Solanum nigrum-derived nanovesicles as novel nanotherapeutics suppressing prostate cancer progression via senescence-based antitumor activity

doi:10.21203/rs.3.rs-7617628/v1

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Research Article

Solanum nigrum-derived nanovesicles as novel nanotherapeutics suppressing prostate cancer progression via senescence-based antitumor activity

https://doi.org/10.21203/rs.3.rs-7617628/v1

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Background

Current prostate cancer (PCa) therapies often encounter significant challenges due to adverse effects and eventual drug resistance, highlighting the need for novel therapeutic approaches. Here, we first isolated Solanum nigrum-derived nanovesicles (SDNVs) and explored their antitumor effects and underlying mechanisms against prostate cancer.

Methods

SDNVs were isolated via ultracentrifugation combined with sucrose gradient purification and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), as well as protein and RNA analyses. Cellular uptake, antitumor activity, and safety of SDNVs were evaluated in prostate cancer PC-3 cells and normal prostate epithelial RWPE-1 cells. A subcutaneous PC-3 xenograft mouse model was established to determine in vivo efficacy and biodistribution. Transcriptomic sequencing, qPCR, Western blotting, and rescue experiments with the p53 inhibitor PFT-α were performed to clarify the molecular mechanisms.

Results

SDNVs were efficiently internalized by PC-3 cells, significantly inhibiting cell viability, proliferation, and migration while promoting apoptosis. Transcriptomic analysis and experimental validation demonstrated that SDNVs selectively triggered cellular senescence in PC-3 cells through activation of the p53/p21 signaling pathway. Importantly, SDNVs did not induce senescence or cytotoxicity in normal RWPE-1 cells, suggesting favorable tumor-selective properties. Oral administration of SDNVs markedly suppressed tumor growth in vivo, with no obvious toxicity in major organs.

Conclusion

SDNVs are biocompatible, plant-derived nanotherapeutics capable of selectively inhibiting prostate cancer growth through senescence induction. This study supports the further development of SDNVs as a safe and effective antitumor strategy targeting tumor-specific senescence pathways.

plant-derived nanovesicles

senescence

prostate cancer

P53

herbal medicine

Prostate cancer (PCa) remains one of the most common malignancies affecting elderly men worldwide, representing a significant global health challenge. According to the 2022 global cancer statistics report, prostate cancer accounted for approximately 1.4 million new cases and 375,000 deaths, making it a major contributor to cancer-related morbidity and mortality [1]. Although several therapeutic strategies, including chemotherapy, radiation therapy, hormone therapy, surgery, and cryotherapy, are currently used for treating PCa, these approaches are limited by substantial side effects, drug resistance, poor tumor selectivity, and high costs [2, 3]. Therefore, developing novel therapeutic options with higher efficacy, fewer adverse effects, and improved selectivity is urgently needed.

Cellular senescence is a well-established tumor-suppressive mechanism characterized by irreversible growth arrest, increased lysosomal activity, DNA damage accumulation, and secretion of numerous bioactive factors. It is primarily mediated by signaling pathways such as p53/p21 and p16INK4a/Rb [4]. Senescent cells also exhibit hallmark features including elevated lysosomal content detectable by senescence-associated β-galactosidase (SA-βgal) activity, distinct morphological changes, epigenetic alterations such as senescence-associated heterochromatic foci, and nuclear membrane remodeling [5]. In cancer, senescence plays a dual role—suppressing tumor cells while potentially promoting progression when present in the tumor microenvironment [6]. In prostate cancer, p53-mediated senescence is critical for restraining tumor growth and overcoming therapy resistance. Loss of PTEN in prostate epithelium activates a p53-dependent senescence program, preventing malignant transformation [7]. In Pten-deficient mice, early lesions such as prostatic intraepithelial neoplasia exhibit stabilized p53 and growth arrest with senescence features [8]. These findings support senescence induction via p53 as a promising therapeutic strategy in prostate cancer.

Recently, plant-derived nanovesicles (PDNVs) have emerged as a promising class of natural nanoparticles with therapeutic potential [9]. PDNVs are extracellular vesicle-like structures naturally enriched with diverse bioactive components, including microRNAs, proteins, lipids, and secondary metabolites [10]. Their inherent biocompatibility, low immunogenicity, and ability to cross biological barriers have made them attractive candidates for therapeutic development. Emerging studies have demonstrated that PDNVs exhibit beneficial effects in various disease contexts, including inflammatory disorders, cardiovascular disease, and cancer [11–13]. In oncology, PDNVs have shown promise in suppressing tumor progression through multiple mechanisms, such as inducing apoptosis, modulating immune responses, and disrupting angiogenesis [14].

Solanum nigrum is a traditional medicinal herb rich in bioactive compounds such as alkaloids, polyphenols, polysaccharides, and steroidal glycoalkaloids. Its anticancer effects—primarily through inhibiting tumor proliferation and inducing apoptosis—have been documented in various studies [15, 16]. However, the potential of Solanum nigrum-derived nanovesicles (SDNVs) in cancer therapy, particularly for prostate cancer, remains largely unexplored. Here, we propose that SDNVs may selectively suppress prostate cancer progression by inducing tumor cell senescence via the p53/p21 pathway, without affecting normal cells. This work presents the first systematic investigation of SDNVs in prostate cancer and aims to provide a novel therapeutic strategy supported by mechanistic evidence.

2.1 Isolating, purifying, and characterizing SDNVs

The dried Solanum nigrum fruits were homogenized using a blender with an appropriate volume of double-distilled water for 3 minutes to obtain the crude extract. The supernatant was collected through differential centrifugation at 300 ×g for 10 minutes, 2,000 ×g for 20 minutes, and 10,000 ×g for 30 minutes sequentially, with the supernatant retained after each step. The resulting supernatant was subjected to ultracentrifugation at 135,000 ×g for 70 minutes. The pellet was resuspended in 20 mmol/L Tris-HCl buffer to yield a yellow suspension enriched in SDNVs. The crude SDNVs were further purified using a discontinuous sucrose gradient. Sucrose solutions of 15%, 30%, 45%, and 60% (w/v) in double-distilled water were prepared. The sample was layered at the bottom of the centrifuge tube, followed by careful overlaying of each sucrose layer in ascending order. After centrifugation at 150,000 ×g for 2 hours, a visible SDNV-enriched band was collected from the 15–30% interface and subjected to a second round of ultracentrifugation at 150,000 ×g for 2 hours. The final pellet was resuspended in Tris-HCl buffer and filtered through a 0.22 µm sterile membrane to obtain a clarified, sterile SDNV suspension.For morphological assessment, SDNVs were negatively stained and examined by transmission electron microscopy (TEM) using a Tecnai G2 Spirit Twin microscope equipped with a Gatan 832.10W camera. Particle size distribution and concentration of SDNVs were determined using nanoparticle tracking analysis (NTA) on a NanoSight NS300 instrument. Total protein content of SDNVs was determined using a BCA assay kit (Biosharp, BL521A) according to the manufacturer’s instructions. Protein identity and purity were further evaluated using Coomassie brilliant blue staining. Total RNA content was quantified using an Agilent 2100 Bioanalyzer.

2.2 Metabolomics analysis

Metabolomic profiling of SDNVs was performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Briefly, SDNV samples, water decoction of Solanum nigrum (SWD), and selected reference standards (Solasonine) were treated with methanol for protein precipitation, followed by centrifugation to collect the supernatants. These were subsequently analyzed by UHPLC-MS/MS. All analyses were carried out on a Vanquish UHPLC system coupled to an Orbitrap Q Exactive™ HF or HF-X mass spectrometer (Thermo Fisher Scientific, Germany) at Novogene Co., Ltd. (Beijing, China). Raw data were processed using Compound Discoverer software (version 3.3, Thermo Fisher Scientific), including peak extraction, retention time alignment, compound identification, and relative quantification.

2.3 Labeling of SDNVs

For fluorescent labeling, SDNVs (equivalent to 100 mg total protein in 1 mL PBS) were incubated with 1 mL of DiR (Macklin, D909616), DiI (Macklin, D82309), or DiO dye (Zeta Life, DiO-10), each at a concentration of 1 mM in DMSO, at 37°C for 30 minutes. After labeling, excess unbound dye was removed by ultracentrifugation at 135,000 ×g for 70 minutes. The labeled SDNV pellet was washed and resuspended in sterile PBS.

2.4 Cell culture

The human prostate cancer cell line PC-3 and normal human prostate epithelial cell line RWPE-1 were obtained from ICELL (Shanghai, China). PC-3 cells were cultured in RPMI-1640 medium (Gibco, 11875-093) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10099141) and 1% penicillin-streptomycin (Gibco, 15140122). Cells were maintained at 37°C in a humidified incubator with 5% CO₂. RWPE-1 cells were cultured in Keratinocyte Serum-Free Medium (K-SFM) (Gibco, 17005042), supplemented with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL recombinant human epidermal growth factor (EGF), according to the manufacturer's protocol. Cells were maintained under the same incubation conditions. Cells were routinely subcultured at 70–80% confluence and used for experiments within 10 passages.

2.5 uptake of SDNVs by PC-3 cells

To evaluate cellular uptake, PC-3 cells were seeded in 6-well plates or glass-bottom confocal dishes and cultured until 70–80% confluence. For confocal microscopy, cells were incubated with DiI-labeled SDNVs (1 × 10⁹ particles/mL) for 6 hours, then washed with PBS, fixed with 4% paraformaldehyde, and stained with DAPI. Internalization and intracellular localization were visualized using laser scanning confocal microscopy (LSM800, Zeiss, Germany). To assess uptake efficiency, PC-3 cells were incubated with DiO-labeled SDNVs for 0, 1, 3, 6, 12, and 24 hours, respectively. After incubation, cells were harvested, washed, and analyzed by flow cytometry (BD FACSCanto II) using the FITC channel. All experiments were performed in triplicate.

2.6 Cell viability assay

PC-3 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere overnight. Cells were then treated with various concentrations of SDNVs (0, 12, 18, 24, 30, 36, 48 µg/mL) for 24 hours. After treatment, 10 µL of Cell Counting Kit-8 (CCK-8, Dojindo, CK04) solution was added to each well and incubated for 3 hours at 37°C. Absorbance was measured at 450 nm using a microplate reader (BioTek, Synergy H1). Cell viability was calculated relative to the untreated control group. All assays were conducted in triplicate..

2.7 Cell proliferation assay

To further evaluate the antiproliferative effects of SDNVs on PC-3 cells, an EdU incorporation assay was performed using the Cell-Light™ EdU Apollo 488 kit (Beyotime, C0078S). PC-3 cells were seeded into 6-well plates containing sterile coverslips and treated with SDNVs (36 µg/mL) for 24 hours. EdU was diluted 1:500 from a 10 mM stock solution into complete culture medium to prepare a 2× working solution (20 µM), which was added in equal volume to the wells to yield a final concentration of 10 µM EdU. Cells were incubated at 37°C for 2 hours. After incubation, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, then washed three times with PBS containing 3% bovine serum albumin (BSA). Permeabilization was performed using 0.3% Triton X-100 in PBS for 15 minutes, followed by another PBS/BSA wash. The “click” reaction mixture was freshly prepared by combining the following components per 1 mL: 860 µL Click Reaction Buffer, 40 µL CuSO₄, 2 µL Azide 488, and 100 µL Click Additive Solution. A total of 100 µL of the reaction cocktail was added to each well and incubated in the dark at room temperature for 30 minutes. After three washes, nuclear staining was performed using Hoechst 33342 (1:1000 dilution in PBS) for 10 minutes. EdU-positive cells (green fluorescence) and total nuclei (blue fluorescence) were visualized using a fluorescence microscope. The proliferation rate was calculated as the ratio of EdU-positive cells to total nuclei. All experiments were performed in triplicate.

2.8 Cell migration assay

The effect of SDNVs on PC-3 cell migration was evaluated using a Transwell assay. PC-3 cells were harvested using 0.25% trypsin, washed, and resuspended in serum-free medium. A total of 1.5 × 10⁴ cells in 100 µL serum-free medium were seeded into the upper chamber of a Transwell insert (8.0 µm pore size; Corning, USA). The lower chamber was filled with 400 µL of complete medium containing 10% fetal bovine serum as a chemoattractant. The plates were incubated at 37°C in a humidified incubator with 5% CO₂ for 12 hours. After incubation, non-migrated cells on the upper surface of the membrane were gently removed with a cotton swab. The inserts were rinsed twice with PBS, and the migrated cells on the lower surface were fixed with 4% paraformaldehyde for 10 minutes. After washing with distilled water, cells were stained with 1% crystal violet solution (filtered through a 0.45 µm filter) for 10 minutes, followed by PBS washes to remove excess stain. Migrated cells were imaged and quantified under an inverted microscope (Olympus CKX53). Five random fields per membrane were captured and counted to evaluate the average number of migrated cells. All experiments were conducted in triplicate.

2.9 Cell apoptosis assay

Apoptosis of PC-3 cells was assessed using an Annexin V-AbFluor™ 488/PI apoptosis detection kit (Abbkine, KTA0002) according to the manufacturer’s protocol. After treatment with SDNVs (36 µg/mL) for 24 hours, cells were harvested using trypsin without EDTA and centrifuged at 300 ×g for 5 minutes at 4°C. The cell pellet (1–2 × 10⁵ cells) was washed twice with pre-chilled PBS and resuspended in 1× binding buffer. Annexin V-AbFluor™ 488 and propidium iodide (PI) were added to the cell suspension and incubated for 15 minutes in the dark at room temperature. Samples were analyzed within 30 minutes using flow cytometry (BD FACSCanto II). The percentage of apoptotic cells (early + late) was quantified. All experiments were conducted in triplicate.

2.10 Nude mouse xenograft model:

Male BALB/c nude mice (8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under specific pathogen-free (SPF) conditions with ad libitum access to food and water. After a one-week acclimatization period, each mouse was subcutaneously injected with 1 × 10⁷ PC-3-Red-Fluc prostate cancer cells suspended in 200 µL of PBS into the right flank. When the tumor volume reached approximately 20 mm³, mice were randomly divided into four groups (n = 6 per group): (1) Control group: gavaged with 100 µL PBS; (2) Low-dose SDNV group: gavaged with 100 µL of SDNV suspension (0.18 µg/µL); (3) Medium-dose SDNV group: gavaged with 100 µL of SDNV suspension (0.54 µg/µL); (4) High-dose SDNV group: gavaged with 100 µL of SDNV suspension (1.62 µg/µL). Oral administration was performed once daily for 28 consecutive days. At the end of the treatment period, mice were euthanized, and tumor tissues were excised, weighed, and processed for subsequent histological and molecular analyses. Tumor volume was measured every three days using a caliper and calculated with the formula: V = ½ × length × width².

2.11 Tumor histopathology and immunohistochemical analysis

Hematoxylin and eosin (H&E) staining was performed to evaluate tumor architecture, necrosis, and cellular morphology. Apoptotic cell death was assessed using the TUNEL assay. Cell proliferation was evaluated via immunohistochemical staining for Ki-67. After deparaffinization and rehydration, antigen retrieval was performed in citrate buffer (pH 6.0) at 95°C for 15 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, followed by incubation with anti-Ki-67 primary antibody (Servicebio, GB111499; 1:500 dilution) overnight at 4°C. After incubation with HRP-conjugated secondary antibody, staining was developed using DAB substrate, and sections were counterstained with hematoxylin. Positive Ki-67 staining was visualized as brown nuclear staining and quantified as the percentage of positive nuclei among total nuclei in five random fields.

2.12 Biodistribution of Orally Administered SDNVs

The experimental approach employed for assessing the in vivo distribution of PDNVs builds upon prior research conducted by our team [12]. When tumors reached approximately 20 mm³, DiR-labeled SDNVs were administered by oral gavage once daily at a dose of 1 × 10¹¹ particles/kg for 7 consecutive days. Six hours after the final gavage, the animals were euthanized, and major organs—including the heart, liver, spleen, lungs, kidneys, and tumor—were harvested for ex vivo fluorescence imaging. Tissue fluorescence was visualized and quantified using a Tanon ABLX3 in vivo imaging system (Tanon, Shanghai, China). The intensity of DiR signal in different organs was used to evaluate the biodistribution pattern of orally administered SDNVs.Tissue

2.13 Biosafety evaluation of Orally Administered SDNVsAt the end of the treatment period, major organs including the heart, liver, spleen, lungs, and kidneys were harvested from each mouse, rinsed with PBS, and fixed in 4% paraformaldehyde for 24 hours. Fixed tissues were dehydrated, embedded in paraffin, and sectioned at 5 µm thickness. Hematoxylin and eosin (H&E) staining was performed following standard protocols. Histopathological changes were examined under a light microscope to evaluate potential tissue toxicity.To assess systemic biosafety, body weight of mice was recorded To To assess systemic biosafety, body weight of mice was recorded every 3 days during the 4-week treatment period. Blood samples were collected by cardiac puncture immediately after euthanasia. Serum was isolated by centrifugation at 1,500 ×g for 15 minutes and subjected to biochemical analysis using an automatic biochemical analyzer to measure liver and kidney function markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE).

2.14 Transcriptomic analysis of SDNV-treated cells

Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA libraries were constructed using the NEBNext® Ultra™ Directional RNA Library Prep Kit (NEB, USA) and sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads). Raw reads were quality-filtered using fastp and mapped to the human reference genome (GRCh38) using HISAT2. Gene expression was quantified using featureCounts and normalized to fragments per kilobase per million mapped reads (FPKM). Differential expression analysis was conducted with the DESeq2 R package. Genes with |log₂(fold change)| ≥ 1 and adjusted p-value < 0.05 were considered significantly differentially expressed. KEGG pathway enrichment analysis was performed using clusterProfiler (v4.6.2). Gene Set Enrichment Analysis (GSEA) was conducted using the GSEA software (v4.3.2) with the MSigDB c2.cp.kegg.v7.5.1 gene set database.

2.15 β-Galactosidase Staining Assay

Senescence-associated β-galactosidase (SA-β-gal) staining was performed to evaluate the induction of cellular senescence in PC-3 cells following SDNV treatment. Cells were seeded into 6-well plates and treated with SDNVs (50 µg/mL) or PBS (control) for 24 hours. After treatment, the culture medium was removed, and cells were washed once with PBS. Cells were then fixed with 1 mL of β-galactosidase fixative solution (Beyotime, C0602) for 15 minutes at room temperature. Following fixation, cells were washed three times with PBS (3 minutes per wash), and 1 mL of freshly prepared staining working solution was added to each well. The staining solution was composed of 10 µL of solution A, 10 µL of solution B, 930 µL of solution C, and 50 µL of X-Gal stock solution (20 mg/mL). Plates were sealed with parafilm and incubated overnight at 37°C in a dry incubator (without CO₂). The following day, SA-β-gal-positive cells exhibiting blue-green cytoplasmic staining were observed under a light microscope. Senescent cells were quantified in five randomly selected fields per well. Experiments were performed in triplicate.

2.16 Quantitative real-time PCR

Total RNA was extracted from PC-3 cells using Total RNA Extraction Reagent (Vazyme, R401-01) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using a reverse transcription premix kit (AG11728, Accurate Biology, China). Quantitative real-time PCR (qPCR) was performed using the SYBR Green Pro Taq HS Premixed qPCR Kit (AG11701, Accurate Biology, China) on a LightCycler 480 system (Roche, Switzerland). The expression levels of senescence-related genes including TP53, P21, and P16 were analyzed. ACTB was used as the internal reference. Relative gene expression was calculated using the 2⁻^ΔΔCt^ method. Each sample was analyzed in triplicate, and data were expressed as mean ± standard deviation (SD). The primer sequences are listed in Additional file 1: Table S1.

2.17 Western Blot Analysis

Total protein was extracted using RIPA lysis buffer (Beyotime, P0013B) supplemented with protease and phosphatase inhibitors (Beyotime, P1045). Protein concentration was determined using a BCA Protein Assay Kit (Biosharp, BL521A). Equal amounts of protein (20–30 µg) were separated by SDS-PAGE on 10% polyacrylamide gels and transferred onto PVDF membranes (Millipore, IPVH00010). Membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 hour and then incubated overnight at 4°C with primary antibodies: anti-P53 (ZENBIO, 345567, 1:1000), anti-P21 (ZENBIO, 381102, 1:1000), and anti-GAPDH (Proteintech, 60004-1-Ig, 1:5000). After washing, membranes were incubated with HRP-conjugated secondary antibodies (EARTH, E030110-01, 1:10000; HUABIO, HA1001, 1:10000) for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Biosharp, BL520A), and images were captured using a Tanon 5200 imaging system. The relative expression levels of target proteins were quantified by densitometric analysis using ImageJ software and normalized to GAPDH.

2.18 Statistical Analysis

All experimental data are expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism software (version 9.0, GraphPad Software, USA). Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests, while one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for comparisons among three or more groups. A p-value < 0.05 was considered statistically significant. Significance levels in figures are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and ns for non-significant differences (p > 0.05).

3.1 Isolation and characterization of SDNVs

Nanovesicles were successfully isolated from Solanum nigrum fruits using an optimized protocol combining differential ultracentrifugation with sucrose gradient centrifugation. Under TEM, these vesicles exhibited typical round or oval morphology with bilayer membranes and diameters between 50 and 120 nm (Fig. 1A). NTA analysis showed a narrow size distribution with an average diameter of 93.4 ± 0.2 nm and particle concentration of (4.22 ± 0.43) × 10¹¹/mL (Fig. 1B). Coomassie Brilliant Blue staining demonstrated distinct protein bands primarily distributed in the 15–35 kDa range (Fig. 1C), indicating that SDNVs carry diverse endogenous proteins. Agilent Bioanalyzer analysis confirmed the presence of small RNAs in SDNVs (Fig. 1D). In addition, metabolomic profiling via LC-MS revealed that the chemical fingerprint of SDNVs was clearly different from both the water decoction of S. nigrum and purified solanine (Fig. 1E), suggesting that SDNVs may represent a distinct, bioactive fraction of the plant not captured by traditional extraction methods. Together, these findings demonstrate that SDNVs are nanoscale, bilayered vesicles enriched with functional proteins, small RNAs, and unique metabolites, laying the foundation for further biological evaluation.

3.2 In vivo biodistribution of orally administered SDNVs

To evaluate the biodistribution of SDNVs following oral administration, DiR-labeled SDNVs (DiR-SDNVs) were gavaged into male nude mice. Compared with the PBS group, the fluorescence intensity of DiR-SDNV group in the liver, spleen, tumor and lungs was significantly increased. This indicates that SDNVS can be absorbed into the blood through the gastrointestinal tract and enter various tissues and organs (Fig. 2). At the same time, it has a certain tendency to accumulate in tumors, suggesting that SDNVS has a targeted regulatory effect on tumor intervention.

3.3 SDNVs inhibit tumor growth in a xenograft nude mouse model

To evaluate the in vivo antitumor efficacy of SDNVs, a prostate cancer xenograft mouse model was established, and tumor growth was assessed at both macroscopic and microscopic levels. As shown in the gross morphology of subcutaneous tumors, obvious differences in tumor size were observed between the model group and the groups treated with different doses of SDNVs (low [L], medium [M], and high [H]) (Fig. 3A). Quantitative analysis of tumor volume revealed that SDNV treatment significantly reduced tumor growth compared to the model group, although no clear dose-dependent trend was observed (Fig. 3B). Consistently, tumor weight measurements showed a similar reduction pattern (Fig. 3C), further supporting the in vivo antitumor potential of SDNVs.

H&E staining revealed that tumors in the model group exhibited high cellular density, disorganized architecture, and prominent nuclear atypia. In contrast, SDNV-treated tumors displayed decreased cell density, nuclear pyknosis, and more orderly arrangement, indicating a reduced malignancy, with the effect becoming more pronounced at higher doses (Fig. 4A). To further explore the underlying mechanism, TUNEL staining was performed to assess tumor apoptosis. The results demonstrated a significant increase in apoptotic cells in the SDNV-treated groups, particularly in the medium- and high-dose groups, suggesting that SDNVs effectively induced programmed cell death in prostate cancer cells (Fig. 4B). In addition, Ki-67 immunohistochemical staining was used to evaluate tumor cell proliferation. Ki-67-positive cells were widely distributed in the model group, whereas their expression was markedly reduced following SDNV treatment, indicating potent anti-proliferative activity (Fig. 4C). These results indicate that SDNVs effectively suppress prostate tumor growth in vivo by promoting apoptosis and reducing proliferation.

3.4 Uptake and Internalization of SDNVs by PC-3 Cells

To further investigate the antitumor effects of SDNVs at the cellular level, we examined their uptake and intracellular distribution in PC-3 prostate cancer cells. SDNVs were labeled with the fluorescent dyes DiI and DiO to generate DiI-SDNVs and DiO-SDNVs, respectively. Confocal laser scanning microscopy revealed distinct red fluorescence within the cytoplasm of PC-3 cells after incubation with DiI-SDNVs, indicating efficient cellular internalization (Fig. 5A). To quantitatively assess the uptake kinetics, DiO-SDNVs were incubated with PC-3 cells and analyzed by flow cytometry at various time points. The results showed that cellular uptake began as early as 3 hours and increased progressively over time, displaying a clear time-dependent pattern (Fig. 5B). By 24 hours, nearly all PC-3 cells had internalized DiO-SDNVs. These findings demonstrate that SDNVs are efficiently and stably taken up by PC-3 cells in a time-dependent manner, providing a cellular basis for their subsequent antitumor activity.

3.5 In Vitro Antitumor Effects of SDNVs on PC-3 Cells

Although SDNVs exhibited clear tumor-inhibitory effects in vivo, we further conducted in vitro experiments to determine whether these effects were due to a direct action on cancer cells and to clarify the specific cellular processes involved. By examining PC-3 cell viability, proliferation, migration, and apoptosis, we aimed to define the functional basis of SDNV-mediated tumor suppression. CCK-8 assays showed that SDNVs significantly reduced PC-3 cell viability in a dose-dependent manner. At a concentration of 36 µg/mL, viability decreased to approximately 58.64% (Fig. 6A). EdU incorporation assays further confirmed this antiproliferative effect, with a marked reduction in DNA synthesis in SDNV-treated cells compared to controls (Fig. 6B). In addition, transwell migration assays revealed a significant inhibition of cell motility following SDNV treatment, suggesting a suppressive effect on invasive potential (Fig. 6C). Flow cytometry analysis using Annexin V-FITC/PI staining demonstrated increased apoptosis in the treated group (Fig. 6D–E), indicating that SDNVs also activate programmed cell death pathways. These in vitro findings complement the in vivo results and demonstrate that SDNVs directly inhibit prostate cancer cell growth through a combination of antiproliferative, antimigratory, and pro-apoptotic effects.

3.6 Transcriptomic analysis suggests activation of p53/p21-associated senescence pathways

To explore the molecular mechanisms underlying the antitumor effects of SDNVs, we performed transcriptomic profiling of PC-3 cells treated with SDNVs. Principal component analysis (PCA) showed that SDNV-treated samples clustered closely together, indicating good reproducibility and a distinct transcriptional profile compared to controls (Fig. 7A). Differential expression analysis identified 5,058 differentially expressed genes (DEGs), including 2,491 upregulated and 2,567 downregulated transcripts (Fig. 7B). A volcano plot highlighted significant gene expression shifts, while unsupervised hierarchical clustering revealed clear segregation between SDNV-treated and control groups, reflecting widespread transcriptional reprogramming (Fig. 7C). KEGG pathway enrichment analysis demonstrated that DEGs were significantly associated with cellular senescence, the p53 signaling pathway, and prostate cancer-related pathways (Fig. 7D). Consistently, gene set enrichment analysis (GSEA) confirmed the activation of both the cellular senescence program and the p53 pathway in SDNV-treated cells. These results suggest that SDNVs exert their antitumor effects, at least in part, by activating the p53-depended senescence in prostate cancer cells. Further experiments were designed to validate these transcriptional changes at the phenotypic and protein levels.

3.7 SDNVs induce cellular senescence in PC-3 cells via the p53/p21 pathway

Based on transcriptomic sequencing and pathway enrichment analysis, SDNVs were hypothesized to exert antitumor effects through the induction of cellular senescence. To validate this, a senescence-associated β-galactosidase (SA-β-gal) staining assay was conducted. Compared with the NC group, SDNV-treated PC-3 cells exhibited markedly increased blue staining and a significantly higher proportion of SA-β-gal–positive cells, indicating the induction of a senescent phenotype (Fig. 8A).To further assess pathway activation, quantitative PCR was performed to measure the mRNA levels of P53 and its downstream senescence-related targets P21 and P16. SDNV treatment significantly upregulated the transcription of all three genes (Fig. 8B), suggesting that the P53 signaling pathway plays a central regulatory role in SDNV-induced senescence.Consistently, Western blot analysis confirmed increased protein levels of P53 and P21 in the SDNV-treated group compared to the NC group (Fig. 8C), further supporting activation of the P53 pathway at the translational level.

3.8 PFT-α inhibits SDNV-induced senescence via p53 pathway suppression

To further confirm whether SDNV-induced cellular senescence in PC-3 cells is mediated through the P53 signaling pathway, we employed PFT-α (Pifithrin-α), a well-established reversible inhibitor of P53-dependent transcriptional activity. PC-3 cells were co-treated with SDNVs and PFT-α (20 µM), and senescence-associated β-galactosidase (SA-β-gal) staining was used to assess the senescence phenotype. Compared to the SDNV-only group, the SDNVs + PFT-α group exhibited a marked reduction in blue-stained senescent cells under light microscopy, indicating that PFT-α effectively attenuated the pro-senescent effect of SDNVs (Fig. 9A).To investigate whether this effect was mediated through suppression of P53 downstream targets, we examined the expression levels of key pathway components. qPCR analysis revealed that mRNA levels of P21 and P16 were significantly reduced in the SDNVs + PFT-α group compared to the SDNVs group (Fig. 9B). Similarly, Western blot analysis showed decreased protein expression of P53 and P21 following PFT-α treatment (Fig. 9C).These results demonstrate that inhibition of P53 by PFT-α partially reverses SDNV-induced cellular senescence and suppresses the transcription and translation of key downstream genes in the P53/P21 pathway.

3.9 In Vivo and In Vitro Safety Evaluation of SDNVs

To assess the biosafety of SDNVs, both in vivo and in vitro toxicity evaluations were performed. In the in vivo setting, mice were orally administered SDNVs at varying doses. Hematoxylin and eosin (H&E) staining of major organs—including the heart, lung, liver, spleen, and kidney—showed no observable pathological changes or tissue damage across all dose groups, indicating that SDNVs did not induce histological toxicity (Fig. 10A). Furthermore, serum levels of liver and kidney injury markers, including CR, ALT, and AST, remained within normal ranges following SDNV administration, with no statistically significant differences observed compared to the control group (Fig. 10B). These findings confirm that SDNVs do not cause hepatotoxicity or nephrotoxicity in vivo. In vitro safety was evaluated using RWPE-1 normal human prostate epithelial cells. CCK-8 assay results showed that SDNVs had no significant effect on cell viability (Fig. 10C). Additionally, qPCR analysis demonstrated that the mRNA expression levels of senescence-related genes—P53, P21, and P16—were not significantly altered following SDNV treatment (Fig. 10D).Together, these results demonstrate that SDNVs exhibit good biocompatibility, showing no observable toxicity in major organs or normal prostate epithelial cells, thereby supporting their potential as a safe nanotherapeutic platform for further application.

This study provides the first evidence that SDNVs exert selective antitumor effects against prostate cancer. Orally administered SDNVs significantly inhibited tumor growth and induced cellular senescence in PC-3 cells without affecting normal prostate epithelial cells. Mechanistic analysis revealed activation of the p53/p21 pathway as a key mediator. These findings highlight SDNVs as a safe and biocompatible nanoplatform with promising therapeutic potential in prostate cancer.

Plant-derived nanovesicles (PDNVs) have emerged as promising therapeutic agents owing to their intrinsic biocompatibility, low immunogenicity, and ability to carry diverse bioactive components. Recent studies have demonstrated that PDNVs derived from edible or medicinal plants can exert antitumor effects through various mechanisms, including induction of apoptosis, suppression of epithelial-mesenchymal transition, and modulation of the immune response. For instance, ginger-derived nanovesicles were shown to induce mitochondrial damage and trigger apoptosis in breast cancer cells [17]. Aloe vera-derived vesicles have been reported to promote pyroptotic cell death and activate immune pathways in colon cancer models [18]. In addition, citrus- and tomato-derived vesicles have demonstrated the ability to cross biological barriers and accumulate in tumor tissues following oral administration, underscoring their translational potential for drug delivery [19]. Despite these advances, few studies have explored the role of PDNVs in prostate cancer, and none have focused on cellular senescence as a therapeutic mechanism. Most existing approaches emphasize cytotoxicity-based interventions, while strategies that selectively activate tumor suppressive senescence pathways remain underdeveloped. In this study, our findings provide the first evidence that Solanum nigrum-derived nanovesicles (SDNVs) can inhibit prostate cancer progression by inducing p53/p21-mediated cellular senescence without affecting normal cells. This non-cytotoxic mode of action represents a novel perspective in PDNV-based therapy, potentially reducing adverse effects associated with traditional chemotherapeutics.

Cellular senescence is characterized by stable, irreversible cell-cycle arrest triggered by various stresses such as DNA damage, oxidative stress, or oncogenic signaling. Key features include activation of tumor-suppressive pathways, particularly p53/p21 and p16/Rb, elevated SA-β-gal activity, chromatin remodeling, and the secretion of SASP factors [4, 20]. In cancer biology, cellular senescence plays dual roles: initially restricting tumor progression through growth arrest and immune-mediated clearance, but potentially driving tumorigenesis through prolonged SASP-induced inflammation and immune evasion [6]. Recent studies have identified PDNVs as novel regulators of senescence in various biological systems [21]. Notably, Semen Sinapis alba-derived nanovesicles have been shown to alleviate endothelial cell senescence and vascular remodeling in spontaneously hypertensive rats by delivering miR393a to suppress CD38 expression and downregulate the p53/p21 axis [13]. Similarly, vesicles isolated from Ecklonia cava reduced oxidative stress and markers of aging by upregulating HSP70 and downregulating TNF-α, MAPK, and NF-κB expression in keratinocytes and aging mouse models [22]. Exosomes from the medicinal fungus Phellinus linteus have also demonstrated protective effects against UV-induced skin aging [23]. In our previous research, nanovesicles derived from Dendrobium were found to enhance wound healing, likely by mitigating cellular senescence [24, 25].

In this study, transcriptomic analysis highlighted significant activation of senescence-related genes and the p53 signaling pathway in SDNV-treated PC-3 cells. Functional validation confirmed typical senescence phenotypes, including increased SA-β-gal activity, altered cell morphology, and upregulated expression of p53, p21, and p16. The tumor-suppressive role of p53 is well documented, with loss of its function being closely linked to aggressive behavior, recurrence, and poor prognosis in prostate cancer [26]. Both p21 and p16 serve as key cyclin-dependent kinase inhibitors that mediate G1-phase arrest and enforce stable senescence [27, 28]. Importantly, pharmacological inhibition of p53 using PFT-α significantly reversed the senescent phenotype, indicating that SDNVs exert their effects primarily through activation of the p53/p21 axis. To our knowledge, this is the first direct demonstration that PDNVs can induce therapy-related senescence in cancer cells, offering a non-cytotoxic mechanism of action and broadening the potential application of PDNVs in oncological treatment strategies.

Although our findings provide strong evidence for the antitumor activity of SDNVs through induction of cellular senescence, several limitations should be acknowledged. Firstly, the exact bioactive components within SDNVs responsible for triggering senescence remain unidentified. Metabolomic analyses indicated unique chemical profiles distinct from traditional aqueous extracts, yet further studies are necessary to pinpoint active metabolites or functional RNAs contributing to the observed effects. Secondly, our animal model utilized subcutaneous xenografts, which do not fully replicate the tumor microenvironment, immune interactions, or metastatic features typical of human prostate cancer. Future studies incorporating orthotopic or genetically engineered mouse models would provide more clinically relevant insights. Finally, detailed investigation into the interactions between senescent cancer cells and immune components—particularly regarding the SASP—is warranted to better understand how SDNV-induced senescence may influence tumor progression in immunocompetent hosts. Despite the aforementioned limitations, our findings highlight SDNVs as a promising candidate for clinical translation due to their selective antitumor activity, oral bioavailability, and excellent biosafety profile. Unlike conventional cytotoxic therapies, SDNV-mediated induction of cellular senescence provides a non-lethal strategy that could reduce the adverse effects commonly associated with chemotherapy and radiotherapy.

In summary, our study demonstrates for the first time that SDNVs selectively inhibit prostate cancer progression by inducing cellular senescence through the p53/p21 signaling pathway. Oral administration of SDNVs effectively suppressed tumor growth in vivo and exhibited excellent safety and biocompatibility, highlighting their potential as a novel, non-cytotoxic therapeutic strategy. These findings expand the therapeutic applications of plant-derived nanovesicles and provide a foundation for future clinical translation in prostate cancer treatment.

SDNVs

Solanum nigrum-derived nanovesicles

PDNVs

plant-derived nanovesicles

PCa

prostate cancer

TEM

transmission electron microscopy

NTA

nanoparticle tracking analysis

RNA

ribonucleic acid

qPCR

quantitative polymerase chain reaction

SA-β-gal

senescence-associated β-galactosidase

SASP

senescence-associated secretory phenotype

UHPLC-MS/MS

ultra-high-performance liquid chromatography–tandem mass spectrometry

PBS

phosphate-buffered saline

DMSO

dimethyl sulfoxide

CCK-8

Cell Counting Kit-8

EdU

5-ethynyl-2'-deoxyuridine

BSA

bovine serum albumin

DAPI

4′,6-diamidino-2-phenylindole

propidium iodide

HRP

horseradish peroxidase

ECL

enhanced chemiluminescence

PVDF

polyvinylidene difluoride

SDS-PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

FBS

fetal bovine serum

H&E

hematoxylin and eosin

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CRE

creatinine

FPKM

fragments per kilobase of exon per million mapped reads

DEGs

differentially expressed genes

KEGG

Kyoto Encyclopedia of Genes and Genomes

GSEA

Gene Set Enrichment Analysis

PCA

principal component analysis

negative control

PFT-α

pifithrin-α

ANOVA

analysis of variance

standard deviation.

Data availability

All data necessary to evaluate the conclusions in the paper are presented within the paper and/or the Supplementary Materials. Additional data can be requested from the authors.

Acknowledgments

This study was financially supported by the Natural Science Foundation of Guangdong Province of China (2025A1515010438, 2022A1515011710), the China Postdoctoral Science Foundation (2024M762709, 2025T180649), the Science and Technology Project of Shenzhen (JCYJ20240813153620027, JCYJ20240813153619026, JCYJ20240813113011016) , the Sanming Project of Medicine in Shenzhen (SZZYSM202206001), Shenzhen Bao'an High-Quality Development Project (YNXM2024079, YNXM2024078) , the Shenzhen Bao’an Chinese Medicine Hospital Research Program (Grant No. BAZYY20220702), Guangzhou University of Traditional Chinese Medicine " Guben " project first-level discipline ability improvement project (ZY2025GB0929), and the Huixin Lifetech Chinese Herbal Medicine-derived Extracellular Vesicles Research Project.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Author contribution statement

Miao Gan: Conceptualization, Methodology, Investigation, Writing – Original Draft, Visualization; Yufei Feng: Data Curation, Validation, Formal Analysis, Writing – Review & Editing; Luhua Xu: Methodology, Investigation, Software, Visualization; Zetao Chen: Validation, Resources, Investigation; Fanjia Zhong: Data Curation, Resources, Project Administration; Na Liu: Formal Analysis, Investigation; Jiamei Huang: Software, Data Curation; Huizhen Zhang: Resources, Supervision; Chao Yang: Conceptualization, Methodology, Supervision, Writing – Review & Editing; Rongfeng Yang: Conceptualization, Writing – Original Draft, Supervision, Funding Acquisition; Fengxia Lin: Project Administration, Funding Acquisition, Writing – Review & Editing, Supervision.

Informed consent

Not applicable.

Ethics statement

The animal experiments were conducted in accordance with the ethical standards of Shenzhen Glorybe Biotechnology Co., Ltd. and were approved by the Animal Ethics Committee of Shenzhen Glorybe Biotechnology Co., Ltd. (Approval No.: RW-IACUC-24-0021).

Use of AI statement

Not applicable.

Electronic Supplementary Material

The Electronic Supplementary Material for this manuscript includes Table S1, detailing the primer sequences used for quantitative RT-PCR analysis.

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GraphicalAbstract.png
Graphical Abstract The graphical abstract summarizes how Solanum nigrum-derived nanovesicles induce senescence in prostate cancer through activation of the P53/P21 signaling pathway (By Figdraw).
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Solanum nigrum-derived nanovesicles as novel nanotherapeutics suppressing prostate cancer progression via senescence-based antitumor activity

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1 Isolating, purifying, and characterizing SDNVs

2.2 Metabolomics analysis

2.3 Labeling of SDNVs

2.4 Cell culture

2.5 uptake of SDNVs by PC-3 cells

2.6 Cell viability assay

2.7 Cell proliferation assay

2.8 Cell migration assay

2.9 Cell apoptosis assay

2.10 Nude mouse xenograft model:

2.11 Tumor histopathology and immunohistochemical analysis

2.12 Biodistribution of Orally Administered SDNVs

2.14 Transcriptomic analysis of SDNV-treated cells

2.15 β-Galactosidase Staining Assay

2.16 Quantitative real-time PCR

2.17 Western Blot Analysis

2.18 Statistical Analysis

3. Results

3.1 Isolation and characterization of SDNVs

3.2 In vivo biodistribution of orally administered SDNVs

3.3 SDNVs inhibit tumor growth in a xenograft nude mouse model

3.4 Uptake and Internalization of SDNVs by PC-3 Cells

3.5 In Vitro Antitumor Effects of SDNVs on PC-3 Cells

3.6 Transcriptomic analysis suggests activation of p53/p21-associated senescence pathways

3.7 SDNVs induce cellular senescence in PC-3 cells via the p53/p21 pathway

3.8 PFT-α inhibits SDNV-induced senescence via p53 pathway suppression

3.9 In Vivo and In Vitro Safety Evaluation of SDNVs

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1