ELNs were isolated from the juice found in the pomegranate peel. A total of ~50 g of pomegranate peel was washed with ultra-pure water, ground with phosphate-buffered saline (PBS) buffer, and filtered through a strainer to collect the leaching filtrate solution. The filtrate was then centrifuged at 500 × g for 10 min, 2,000 × g for 20 min, and 12,000 × g for 60 min to remove dead cells and cell debris (Fig. 1A). The supernatant was subsequently ultra-centrifuged at 100,000 × g for 70 min at 4°C (Thermo Fisher Scientific, Inc.). The pellet was resuspended in PBS and then ultra-centrifuged again for 70 min. Finally, the PELNs were resuspended in PBS and stored at −80°C until further analysis.

PELN size and concentration were measured using a Nanoparticle Tracking Analysis (NTA) instrument (ZetaView; Particle Metrix GmbH). PELNs were then stained with 2% uranyl acetate for 1 min at room temperature, and images of their morphology structure were captured using transmission electron microscopy (TEM; Hitachi HT-7700; JEOL, Ltd.).

The CRC cell line SW480 utilized in the present study was obtained from Procell Life Science & Technology Co., Ltd. SW480 cells were cultured in Dulbecco's Modified Eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; HyClone™; Cytiva) and 1% penicillin/streptomycin (PS) solution in a 37°C cell culture incubator with 5% CO₂.

Next, 50 µl of PELNs was mixed with 4 µl of PKH26 red fluorescent cell membrane dye (MilliporeSigma) in 2 ml of PBS. The mixture was incubated at room temperature for 5 min and 2 ml of FBS was added to stop the staining reaction. The mixtures were subsequently ultra-centrifuged at 100,000 × g for 70 min at 4°C. The pellet was re-suspended in PBS buffer and then ultra-centrifuged again for 70 min. Finally, the PKH26-labeled PELNs were re-suspended in PBS for future analysis.

SW480 cells were seeded in 24-well plates and treated with PKH26-labeled PELNs for 5 h. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, stained with DAPI blue-fluorescent dye for 15 min at room temperature, and images were captured using confocal microscopy (TCS SP8; Leica Microsystems GmbH).

The effect of PELNs on SW480 cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8; Beijing Solarbio Science & Technology Co., Ltd.). SW480 cells were seeded in 96-well plates and treated with PBS (Control group) or PELNs (PELN group) for 0, 24, 48 and 72 h. Next, 10 µl of CCK-8 reagent was added to each well and further cultured for 1 h at 37°C. The absorbance was measured at 450 nm using an enzymatic reader (Bio-Rad Laboratories, Inc.).

SW480 cells were seeded in 6-well plates and treated with PBS or PELNs. After 14 days, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with crystal violet staining solution (cat. no. C0121; Beyotime Biotechnology) for 15 min at room temperature. The areas of cell colony formation were quantified using ImageJ software (v1.46r; National Institutes of Health).

SW480 cells were seeded in 6-well plates and treated with PBS or PELNs. The cells were harvested using trypsin upon reaching 80% confluency, resuspended in PBS, and stained with 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min in the dark. Apoptotic cells were then quantified using a flow cytometer (MoFLO XDP; Beckman Coulter, Inc.) and CytExpert software (v2.3.1.22; Beckman Coulter, Inc.).

SW480 cells were seeded in 6-well plates until reaching 90% confluency. Then, straight scratches were made in each well using a 1-ml sterile pipette tip. After removing cell debris with a PBS wash, cells were cultured for 0, 24, or 48 h in 2 ml of DMEM with 1% FBS and 1% PS solution, containing PBS or PELNs (1.0×109 particles/ml), at 37°C in a 5% CO2 incubator. Wound healing was observed using a light microscope (bright-field). The migration distance was quantified by measuring the wound width using ImageJ software.

SW480 cells were seeded in the upper chamber of a 24-well Transwell plate in 200 µl of DMEM containing PBS or PELNs. The lower chamber was filled with 500 µl of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature after treatment for 24 h and stained with crystal violet staining solution (cat. no. C0121; Beyotime Biotechnology) for 15 min. The areas of cell migration were quantified using ImageJ software.

The total RNA samples from SW480 cells treated with PBS or PELNs were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). TRIzol reagent was added to the sample, and the mixture was incubated for 5 min and mixed with chloroform, phenol, and isopropanol for 3 min according to the manufacturer instructions. The RNA samples were collected with DEPC water by centrifugation at 12,000 × g for 15 min at 4°C. RNA quality was assessed prior to library construction. Purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.) and a Qubit 4.0 fluorometer (Thermo Fisher Scientific, Inc.), with a 260/280 nm ratio >1.8 required for sample acceptance. The integrity and quantity of the extracted RNA were measured using the Agilent 2100 system (Agilent Technologies, Inc.).

The SW480 cells were seeded in 6-well plates and treated with PELNs or PBS for 24 h for RNA-sequencing (RNA-Seq) analysis. Total RNA was extracted from the CRC cell samples, and mRNA was purified using the HieffNGS^® mRNA isolation master kit (cat. no. 12603; Shanghai Yeasen Biotechnology Co., Ltd.). First-strand cDNA was synthesized using random hexamers, followed by second-strand synthesis with DNA polymerase I and RNase H. Double-stranded cDNA was end-repaired, adenylated, and ligated to sequencing adaptors. For purification and fragment selection, Hieff NGS^® DNA Selection Beads (cat. no. 12601ES56; Yeasen Biotechnology Co., Ltd.) were used. After adapter ligation, bead-based size selection was performed using 0.6× and 0.2× ratios to enrich for fragments of the desired size. The final libraries were purified using a 0.8 × bead ratio and subsequently amplified by PCR. The purified libraries were quantified using a Qubit fluorometer (Thermo Fisher Scientific, Inc.) and initially measured in ng/µl. The molar concentration was then calculated based on the average fragment size determined by an Agilent 2100 system (Agilent Technologies, Inc.). Library circularization was performed using the MGIEasy Dual Barcode Circularization Module (cat. no. A0508; MGI Tech Co., Ltd.) prior to sequencing. Finally, the libraries were sequenced on the DNBSEQ-T7 platform (MGI Tech Co., Ltd.) using the DNBSEQ-T7RS High-throughput Sequencing Set (App-D FCL PE150, V3.0; cat. no. Group 100-000016-00; MGI Tech Co., Ltd.) in paired-end 150 bp (PE150) mode, following the manufacturer's instructions.

StringTie (v2.1.5; http://ccb.jhu.edu/software/stringtie/index.shtml) was used to compare the read count values for each gene as the original expression level. Fragments per kilobase of transcript per million mapped reads were then utilized to standardize the expression. Differentially expressed genes (DEGs) were then analyzed using DESeq2 (v1.30.1; http://bioconductor.org/packages//release/bioc/html/DESeq2.html), with screening conditions set as follows: |log₂FoldChange| >1 and adjusted P-value (padj) ≤0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of DEGs were performed using clusterProfiler (v3.8.1; http://github.com/GuangchuangYu/clusterProfiler). Significant enrichment results (P-adj ≤0.05) were selected to identify connections between genes and pathways.

Statistical significance was analyzed using GraphPad Prism 5.0 (Dotmatics) and Microsoft Excel (2010) software. All data are presented as the mean ± standard deviation (mean ± SD). Paired Student's t-test was performed to compare the means of two data sets. P<0.05 was considered to indicate a statistically significant difference.

PELNs were isolated and purified from Punica granatum L. using filtration and differential ultra-centrifugation (Fig. 1A). TEM analysis revealed that the isolated particles exhibited a cup-shaped nanostructure with exosome-like characteristics (Fig. 1B). NTA analysis revealed that PELNs had an average diameter of 152.0 nm, a concentration of 4.2×10¹⁰ particles/ml, and a zeta potential of −26.14 mV (Fig. 1C and D).

An internalization experiment was carried out to investigate the biological function of PELNs in the anti-CRC mechanism. After treating SW480 cells with PKH26-labeled PELNs or PBS, red fluorescence was only observed in the PELN-treated group and located around the blue fluorescence, but not in the control group (Fig. 1E). These results indicated that PELNs were effectively received by the CRC cells.

CCK-8 and colony formation assays were performed to further evaluate the effect of PELNs on CRC cell proliferation. The proliferation ability of CRC cells treated with PELNs for 24, 48 and 72 h was significantly reduced compared with that of the control group (P<0.001; Fig. 2A and B). In the colony formation assay, the areas of SW480 cells stained with crystal violet in the PELN group were significantly decreased (P<0.01; Fig. 2C and D).

Furthermore, SW480 cells were treated with PELNs for 24 h to investigate whether PELNs affect CRC cell apoptosis. Early and late apoptotic cells were labeled with Annexin V-FITC/PI. Flow cytometric analysis indicated that the PELN treatment significantly increased the rate of CRC cell apoptosis (P<0.01; Fig. 2E and F).

Wound healing and Transwell migration assays were carried out to evaluate the effect of PELNs on the migration ability of CRC cells. SW480 cells were treated with PELNs after generating scratch wounds in the plate wells. The changes in wound width were observed at 0, 24 and 48 h. The wound width in the PELN-treated group was significantly increased compared with that in the control group (P<0.001; Fig. 3A and B).

SW480 cells were incubated with PELNs in the upper chamber of Transwell plates for 24 h. The migratory cells were then quantified. Crystal violet-stained areas in the PELN group demonstrated a significant reduction (P<0.05; Fig. 3C and D). These results indicated that PELNs significantly inhibit CRC cell migration in vitro.

The sample correlation analysis heat map indicated that the correlation between repeated samples was strong, confirming experimental reproducibility (Fig. 4A). DEGs of SW480 cells after treatment with PBS (Control-1, Control-2 and Control-3) or PELNs (PELNs-1, PELNs-2 and PELNs-3) were statistically analyzed. The results of a volcano plot and cluster analysis showed that there were 2,303 upregulated and 3,295 downregulated genes (Fig. 4B and C). KEGG pathways and GO terms related to DEGs were subsequently analyzed based on the functional annotation results in the database.

Statistical analysis of KEGG pathway enrichment (top 20) indicated that multiple enriched pathways were associated with signal transduction and cancer, including ‘MAPK signaling pathway’ (ID: map04010), ‘Hippo signaling pathway’ (ID: map04390), ‘NF-kappa B signaling pathway’ (ID: 04064), ‘TNF signaling pathway’ (ID: map04668), ‘Wnt signaling pathway’ (ID: map04310), ‘Estrogen signaling pathway’ (ID: map04915), ‘Cellular senescence’ (ID: map04218), ‘p53 signaling pathway’ (ID: map04115), ‘Cell cycle’ (ID: map04110), ‘FoxO signaling pathway’ (ID: map04068), ‘ECM-receptor interaction’ (ID: map04512), ‘MicroRNAs in cancer’ (ID: map05206) and ‘Transcriptional misregulation in cancer’ (ID: map05202) (Fig. 4D, Table SI).

GO enrichment analysis of the top 20 molecular function terms demonstrated significant enrichment for processes directly related to cell cycle regulation and signal transduction, including ‘MAP kinase tyrosine/serine/threonine phosphatase activity’ (GO: 0017017), ‘protein kinase activity’ (GO: 0004672), ‘protein serine/threonine kinase activity’ (GO: 0004674), ‘DNA replication’ (GO: 0006260), ‘protein phosphorylation’ (GO: 0006468), ‘signal transduction’ (GO: 0007165), ‘signaling receptor binding’ (GO: 0005102), and ‘DNA replication initiation’ (GO: 0006270) (Fig. 4E, Table SII). Transcriptome sequencing analysis results indicated that the DEGs associated with PELNs specifically affected the proliferation and migration abilities of CRC cells.