%) was calculated as the OD value of that group/OD value of the negative control group × 100%^[45].

RS-EVLPs were isolated from rose petals by ultracentrifugation [Figure 1A] and observed by transmission electron microscopy, which revealed a double-layered, elliptical structure of various sizes [Figure 1B]. Protein analysis revealed a molecular weight of 25-35 kDa, while nanoparticle tracking analysis showed a particle size distribution of 70-100 nm. Membrane integrity was confirmed by treatment with Triton X-100, with an increased detergent concentration indicating a higher degree of vesicle cleavage and purity of up to 83% [Figure 1C-E]. Metabolomics analysis revealed that the RS-EVLP contained high proportions of lipids and lipid-like molecules (38%), followed by organic acids (14%) and phenylpropanoids (9%). These compounds are involved in plant defense and signaling. Other components included phenylpropanoids and polyketides (9%), which have antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Further analysis of lipid subclasses revealed 20% fatty acids, 15% glycerophospholipids, and 13% isoprenoid lipids and flavonoids (with antioxidant properties) [Figure 1F and G]. Notably, RS-EVLP contains six bioactive compounds abundant in roses: quercetin, luteolin, apigenin, kaempferol, ellagic acid, and procyanidin B3 [Figure 1H and I].

The results show that the 47 active RS-EVLP compounds are linked to hyperpigmentation, with a subset of 159 gene targets. The quantitative relationships are presented as a Venn diagram [Figure 2A and B]. Additionally, the interactions among the active compounds of the RS-EVLP and the hyperpigmentation-related intersecting targets were demonstrated with a network pharmacology map and a protein-protein interaction network [Figure 2C and D]. The active compounds of the RS-EVLP are linked to the melanogenesis-related biological pathway [Figure 2E]. GO analysis revealed various molecular functions associated with RS-EVLP, including protein kinase activity, growth factor receptor binding, and oxidoreductase activity [Figure 2F].

The results showed that quercetin, luteolin, apigenin, kaempferol, ellagic acid, and procyanidins could all bind to the allosteric sites of MITF and TRP-1 proteins, with predicted binding energies of -6.22, -5.06, -5.08, -4.73, -4.98, and -4.90 for MITF, and -5.85, -5.84, -5.60, -5.58, -5.69, and -7.47 for TRP-1, respectively. This indicates that the luteolin and procyanidins in RS-EVLP can form strong binding affinities with the allosteric sites of the MITF and TRP-1 proteins [Figure 3A-D and Supplementary Figure 1A-K]. It is hypothesized that these active RS-EVLP ingredients can inhibit melanogenesis by binding to the amino acid residues of the MITF and TRP-1 proteins.

The network pharmacological analysis (Section “Molecular docking results of RS-EVLP key compounds”) and molecular docking results revealed an association of RS-EVLP with the melanogenic pathway. Two sets of complexes with stronger binding energy (procyanidins/luteolin-MITF/TRP-1) were screened for validation. We found that procyanidins bind to MITF and TRP-1 proteins more stably, as shown by lower RMSD values, higher structural tightness (lower “solvent accessible surface area” and “radius of gyration” values), fewer flexibility changes (lower “root mean square fluctuation” values), and more hydrogen bonds [Figure 3E-H]. Binding free energy analysis showed that procyanidins had lower binding energies (mainly dependent on van der Waals forces), whereas luteolin was weakly bound [Figure 3I]. Procyanidins were also expressed at high abundance in RS-EVLP, suggesting that they influence melanogenesis by stabilizing the protein structure and providing theoretical support for the potential application of RS-EVLP. SPR analysis showed that both procyanidins and luteolin specifically bound to MITF and that the binding process was consistent with the 1:1 model. These results provide a quantitative basis to investigate the roles of procyanidins and luteolin in biological processes, such as melanogenesis [Figure 3J].

To assess the interaction between RS-EVLP and target cells, we first confirmed that RS-EVLP did not exhibit an antiproliferative effect on B16 cells at concentrations below 46 µg/mL. Two non-cytotoxic concentrations were therefore selected for uptake studies: 4.6 and 23 µg/mL. Fluorescein-labeled RS-EVLP exhibited time- and dose-dependent cellular uptake in B16 cells. Flow cytometry showed maximum cellular uptake (94.76% positive cells) at 23 µg/mL after 8 h. Confocal microscopy further confirmed that the RS-EVLPs were efficiently taken up into the cytoplasm of B16 cells, with fluorescence intensity increasing proportionally with incubation time [Figure 4A-C].

B16 cells were stimulated with 200 ng/mL of α-MSH and then treated with various concentrations of RS-EVLP (0.23, 4.6, 10, 20, and 23 µg/mL) or arbutin (250 µg/mL; positive control). After 48 h, microscopic analysis revealed that α-MSH had successfully induced melanin synthesis, whereas RS-EVLP showed an inhibitory effect at 4.6 and 23 µg/mL, as well as arbutin (positive control) at 250 µg/mL. After 48 h, microscopic analysis showed that α-MSH had successfully induced melanin synthesis, whereas both arbutin and RS-EVLP (4.6 and 23 µg/mL) significantly reduced melanin synthesis as compared to the cell group treated with α-MSH only [Figure 4D]. The NaOH assay showed that the inhibitory effect of the RS-EVLP on melanogenesis was concentration-dependent, with the highest concentration (23 µg/mL) having the most significant inhibitory effect (16% reduction in melanin content; Figure 4E). Furthermore, the RS-EVLP significantly inhibited TYR activity, as demonstrated by the L-DOPA oxidase assay (18% and 26% reductions in the 4.6 and 23 µg/mL groups, respectively; Figure 4F). This is consistent with the inhibitory effect on melanogenesis, suggesting that the RS-EVLP may affect the melanin synthesis pathway by modulating TYR activity.

In order to elucidate the molecular mechanism employed by the RS-EVLP to regulate melanogenesis, B16 cells induced by α-MSH were treated with 23 µg/mL of RS-EVLP for 48 h. Total RNA was then extracted with TRIzol reagent [Figure 5A and B]. Melanin synthesis is closely linked to MITF, the ultimate target of multiple signaling pathways, and TYR, a key enzyme in melanogenesis. Melanin synthesis mainly involves three enzymes in the TYR gene family: TYR, TRP-1, and TRP-2. We therefore examined the mRNA expression of MITF, the core regulator of melanin synthesis, and related downstream effector molecules (TYR, TRP-1, and TRP-2). The experimental results showed that treatment with RS-EVLP at 23 µg/mL significantly suppressed the expression of these genes compared with the α-MSH-induced control group [Figure 5C-F]. Further examination of protein levels with an ELISA revealed that RS-EVLP reduced expression of MITF, TYR, TRP-1, and TRP-2 in a concentration-dependent manner. In the 4.6 µg/mL treatment group, the protein levels of MITF, TYR, TRP-1, and TRP-2 were reduced by 30%, 18%, 25%, and 29%, respectively, while in the 23 µg/mL treatment group, the reduction was even more significant at 41%, 40%, 44%, and 47%, respectively [Figure 5G-J]. These findings suggest a potential mechanism by which RS-EVLP regulates melanogenesis.

This study evaluated the effect of RS-EVLP on melanogenesis in vivo using a wild AB zebrafish embryo model. Different concentrations of RS-EVLP (0.046, 0.23, and 0.46 mg/mL) and a positive control (arbutin at 1 mg) were administered to observe changes to body pigmentation at 24, 48, and 72 h. In vivo microscopic observation showed that RS-EVLP attenuated body surface pigmentation in a concentration-dependent manner [Figure 6A]. Subsequently, zebrafish larvae at 72 h post-treatment were subjected to tissue homogenization, and the relative melanin content was determined using the NaOH method. ImageJ quantitative analysis confirmed that the melanin content in the head was significantly lower in each RS-EVLP-treated group than the NC group [Figure 6B and C]. Notably, none of the tested concentrations exhibited embryotoxicity. The NaOH assay further showed that arbutin reduced melanin content by 15.1%, whereas RS-EVLP at 0.046, 0.23, and 0.46 mg/mL resulted in reductions of 7.0%, 6.0%, and 10.7%, respectively, which were consistent with the phenotypic observations [Figure 6D-F].

Transdermal analysis was conducted to assess the penetration density of the DiI-RS-EVLP group compared to the DiI group over 15 min to 1 h. Permeation density was measured by integrating the fluorescence intensity, which reflects the distribution of DiI-labelled particles in the skin tissue of nude mice. DiI-labelled RS-EVLP showed time-dependent enhancement of skin permeation from 15 min to 1 h. Quantitative fluorescence analysis showed that the penetration density was higher in the RS-EVLP-treated group than in the DiI-only control group, with a progressive increase over time. This may be attributed to the phospholipid bilayer structure of the RS-EVLP, which promotes diffusion and penetration into the skin. The results showed that RS-EVLP exhibited strong penetration ability in skin tissue, entering the stratum corneum and gradually penetrating into the basal layer over time [Figure 7A-C].

Clinical evaluation showed that the 5% RS-EVLP preparation improved skin pigmentation. The L^* value of the RS-EVLP group increased steadily from day 8 (ΔL^* = 1.27, P = 0.0004) to day 21 (ΔL^* = 2.15, P < 0.0001), demonstrating a significantly greater whitening effect than hydroquinone cream (day 21, ΔL^* = 1.29, P = 0.0060). Notably, the RS-EVLP group exhibited a cumulative effect over time, with the difference in L^* values increasing from 1.27 to 2.15. This suggests that pigmentation regulation is achieved through multiple mechanisms, and that the product may be suitable for long-term management of pigmentation issues. Furthermore, the difference in L^* values between the treated and control surfaces of the RS-EVLP group widened over time (0.27 on day 8 to 2.15 on day 21) [Figure 8A and B], suggesting that a cumulative effect suitable for long-term pigmentation management, potentially circumventing the risk of pigment rebound associated with conventional chemical whiteners. Safety analysis showed no serious adverse reactions in any of the subjects, confirming that the RS-EVLP were well tolerated.