Extracellular vesicle delivery of Myricetin suppresses ovarian cancer through mitochondrial dynamics by downregulating the ECM1/NF-κB/TGFβ signaling pathway.

CHO cells were maintained in a sterile environment in a large population (Fig.  1 a), and the EVs released from these cells were isolated to obtain the pellet (Fig.  1 b) were characterized by its size, stability, morphology, and EVs proteins in accordance with guidelines proposed by the MISEV 2023 42 . The Malvern Zeta Sizer instrument was utilized to assess the size and zeta potential of CE. We observed that the average size of the CE was 163.9 nm (Fig.  1 c), and the polydispersity index of CE was 0.199. The stability of CE was determined by the zeta potential of -24.4 mV and a conductivity of 1.12 mS/cm before loading of MYR (Fig.  1 d). We observed the spherical morphology of the CE under SEM with an intact lipid bilayer (Fig.  1 e). In addition, the expression of Alix and ICAM-1, were detected by western blotting from CE (Fig.  1 f). The cytosolic protein Alix, present in EVs, plays a role in cargo packaging in EVs and ICAM-1 serves as a EVs surface protein involved in intercellular communication 43 . Fig. 1 ( a ) CHO cells. Scale bar: 10X. ( b ) CE pellet. Isolation and characterization of CE ( c ) Malvern Zetasizer measurement of the CE depicting the size distribution in nm before loading of MYR. ( d ) Zeta potential of the CE in mV before loading of MYR. ( e ) SEM image of the CE before loading of MYR. Scale bar: 300 nm. ( f ) EVs protein markers ICAM-1 and Alix present in the CE. ( a ) CHO cells. Scale bar: 10X. ( b ) CE pellet. Isolation and characterization of CE ( c ) Malvern Zetasizer measurement of the CE depicting the size distribution in nm before loading of MYR. ( d ) Zeta potential of the CE in mV before loading of MYR. ( e ) SEM image of the CE before loading of MYR. Scale bar: 300 nm. ( f ) EVs protein markers ICAM-1 and Alix present in the CE. MYR is a polyphenolic compound characterized by hexahydroxyl groups with fused benzene rings that interact through hydrogen bonds (Fig.  2 a). The properties determined by the MYR structure showed a molecular formula (C 15 H 10 O 8 ), weight 318.23 g/mol, molar refractivity 75.2 ± 3 cm 3 , molar volume 166.4 ± 3 cm 3 , index of refraction 1.863 ± 0.02, surface tension 133.0 ± 3 dyne/cm, and density 1.912 ± 0.06 g/cm 3 . Exogenous loading in engineered EVs allows the incorporation of specific compounds into isolated EVs via different techniques 44 . CE were loaded with MYR via the sonication method (Fig.  2 b). Fig. 2 ( a ) Chemical structure of MYR together with its properties. ( b ) Diagrammatic representation of drug loading in EVs via the sonication method. ( c ) FTIR spectra of CE, MYR, and CE + MYR. ( a ) Chemical structure of MYR together with its properties. ( b ) Diagrammatic representation of drug loading in EVs via the sonication method. ( c ) FTIR spectra of CE, MYR, and CE + MYR. The spectral peaks observed via FTIR analysis facilitated the identification of the chemical structure of the distinct functional groups present in the CE and MYR. The primary FTIR peaks of MYR were typically found approximately 1028 cm -1 (C-O stretching vibrations), 1159 cm -1 (C–O–C vibrations), 1271 cm -1 (C-O stretching and C–OH bending), 1326 cm -1 (O–H in-plane bending vibrations), 1375 cm -1 (C–C stretching), 1450 cm -1 (C–C stretching of the benzene ring), 1555 cm -1 (C = C stretching), 1648 cm -1 (R–C(= O)-NR stretching vibration), and 1671 cm -1 (R–C(= O)-R stretching vibration) 45 , 46 . It was observed that the infrared spectrum for CE and MYR shows variations in the transmittance peaks across the 400 cm -1 to 1800 cm -1 range, which is referred to as the “Fingerprint” region, as it is unique to each molecule. Peaks were observed around 2924 cm -1 (C-H stretching vibration), 3301 cm -1 (R-NH 2 vibration), 3420 cm -1 (O–H stretching vibration), and the wavenumbers between 2800 cm -1 and 3600 cm -1 , indicating hydrogen bonding in all three groups (CE, MYR, and CE + MYR). Following drug incorporation, MYR peaks between 1300 and 4000 cm -1 were masked, indicating its encapsulation within the CE. Conversely, certain peaks in CE + MYR displayed an overlay combination of peaks specific to MYR and CE in the range of 800–1300 cm -1 in their fingerprint region, indicating that the peaks of both components to remained identifiable (Fig.  2 c). The transmittance peak of CE + MYR at the carbon–oxygen bond at approximately 1028 cm -1 , 1271 cm -1 , and 1321 cm -1 was lower than that of the CE spectrum because of the hydrogen bond formation with the phenol group of MYR. The region between 800 cm -1 and 1500 cm -1 showed a shift in the CE + MYR spectra. Therefore, the FTIR spectrum of CE + MYR resembled that of CE, with certain transmittance peaks of MYR incorporated by the EVs. MYR enclosed in the CE can be transmitted to the recipient cells (Fig.  3 a). To analyze the concentration of MYR present in CE, RP-HPLC was performed. The encapsulation efficiency was 46.419%, and the drug loading capacity was 15.473%, confirming the amount of MYR incorporated inside the CE (Fig.  3 b). EE indicated a substantial amount of MYR is shielded by CE and can be easily delivered to the target cells. DLC shows the ability of CE to hold MYR and release it to ovarian cells. Our study revealed an increase in the average size of the CE from 163.9 nm to 187.8 nm after the loading of MYR inside the CE (Fig.  3 c). The polydispersity index of MYR-loaded CE was 0.179. The zeta potential decreased from -24.4 mV to -24.3 mV, and a conductivity of 1.12 mS/cm because of the mechanical shear force formed by the sonicator probe (Fig.  3 d). Moreover, the SEM image depicted the increased size of the CE by MYR incorporation without distorted morphology of EVs (Fig.  3 e). The incubation of DiO-labeled CE with ovarian cells displayed green fluorescence near the cell membrane, which indicates the communication of CE with the target cells (Fig.  3 f). Collectively, these results suggested that size of CE increased due to the incorporation of MYR and the DiO-labeled CE effectively interacted with the ovarian cells. Fig. 3 ( a ) Diagrammatic representation of the MYR in the CE. ( b ) Statistical analysis of the amount of MYR inside the CE, as a function of its encapsulation efficiency (EE) and drug loading capacity (DLC). Mean ± SEM. ( c ) Zetasizer measurements of the CE showing the size distribution in nm after loading of MYR. (d) Zeta potential of the CE in mV after loading of MYR. ( e ) SEM image of the CE after loading with MYR. Scale bar: 300 nm. (f) DiO-labeled CE interacts with ovarian cells. Scale bar: 10X. ( g ) Viability of CHO and OVCAR3 cells treated with MYR or CE + MYR. Mean ± SEM. ( a ) Diagrammatic representation of the MYR in the CE. ( b ) Statistical analysis of the amount of MYR inside the CE, as a function of its encapsulation efficiency (EE) and drug loading capacity (DLC). Mean ± SEM. ( c ) Zetasizer measurements of the CE showing the size distribution in nm after loading of MYR. (d) Zeta potential of the CE in mV after loading of MYR. ( e ) SEM image of the CE after loading with MYR. Scale bar: 300 nm. (f) DiO-labeled CE interacts with ovarian cells. Scale bar: 10X. ( g ) Viability of CHO and OVCAR3 cells treated with MYR or CE + MYR. Mean ± SEM. The anticancer activity of MYR was assessed in CHO cells and OVCAR3 cells. MYR decreased cell viability in a dose-dependent manner by suppressing the growth of OVCAR3 cells. The cells were treated with varied concentrations of MYR and CE + MYR for 48 h, with maximum viability in no treatment condition. Therefore, the incorporation of MYR into CE increased the delivery efficiency of MYR to target cells, thereby reducing the IC 50 value at 30 μM in the CE + MYR-treated group compared with that of MYR at 42 μM in OVCAR3 cells (Fig.  3 g). The IC 50 values of MYR and CE + MYR were examined in CHO cells, and these compounds were found to be nontoxic. Hence, they were chosen for the following studies. Modifications in the structural anatomy of ovarian cells caused by MYR and CE + MYR were examined under an inverted microscope. The transformation of cell morphology from an epithelial shape to a round structure was observed in the OVCAR3 cells treated with CE + MYR and MYR. There was no change in cell shape in the CE-treated OVCAR3 cells and the control (untreated) cells. The cell adherence was weakened, with a reduced cell count and an increase in cell debris in OVCAR3 cells (Fig.  4 a). CHO cells were unaffected by CE, MYR, and CE + MYR. Nuclear morphology was analyzed via DAPI staining. The control group and CE-treated cells presented normal nuclei that were uniformly stained with no nuclear condensation. MYR and CE + MYR treated OVCAR3 cells showed condensed and fragmented nuclei that underwent cell death (Fig.  4 b). CHO cells showed no change in nuclear morphology after treatment. Fig. 4 ( a ) Morphological changes were observed in OVCAR3 cells treated with CE, MYR, and CE + MYR after 48 h. Scale bar: 10X. ( b ) Nuclear fragmentation depicted by DAPI staining on OVCAR3 cells after treatment with MYR and CE + MYR. Scale bar: 20X. ( a ) Morphological changes were observed in OVCAR3 cells treated with CE, MYR, and CE + MYR after 48 h. Scale bar: 10X. ( b ) Nuclear fragmentation depicted by DAPI staining on OVCAR3 cells after treatment with MYR and CE + MYR. Scale bar: 20X. To understand the cellular mechanism by which MYR affects ovarian cancer cells, resulting in the changes in cell structure and nuclear breakdown, we evaluated the expression of ECM1 and its associated molecules (Fig.  5 a, b). Thus, we investigated the effects of MYR and CE + MYR on the expression of ECM1, which was significantly reduced in OVCAR3 cells (p-value = 0.003) but unaffected in CHO cells (p-value = 0.9625) under the same conditions (Supplementary Table. 3 ). The reduction in ECM1 expression was greater in the CE + MYR group than in the MYR group. Interestingly, the expression of key ECM1-associated mediators for migration and cell proliferation, NF-κB, and TGFβ decreased in the CE + MYR group and subsequently in the MYR group, compared with that in the CE-treated and control groups of OVCAR3 cells. PTEN is activated by CE + MYR and MYR, which inhibits cancer progression and leads to apoptosis. Similarly, MYR and CE + MYR suppressed the mRNA expression of ECM1 in OVCAR3 cells (p-value = 0.0023), with CE + MYR exhibiting a greater reduction; however, this effect was nonsignificant for CHO cells (p-value = 0.8922) (Supplementary Table. 4 ). The expression of growth-associated genes, which include RELA and TGFβ, was also downregulated by exposure to CE + MYR and MYR in tumor cells. However, their mRNA expression in CHO cells remained unchanged (Fig.  5 c, d). In addition, PTEN tumor suppressor expression was increased in both the CE + MYR and MYR-treated OVCAR3 cells. Compared with those in the control group, the expression levels of these molecules in the CE-treated OVCAR3 cells were not significantly different. Additionally, the expression levels of ECM1 and its related molecules across all the groups of CHO cells were found to be nonsignificant, indicating that it is unaffected by the compound. Therefore, these results reveal that MYR and CE + MYR inhibit cell proliferation by downregulating growth-promoting molecules and stimulating the expression of tumor suppressor molecules that target ovarian cancer. Fig. 5 ( a ) Western blot images of the protein levels of ECM1 and growth-associated molecules in ovarian cells after treatment. ( b ) Quantitative analysis of the protein expression of ECM1 and other molecules normalized to that of β-actin, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. ( c ) PCR results depicting the relative mRNA expression of ECM1 and its growth-related molecules in ovarian cells. ( d ) Quantitative analysis revealed a significant change in the mRNA expression of ECM1-related molecules in ovarian cells. The bars indicate the mean ± SEM. ( a ) Western blot images of the protein levels of ECM1 and growth-associated molecules in ovarian cells after treatment. ( b ) Quantitative analysis of the protein expression of ECM1 and other molecules normalized to that of β-actin, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. ( c ) PCR results depicting the relative mRNA expression of ECM1 and its growth-related molecules in ovarian cells. ( d ) Quantitative analysis revealed a significant change in the mRNA expression of ECM1-related molecules in ovarian cells. The bars indicate the mean ± SEM. Clonogenic and wound healing assays were performed to investigate the colony-forming ability and migration of cells treated with CE + MYR. In OVCAR3 cells, treatment with MYR or CE + MYR for 48 h resulted in a significant reduction in cell viability in a dose-dependent manner (Fig.  6 a). The treatment with CE alone resulted in 165 and 134 colonies, MYR treatment alone showed 164 and 112 colonies, while treatment of CE + MYR resulted in 162 and 92 colonies for CHO and OVCAR3 cells, respectively, after 48 h. Conversely, the control group showed 166 and 138 colonies for CHO and OVCAR3 cells, respectively, as illustrated in (Fig.  6 b). No considerable variation in colony count was observed in the CHO cells groups. Our results revealed that the colony-forming ability of OVCAR3 cells was markedly reduced by CE + MYR. Fig. 6 ( a ) CE + MYR suppressed the number of tumor cell colonies formed by OVCAR3 cells after treatment without affecting CHO cells. ( b ) Statistical analysis of colony formation in CHO and OVCAR3 cells, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. ( c ) CE + MYR inhibited the wound healing ability of OVCAR3 cells. Images were taken and are shown for 0 and 48 h. Scale bar: 10X. ( d ) Statistical analysis of wound healing assay in CHO and OVCAR3 cells, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. ( a ) CE + MYR suppressed the number of tumor cell colonies formed by OVCAR3 cells after treatment without affecting CHO cells. ( b ) Statistical analysis of colony formation in CHO and OVCAR3 cells, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. ( c ) CE + MYR inhibited the wound healing ability of OVCAR3 cells. Images were taken and are shown for 0 and 48 h. Scale bar: 10X. ( d ) Statistical analysis of wound healing assay in CHO and OVCAR3 cells, n = 3. (ns- nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001). The bars indicate the mean ± SEM. To determine the influence of MYR on the migration of ovarian cells, we conducted a wound-healing assay in the MYR and CE + MYR-treated groups at different time intervals. The results revealed that the scratched area was recovered in all the groups of CHO cells, and no observable inhibition was observed during migration (Fig.  6 c). However, the rate of cell migration in the OVCAR3 cells treated with the MYR and CE + MYR gradually decreased. Notably, the group treated with CE + MYR demonstrated a more significant reduction in wound closure (Fig.  6 d). As shown in (Supplementary Fig.  1 ), CHO cells migrated and covered part of the scratch after 6 h of culture, whereas OVCAR3 cell migration was inhibited. These results indicated that CE efficiently delivered MYR, causing a reduction in colony formation and wound healing in ovarian cancer. Mitochondrial function is related to cancer progression. Therefore, we analyzed the morphology, membrane potential, and dynamics of mitochondria in ovarian cells. To investigate the modifications in mitochondrial morphology, ovarian cells were stained with MitoTracker red, and images were captured using a fluorescence microscope. The normal structure of mitochondria is a mixed reticulum that includes both tubular and rounded forms. In the CHO groups, treatment with CE, MYR, and CE + MYR effectively maintained the relatively normal morphology of the majority of mitochondria (Fig.  7 a). However, the OVCAR3 cells treated with MYR and CE + MYR exhibited mitochondrial elongation and fusion when compared to the control and CE-treated groups. Our results indicated that MYR and CE + MYR strongly influenced mitochondrial shape by the fusion of mitochondria and promoting mitochondrial fusion in tumor cells. Fig. 7 ( a ) The alteration of mitochondrial morphology observed in OVCAR3 cells by MitoTracker™ Red. Mitochondrial shape was unaffected in CHO cells. Scale bar: 10X. ( b ) Mitochondrial membrane dysfunction and mitochondrial permeability changes occurred in OVCAR3 cells after treatment; in contrast, in CHO cells, MMP remained unchanged. Scale bar: 10X. ( c ) Statistical analysis of JC-10 dye Red/Green Fluorescence intensity in CHO and OVCAR3 cells. The bars indicate the mean ± SEM. ( a ) The alteration of mitochondrial morphology observed in OVCAR3 cells by MitoTracker™ Red. Mitochondrial shape was unaffected in CHO cells. Scale bar: 10X. ( b ) Mitochondrial membrane dysfunction and mitochondrial permeability changes occurred in OVCAR3 cells after treatment; in contrast, in CHO cells, MMP remained unchanged. Scale bar: 10X. ( c ) Statistical analysis of JC-10 dye Red/Green Fluorescence intensity in CHO and OVCAR3 cells. The bars indicate the mean ± SEM. While investigating mitochondrial dynamics, we also focused on the mitochondrial membrane potential (MMP) (ΔΨm) after treatment with MYR and CE + MYR. A decrease in the mitochondrial potential was observed in the groups treated with MYR and CE + MYR compared with the control and CE-treated groups of OVCAR3 cells (Fig.  7 b). The control and CE-treated cells had high MMP with JC-10 dye in dimeric form, showing red fluorescence inside the mitochondria. On the other hand, the OVCAR3-treated cells with MYR and CE + MYR presented a weak MMP, resulting in the reversal of JC-10 dye to monomeric green fluorescence in the cytosol. The ratios of red to green fluorescence intensity of JC-10 dye in the MYR and CE + MYR groups exhibited a significant decrease when compared to the control and CE-treated groups, suggesting a loss of MMP in OVCAR3 cells (Fig.  7 c). Conversely, there was no change in the membrane potential of CHO-treated cells. The expression of mitochondrial dynamics-related molecules in ovarian cells was analyzed after treatment with MYR or CE + MYR. The treatment with MYR and CE + MYR resulted in a reduction of DRP1 and FIS1 expression levels, while simultaneously increasing the levels of MFN1 and MFN2, in comparison to the CE-treated and control groups in OVCAR3 cells. CE + MYR treatment caused a greater decrease in the expression of DRP1 and FIS1 in OVCAR3 cells, particularly in comparison with the MYR-treated group. In addition, the levels of the mitochondrial fusion proteins MFN1 and MFN2 were notably greater in the CE + MYR group than in the MYR-treated group of OVCAR3 cells. However, we found nonsignificant differences in the expression of mitochondrial dynamics-related molecules of all the CHO cell groups (Fig.  8 a, b). Furthermore, the mRNA expression of mitochondrial fission and fusion molecules was assessed in MYR-treated cells. Compared with the control group, treatment with MYR and CE + MYR downregulated the transcription of DRP1 and FIS1 in OVCAR3 cells. The mRNA expression of the mitochondrial fusion molecules MFN1 and MFN2 was greater in the CE + MYR group than in the MYR-treated groups of OVCAR3 cells. There was no change in the mRNA expression level of mitochondrial dynamics-related molecules in CHO cells (Fig.  8 c, d). These results indicate that CE + MYR alters mitochondrial morphology, reduces the membrane potential, and increases the mitochondrial fusion events in tumor cells. Fig. 8 ( a ) Mitochondrial protein levels were evaluated by western blot analysis after 48 h of treatment with CE, MYR, and CE + MYR. ( b ) Quantitative analysis of the protein expression of mitochondrial dynamics-related molecules normalized to that of β-actin. The bars indicate the mean ± SEM. ( c ) The expression of mitochondrial dynamics-related molecules DRP1, MFN1, MFN2, and FIS1 was determined via PCR on an agarose gel. ( d ) Quantitative analysis showing a significant change in the mRNA expression of mitochondrial dynamics-related molecules in CHO cells and OVCAR3 cells after treatment with CE, MYR, and CE + MYR. The bars indicate mean ± SEM. ( a ) Mitochondrial protein levels were evaluated by western blot analysis after 48 h of treatment with CE, MYR, and CE + MYR. ( b ) Quantitative analysis of the protein expression of mitochondrial dynamics-related molecules normalized to that of β-actin. The bars indicate the mean ± SEM. ( c ) The expression of mitochondrial dynamics-related molecules DRP1, MFN1, MFN2, and FIS1 was determined via PCR on an agarose gel. ( d ) Quantitative analysis showing a significant change in the mRNA expression of mitochondrial dynamics-related molecules in CHO cells and OVCAR3 cells after treatment with CE, MYR, and CE + MYR. The bars indicate mean ± SEM. To further validate the induction of apoptosis by MYR in ovarian cells, dual staining with AO/EtBr was performed. Our results revealed more apoptotic OVCAR3 cells in the CE + MYR and MYR groups than in the CE-treated and control groups. The orange and red-stained nuclei in the treated cells revealed that the apoptosis caused the loss of membrane integrity (Fig.  9 a). There were fewer apoptotic CHO cells in all the treated groups. Fig. 9 CE + MYR triggers apoptosis. ( a ) Cell death analysis via the AO/EtBr assay using fluorescence microscopy, and merged images of ovarian cells after treatment with MYR and CE + MYR. Scale bar: 10X. ( b ) Apoptosis-related protein expression in ovarian cells after treatment. ( c ) Graphical representation of relative protein expression in each group. The bars indicate mean ± SEM. ( d ) Representative mRNA expression analysis in ovarian cells after the treatment, as determined by PCR. ( e ) Statistical analysis showing the mRNA expression of apoptosis-related molecules in ovarian cells. The bars indicate mean ± SEM. CE + MYR triggers apoptosis. ( a ) Cell death analysis via the AO/EtBr assay using fluorescence microscopy, and merged images of ovarian cells after treatment with MYR and CE + MYR. Scale bar: 10X. ( b ) Apoptosis-related protein expression in ovarian cells after treatment. ( c ) Graphical representation of relative protein expression in each group. The bars indicate mean ± SEM. ( d ) Representative mRNA expression analysis in ovarian cells after the treatment, as determined by PCR. ( e ) Statistical analysis showing the mRNA expression of apoptosis-related molecules in ovarian cells. The bars indicate mean ± SEM. In the present study, we found that the reduction in ECM1 levels caused by CE + MYR enhanced p53 expression, leading to cell death. The apoptotic effects of MYR and CE + MYR trigger activation of Caspase-3 and Caspase-7 into Cleaved Caspase-3 and Cleaved Caspase-7, respectively. There was a notable rise in the protein levels of apoptosis-promoting phosphorylated-p53, Cleaved Caspase-3, and Cleaved Caspase-7 in tumor cells treated with CE + MYR and MYR alone, in contrast to the control group (Fig.  9 b, c). Moreover, there was a reduction in the antiapoptotic molecule Bcl-xL in both the MYR and CE + MYR groups of OVCAR3 cells. Apoptosis is a systematic process of cell death, marked by morphological alterations, nuclear fragmentation, disintegration of cellular structures, suppression of growth-promoting factors, release of cytochrome c, and increased levels of apoptosis-related molecules, culminating in the formation of apoptotic bodies. The effects of MYR and CE + MYR on OVCAR3 cells included increased mRNA expression of p53 with subsequent upregulation of cytochrome c, BID, caspase-3, and caspase-7, while CHO cells were unaffected. CE + MYR and MYR significantly suppressed Bcl-2 expression in OVCAR3 cells. Therefore, these expression profiles revealed that, in contrast with MYR alone, CE + MYR had a greater influence on tumor target cell molecules, resulting in the activation of apoptosis (Fig.  9 d, e). CE + MYR results in p53 activation, which is associated with the apoptotic signaling pathway in ovarian cancer cells.

MYR was purchased from TCI Chemicals (Tokyo, Japan); MTT (3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide), paraformaldehyde, 3,3ʹ-dioctadecyloxacarbocyanine perchlorate (DiO), Triton X-100, and Triazol reagent were obtained from Sigma-Aldrich Chemicals (Burlington, USA). Crystal violet, Acridine orange (AO), and Ethidium bromide (EtBr) were purchased from HiMedia Laboratories Private Limited (Mumbai, India). Fetal bovine serum (FBS), exosome-depleted FBS, and the PCR Master Mix were purchased from Thermo Fisher Scientific (Waltham, USA). DAPI (4′,6-diamidino-2-phenylindole) and MitoTracker™ Red were purchased from Invitrogen (Waltham, USA), and the JC-10 mitochondrial membrane potential assay and reverse transcriptase kit were obtained from G-Biosciences (St. Louis, USA). Amersham ECL western blotting detection reagent was purchased from Cytiva (Marlborough, USA), and ExoQuick-TC™ Tissue Culture media Exosome Precipitation Solution (EXOTC10A-1) was purchased from System Biosciences (Palo Alto, USA). Tissue culture flasks (T25, T75) and tissue culture plates (96 wells, 6 cells, 60 mm, and 100 mm) were purchased from HiMedia Laboratories Private Limited. Chinese hamster ovary (CHO) cells and OVCAR3 cells were purchased from the American Type Culture Collection (ATCC). CHO cells are normal ovarian cells used in research and are the most commonly employed cell type 22 . OVCAR3 is a high-grade serous ovarian adenocarcinoma cell used for studying cytotoxicity assays and complex mechanisms in ovarian cancer 23 . They were cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum in a humidified incubator with 5% CO 2 at 37 °C. EVs were isolated from CHO cells using the isolation kit under sterile conditions, following the protocol with slight modifications 24 . CHO cells were grown in media at ~ 90% confluency and replaced with RPMI media containing exosome-depleted FBS (10%) for a few days (~ 4 to 5 days) to obtain a greater yield of EVs. EVs were acquired from the media by centrifugation at 4725 rpm for the first round to eliminate cell debris. The supernatant was added to 1.2 ml of isolation buffer per 6 ml of media under a laminar airflow cabinet to maintain sterility and incubated at 4 °C overnight in the sealed Falcon tube. A second round of centrifugation was carried out at 3341 rpm for 30 min at 4 °C. After the supernatant was discarded, the EVs pellet was resuspended in autoclaved Phosphate buffer saline (PBS) and stored at -80 °C. CE are recognized as an industrially relevant and extensively used cell host for the production of biopharmaceutical proteins 25 . Therefore, in this study, CE were employed as a MYR delivery vehicle for ovarian cancer therapy. Moreover, the quantification of isolated EVs was performed using a Nanodrop spectrophotometer (NanoDrop™ One Microvolume UV–Vis Spectrophotometer, 13–400-518, Thermo Scientific™, Massachusetts, USA), where approximately 1 µl of the sample was analyzed at 280 nm to assess protein content 26 . The fundamental parameters of EVs were measured via a Zetasizer instrument (NANO ZS, MPT-2, Malvern Panalytical, Worcestershire, UK). The size of EVs was determined in nm by diluting with cold PBS (Viscosity 0.8882 cP at 25 0 C) measured in a cuvette 27 . Stability was defined by the zeta potential in mV by the dilution with water (Viscosity 0.8872 cP at 25 0 C) measured in a Folded Capillary Zeta Cell. The sample size and zeta potential were measured before and after the loading of MYR in the CE. The EVs were fixed in 1% paraformaldehyde at 4 °C and subjected to successive centrifugation washes with absolute ethanol (70%, 80%, and 100%). The samples were loaded on a glass coverslip and air-dried for 5 min. The sample was kept on a microscope stage sputter-coated with gold (SPI- Module Sputtering, with Argon gas) for the final analysis. The morphology of EVs was examined using a scanning electron microscope (LEO, 435VP, Carl Zeiss, Cambridge, UK) 28 . ChemSketch freeware (ACD/Labs 2021, 2.0, File version C35E41, Build 125,843, Toronto, Canada) 29 was utilized to develop the structure of the MYR. The chemical conformation was set to standardize the bond length and angle. The properties of the MYR are predicted by its structure, which includes molecular formula, its weight, composition, molecular refractivity, molar volume, index of refraction, surface tension, and density using the software tools. Earlier studies indicated that sonication is an effective method of drug loading into EVs. Therefore, MYR was incorporated into CE through a mild sonication method with slight modifications 30 – 33 . For this, 1.5 mg of CE was loaded with 500 µg of MYR mixed with 5 ml of cold PBS. The sample was sonicated for 2 s on/off (15 cycles, 20% power amplitude) on ice by the Probe Sonicator (Fisherbrand Sound Enclosure for Model 50 and 120 Sonic Dismembrator, Fisher Scientific, Pittsburgh, USA). The sample was incubated at room temperature for 30 min to recover the lipid bilayer, then transferred to an Amicon filter and centrifuged at 13,000 rpm for 30 min to remove excess unloaded drug. CE + MYR formulation was stored at -20 °C. CE + MYR was dissolved in methanol to release the MYR from CE to determine the amount loaded in EVs. The absorbance of MYR incorporated in the CE was assessed using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) at 369 nm. The mobile phase consisted of acetonitrile with a 0.1% acetic acid gradient, eluted at a flow rate of 1 ml/min using a C18 reverse-phase column. The Encapsulation efficiency (%EE) and Drug Loading capacity (%DLC) of the nanoparticles were evaluated 34 . EE reveals the percentage of the drug amount that is encapsulated in EVs to the amount of drug taken for loading in the formulation. DLC signifies the percentage of the weight of the drug that is loaded into the EVs relative to the EVs used in the process 34 . Fourier transform infrared spectroscopy (FTIR) was employed to examine the chemical constitution of MYR and functional group adsorption on the surface of the CE. The samples were prepared in PBS and placed on a transparent crystal for transmittance analysis in the wavenumber range from 400 to 4000 cm -1 via the FTIR instrument (Tensor II Bruker, Massachusetts, USA). DiO staining analysis was used to determine the interaction of EVs with ovarian cell lines by staining the EVs bilayer 35 . In this study, 2 µg of DiO stain was mixed with CE and PBS in the media and further incubated at 4 °C in the dark. The excess DiO stain was removed by centrifugation at 4725 rpm for 15 min. These labeled EVs were added to the adherent cells and visualized under a fluorescence microscope (Olympus, DP73 Digital Camera, Microscope, Tokyo, Japan). Cytotoxicity analysis was carried out for MYR and CE + MYR on ovarian cells with slight modifications to the protocol 35 . The cells were seeded in 96-well tissue culture plates (5,000 cells per well) and treated with (5 µM, 10 µM, 25 µM, 40 µM, 50 µM, and 100 µM) MYR or CE + MYR for 48 h. MTT stock solution (5 mg/ml) was added, and the mixture was incubated for 3 h. The media was discarded, 100 µl of DMSO was added, and the mixture was incubated for 30 min in the dark. Finally, the absorbance was measured at 570 nm on a microplate reader (Agilent BioTek Epoch 2 microplate spectrophotometer, Agilent Technologies, Santa Clara, CA, USA). Half maximal inhibitory concentration (IC 50 ) serves as an indicator of the potency of a drug in reducing biological function by 50% within the cells. It is evaluated by developing a dose–response curve, analyzing the impact of various MYR concentrations on cell viability, and identifying the concentration required to inhibit half of the biological activity 36 . Cells were seeded at a density of 3 × 10 5 per well into 6-well tissue culture plates and were allowed to adhere and attain their morphology. The cells were treated with CE (1.5 mg) as per the quantity of EVs used in preparing the formulation and the IC 50 concentration of MYR, and CE + MYR in RPMI media for 48 h. After the samples were washed with PBS, images were taken under an inverted microscope (Compact Cell Culture Microscope, CKX53, Olympus, Tokyo, Japan). Cells were seeded at a density of 1.5 × 10 5 on glass coverslips placed in 6-well tissue culture plates and treated with CE, MYR, and CE + MYR for 48 h. After treatment, the cells were washed with PBS and treated with paraformaldehyde and Triton X-100. DAPI stain mountant was added to the samples in the dark for 5 min, and the coverslip was placed on a glass slide. The images were taken under a fluorescence microscope 37 . Cells were cultured at a density of 7.5 × 10 4 cells on 60 mm tissue culture plates and treated with CE, MYR, and CE + MYR for 48 h. The treated media was replaced with fresh complete RPMI media, and the plates were kept in a CO 2 incubator at 37 °C, with the media being replenished every 3 to 4 days over a period of 10 days to allow the cells to form colonies. The cells were washed with PBS, and cell fixation solution was added to each plate for 20 min. The plates were incubated with 0.5% crystal violet solution for 5 min and then washed with Milli-Q water. The plates were air-dried, colonies were counted, and images were taken 38 . The cells were grown at a density of 5 × 10 5 cells on 6-well tissue culture plates for 24 h. When the cells reached 100% confluence, the monolayer was scratched with a straight line using a 10 µl pipette tip. The cells were washed with PBS to remove cell debris and were treated with CE, MYR, and CE + MYR. Images were taken under an inverted microscope at 0 h, 6 h, 12 h, 24 h, and 48 hours 38 . The wound healing was determined using the formula: wound healing rate (%) = (width of the wound at 0 h – width of the wound at 48 h) / width of the wound at 0 h X 100. Ovarian cells were seeded at a density of 1.5 × 10 5 per well into 6-well tissue culture plates and treated with CE, MYR, and CE + MYR for 48 h. The cells were washed and incubated with MitoTracker Red in RPMI media without FBS for 45 min at 37 °C. The cells were washed, and images were captured under a fluorescence microscope 39 . The mitochondrial membrane potential (MMP) was estimated using the JC-10 dye by seeding ovarian cells at a density of 1.5 × 10 5 per well into 6-well tissue culture plates and treating them with CE, MYR, and CE + MYR for 48 h. The cells were washed with PBS twice and incubated with JC-10 in phenol-free media for 45 min at 37 °C. The cells were washed with PBS and examined under a fluorescence microscope 40 . The cells were grown at a density of 1.5 × 10 5 per well into a 6-well tissue culture plate and treated with CE, MYR, and CE + MYR for 48 h. The cells were washed with PBS and were treated with AO/EtBr stain (1:1 ratio) in PBS for 5 min in the dark at room temperature. The cells were washed to remove excess stain and observed under a fluorescence microscope 41 . The cells were cultured at a density of 8 × 10 5 per well into 100 mm tissue culture plates and treated with CE, MYR, and CE + MYR for 48 h. The cells were lysed with lysis buffer, scraped, centrifuged to collect the supernatant protein, and stored at -80 °C. The CE protein and cell lysate protein were isolated by incubating the samples with lysis buffer on ice and centrifuging them at 4 °C. The proteins were loaded on an SDS gel for separation and transferred to a PVDF membrane. The membranes were blocked in 3% BSA overnight at 4 °C. The membranes were incubated with primary antibodies for 1 h at room temperature on a rocker. The membranes were washed with TBST, then incubated with secondary antibodies for 1 h at room temperature (Supplementary Table. 1 ). The membranes were washed with TBST using an ECL reagent on the ChemiDoc instrument. The protein bands were quantified via ImageJ software 41 . β-actin was used as an internal control. The ovarian cell lines were grown at a density of 8 × 10 5 per well into 100 mm tissue culture plates and treated with CE, MYR, and CE + MYR for 48 h. RNA was isolated using a triazol reagent followed by chloroform and isopropanol precipitation 41 . The RNA pellet was washed with absolute ethanol, 20 µl of DEPC water was added after pellet drying, and the RNA was stored at -80 °C. A cDNA conversion kit was used, followed by polymerase chain reaction (PCR) of the samples with specific primer sequences on a PCR instrument (SureCycler 8800 Thermal Cycler, Agilent Technologies, California, USA) (Supplementary Table. 2 ). The band results were analyzed on an agarose gel and quantified via ImageJ software. GAPDH was used as an internal control. The results are expressed as the mean ± standard deviations. Statistical significance was determined by a one-way ANOVA using GraphPad Prism 9.0. Graphical data analysis was performed via ImageJ and GraphPad Prism software. A p-value < 0.05 was considered statistically significant.

Myricetin-loaded CHO cell-derived extracellular vesicles (CE + MYR) can induce ovarian cancer cell death, which is regulated by mitochondrial dynamics. Our investigation revealed that, compared with direct MYR treatment, CE + MYR treatment results in better conveyance into target cells. CE + MYR inhibited growth by reducing the level of ECM1 along with NF-κB and TGFβ, thereby impeding the tumorigenic pathway. Furthermore, we found that CE + MYR activated mitochondrial fusion (MFN1, MFN2) and suppressed fission events (DRP1, FIS1), resulting in alterations in mitochondrial structure and increased expression of p53, cleaved caspase-3, and cleaved caspase-7, leading to apoptosis. As a result, internalization of MYR into CE could be a potential therapeutic agent for the treatment of ovarian cancer. However, the study is limited to in vitro testing. Further animal studies are required to evaluate the effectiveness of the formulation.

Different types of ovarian cancer are treated, including surgical procedures, neoadjuvant chemotherapy, hormonal treatments, and immune-based therapies. Nevertheless, the prognosis and survival outcomes remain unfavorable, thereby emphasizing the necessity for an efficient therapeutic approach targeting cancer. EVs-based cancer therapy has emerged as an effective approach for inhibiting the growth of cancer cells. EVs show remarkable biocompatibility and minimal immunogenicity, rendering them suitable as drug delivery vehicles that can effectively target cancer cells while minimizing off-target damage and adverse effects. EVs-based drug delivery systems present a significant ability for the development of future cancer therapies. There is a need to improve the production efficiency of such drug delivery formulation and support clinical translation 47 . The Chinese Hamster Ovary (CHO) cells are the most frequently used cell line, known for their high transfection capabilities, and have set the standard for the manufacturing of approved therapeutic proteins. These cells can be easily adapted to suspension culture, making them ideal for large-scale production in bioreactors 14 ; consequently, their extracellular vesicles (EVs) can be well-suited for bulk production of EVs-drug delivery formulation. The present research focused on the use of EVs obtained from CHO cells as a vehicle for Myricetin (MYR) delivery, to alter signaling pathways in ovarian cancer cells while preserving the integrity of healthy cells. Numerous studies have indicated that MYR has therapeutic potential against various diseases by triggering cardio-cerebrovascular defense mechanisms, strengthening the immune system, and increasing antibacterial activity. The medicinal properties of MYR include cell cycle arrest caused by double-strand breaks in DNA and increased expression of the endoplasmic reticulum stress-related protein GRP-78 and C/EBP homologous proteins, which include apoptosis in ovarian cancer 48 . Moreover, the loading of MYR in EVs effectively protects against oxidative stress 49 . EVs are incorporated with drugs for therapeutic delivery to the recipient cells. Similarly, this study aimed to assess the efficacy of Myricetin-loaded CHO cell-derived extracellular vesicles (CE + MYR) in promoting anticancer activity through mitochondrial dynamics in ovarian cancer. CE was characterized by its average size of 163.9 nm, which then increased to 187.8 nm after the inclusion of MYR by DLS analysis. Research has revealed the divergent exosome sizes ranging from 30 to 200 nm 12 , which is consistent with the CE analyzed in this study. There was a slight reduction in the zeta potential from -24.4 mV to -24.3 mV by sonication due to the mechanical shear force of the probe sonicator. The stabilization of exosomes depicted by the zeta potential in the previous studies also had a negative value, which stabilized the allocation of charge density encircling the nanovesicle 50 . It was observed that the integrity of the lipid bilayer of EVs was preserved even following mild sonication due to the recovery step involved in the process. This was validated through SEM analysis, which revealed that CE exhibited the spherical shape before and after loading of MYR into EVs, with no observed deformities in its morphology. Previous investigations highlighted the protein expression of individual molecules embedded on the lipid bilayer and present in its lumen 51 , 52 . Here, we found the expression of Alix and ICAM-1 proteins in CE. Alix functions as a protein that is integral to ESCRT-dependent exosome biogenesis, aiding the budding machinery located at the endosomal membrane 53 , 54 . ICAM-1 governs the internalization of exosomes into target cells through its correlation with integrins, which is essential for cellular interaction 55 . MYR has the benzo-γ-pyrone structure of the flavonoid family, with fifteen carbon atoms in two aromatic rings and six reactive hydroxyl groups. The properties of MYR align with its structure, revealing results that are consistent with the previous reports on MYR 56 – 58 . FTIR spectrum of CE + MYR was similar to CE, with a few transmittance peaks of MYR included in EVs. The encapsulation efficiency was measured at 46.419%, which is crucial for assessing the performance of drug delivery systems. The drug loading capacity of 15.473% demonstrates the capability of EVs to preserve the MYR and their release. Our study identified CE as a potent conveyance vehicle of MYR that exerts a more efficient response by its targeted delivery than MYR alone. CE + MYR impacts proteins found in the cell membrane, cytoplasm, mitochondria, and nucleus, collectively altering the tumor cell survival and resulting in cell death. The results revealed that normal CHO cells have the lowest cytotoxicity and a lesser influence on proteins under the same treatment with CE, MYR, and CE + MYR, which means that MYR kills tumor cells, and that normal cells are unaffected. These results are consistent with earlier reports regarding the cytotoxicity of MYR in pancreatic cancer 59 . MYR was identified as a potent inducer of pancreatic cell death by blocking PI3-kinase signaling, which caused a significant elevation in the activity of caspase-3 and caspase-9. However, it did not influence the viability of normal pancreatic ductal cells 60 . The incorporation of MYR into CE resulted in enhanced anti-proliferative effects, achieving a lower IC 50 value of 30 µM due to improved delivery to tumor cells. This indicates that a reduced concentration of MYR is required to inhibit 50% of tumor cell viability in the CE + MYR group compared to free MYR. OVCAR3 cells treated with MYR and CE + MYR exhibited a shrinkage of cell size and fragmented nuclear morphology, while CHO cells were not affected by the treatment. Moreover, the colony count was reduced in OVCAR3 cells treated with the CE + MYR group and the MYR group. On the other hand, there was a nonsignificant difference in colony count among all groups in CHO cells. Receptor-mediated signal transduction is a crucial cellular mechanism that facilitates communication between the cell surface and the extracellular environment, which is vital for normal cell function. If any pathway malfunctions, the cell will proliferate uncontrollably, which can lead to tumor formation. There is an increase in the expression of growth-promoting genes, in contrast to a reduction in the expression of tumor suppressor genes, resulting in a loss of cellular control over proliferation, the cell cycle, and differentiation 61 . Regulating these cells is quite difficult since they are normal cells expressing abnormal growth. Research indicated that MYR alleviates the severity of inflammatory lesions and tumor development, while also restoring the normal histological characteristics of cancerous cells by its transition against colon cancer 62 . In this study, MYR has proven to be highly effective in inducing selective apoptotic cell death in ovarian cancer without affecting normal cells. ECM1 is associated with HER + and estrogen receptor-negative breast cancers, causing aberrant vascular proliferation in the tumor microenvironment 63 . Previous reports have shown that increased expression of ECM1 via pathway enrichment and gene ontology enrichment analysis is associated with tumorigenesis in ovarian cancer 64 . ECM1 promotes the migration and angiogenesis associated with activating the NF-κB/AKT mechanism 65 . Furthermore, we demonstrated that the anticancer influence of the ECM1 protein was more pronounced in the CE + MYR group than in the control group, which caused morphological changes in the OVCAR3 cells from an epithelial shape to a round structure. Previous investigations have suggested that MYR modifies the structural anatomy of tumor cells 48 . ECM1 is triggered in cancer conditions along with TGFβ and NF-κB, promoting cell proliferation, migration, and prolonging survival. The suppression of ECM1 by CE + MYR successively downregulated the levels of TGFβ and NF-κB, leading to growth arrest in tumor cells. CE + MYR suppresses the proliferation mechanism in ovarian cancer cells, thus controlling cell migration. Elevated TGFβ triggers Smad2/3 and Snail, which in turn activate the MAPK, PI3K/AKT, and RhoA pathways contributing to EMT during ovarian cancer progression. MYR decreases the number of viable cells, suppresses the activation of phosphorylated ERK and PI3K/AKT triggered by TGFβ, and increases the Bax/Bcl-2 ratio, leading to apoptosis in cancer cells 66 . Moreover, TGFβ signaling plays a role in tumor growth and metastasis via the activation of NF-κB IκB-α subunit phosphorylation in pancreatic cancer. In addition, TGFβ suppressed PTEN promoter activity and increased NF-κB levels to promote cell motility 67 . In the present study, the dysregulation of TGFβ and NF-κB by MYR and CE + MYR resulted in the upregulation of PTEN expression in OVCAR3 cells. The modification of cristae to alter the mitochondrial morphology results in the formation of fused mitochondria, and stimulating mitochondrial fusion increases anticancer activity 68 . The mitochondrial morphological transition occurred due to changes in the expression of fission and fusion molecules affected by MYR. Interestingly, MYR and CE + MYR induced mitochondrial changes in their reticular network organization, which became apparent as a long hyperfused lattice promoting mitochondrial fusion in OVCAR3 cells. JC-10 staining serves as an indicator of mitochondrial membrane potential, revealing that the red/green fluorescence intensity ratio in the MYR and CE + MYR groups was markedly lower in OVCAR3 cells. The result indicated a loss of MMP in OVCAR3 cells. The unbalanced mitochondrial dynamics mechanism in cancer cells affects different cellular functions, including ATP generation, cell proliferation, tissue development, autophagy, and apoptosis. Mitochondrial dynamics are related to ovarian cancer progression, with increased expression of DRP1 found in cancer lesions attributed to the phosphorylation at the Serine 616 residue 69 . The current investigation is based on mitochondrial dynamics, involving two distinct mechanisms: fission and fusion. In OVCAR3 cells, mitochondrial fission was found to be dominant over fusion, resulting in increased expression of the regulatory molecules DRP1 and FIS1, and decreased expression of MFN1 and MFN2. Previous studies revealed that extracellular matrix stiffness increases the phosphorylation of DRP1 causing mitochondrial fission, which facilitates the spread of tumors to the brain, lungs, and liver 70 . In the present study, ECM1 may affect the DRP1 mitochondrial fission mechanism, causing mitochondrial dysfunction in ovarian cancer. MYR reduces Aβ-stimulated mitochondrial dysfunction, leading to neuroprotection against Alzheimer’s disease. It repairs mitochondrial function by enhancing biogenesis, regulating fission–fusion events, and promoting the electron transport chain to increase ATP generation 71 . MYR behaves distinctly under cancer conditions, ultimately leading to the death of cells. Mitochondrial dynamics are disrupted by increased levels of DRP1, which inhibits the MFN1 and MFN2 proteins, leading to lamellipodia formation for the expansion of breast cancer 72 . Ovarian cancer is characterized by increased IL-6 expression, which interacts with DRP1 and ERK1/2 activation, and is related to cell survival 73 . In the study, we found that the treatment of MYR and CE + MYR induces a decline in the expression of DRP1 and FIS1, suppressing mitochondrial fission. Additionally, it results in elevated levels of MFN1 and MFN2, thereby promoting mitochondrial fusion in OVCAR3 cells. Prior reports demonstrated that the ECM1 level was significantly elevated due to p53 mutation in colorectal cancer, as it cooperates with Notch1 and enhances the expression of HES1, which influences a series of oncogenic events associated with cancer progression, metastasis, and angiogenesis 74 . ECM1 inhibits cell death by reducing p53 expression through integrin-focal adhesion kinase signaling, increasing Bcl-2 expression 75 . In this research, CE + MYR affects the level of ECM1 and activates p53 along with different caspases, leading to apoptosis. Moreover, the reports indicated that an increase in p53 expression results in the phosphorylation of DRP1 at serine 637, thereby reducing DRP1 expression and regulating mitochondrial dynamics 76 . Mitochondrial dysfunction plays a key role in CE + MYR-induced tumor cell apoptosis, which is characterized by the loss of the MMP (ΔΨm). Earlier investigations revealed that p53 activates BID and causes cytochrome c release in the cytosol from mitochondria, which activates caspase-3 and caspase-9 77 . MYR exhibited chemosensitivity against esophageal cancer cells, inhibiting cell survival and proliferation by decreasing the levels of survivin and cyclin D1. Additionally, MYR promoted cell apoptosis by elevating the expression of p53 and caspase-3 levels 78 . MYR inhibits migration and invasion, while also promoting apoptosis through the elevation of cleaved caspase-3 and caspase-9 levels in SKOV3 cells 79 . It activates apoptosis in cancer cells resistant to cisplatin by enhancing the enzymatic activities of caspase-3/7 in a p21-independent pathway 80 . In the current research, CE + MYR elevated the expression of p53, BID, and cytochrome c. CE + MYR and MYR activate the caspase-3 and caspase-7 proteins. The treated cells increased the expression of cleaved caspase-3 and cleaved caspase-7 in OVCAR3 cells. The expression of antiapoptotic molecule Bcl-2 was reduced in OVCAR3 cells but not in CHO cells. Thus, our results demonstrated that CE + MYR influences ECM1, which in turn affects cytoplasmic and mitochondrial molecules, leading to the activation of an apoptotic mechanism.

Ovarian cancer is a precarious neoplasm affecting the female reproductive region and has been examined as a menace to their well-being. It is considered the 18 th most predominant rising tumor, with 324,398 cases and 206,839 deaths worldwide 1 . The National Cancer Institute has evaluated approximately 20,890 diagnostic cases and 12,730 deaths from ovarian cancer in the year 2025 2 . According to an Indian report, there are an estimated 49,644 cases of ovarian cancer, which is a serious health issue among females in the year 2025 3 . Risk factors include lineage case history, genetic mutation, adiposity, and endometriosis. Ovarian cancer detection involves a pelvic examination, transvaginal ultrasound, laparoscopy, and color doppler imaging 4 . In the last several decades, there has been ongoing advancement in the exploration and identification of tumor biomarkers, which has substantially advanced medicine and improved the health of cancer patients. This research emphasizes the significance of ECM1 in ovarian cancer. The Extracellular matrix protein 1 (ECM1) is a glycoprotein associated with cell growth, its endurance, epithelial-mesenchymal transition (EMT), and cellular locomotion. It is typically found on the skin’s basement membrane because of its durability and provides a structural framework for the cells. It interacts with various cellular and non-cellular components, allowing the attachment of cells to the basement membrane 5 . Previous studies have shown that ECM1 promotes the growth of tumor masses in patients with colorectal carcinoma exhibiting lymph node metastasis and in vitro cell migration. It regulates the PI3K/AKT/GSK3β/Snail pathway, enabling tumor progression and reduction in cell death 6 . The transcriptome expression profiling revealed an increased expression of ECM1 in human ovarian cancer cell lines, which contributes to cell survival, tumor invasion, and metastasis 7 . Mitochondria are fundamental to cellular energy metabolism and cell signaling; mitochondrial deregulation is recognized as a key indicator for the progression of cancer. Mitochondrial dynamics establish equilibrium amid mitochondrial power generation and apoptosis mechanisms 8 . In the regular cellular milieu, mitochondria undergo a synchronized series of fission and fusion events to sustain organelle size, structure, and allocation in the cytoplasm 9 . The process of fission results in smaller, fragmented mitochondria, whereas an increase in fusion leads to the development of more elongated and interconnected networks. Mitochondrial fission, facilitated by Dynamin-related protein (DRP1), reduces mitochondrial size and elevates their number. On the other hand, Mitochondrial fusion, governed by Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2), counteracts division to uphold mitochondrial integrity 10 . However, an aberration in the basic mechanism results in carcinoma by generating increased dysfunctional mitochondria through mitochondrial fission, accompanied by high levels of DRP1 and lower expression of the MFN1/2 gene, which affects cell longevity 11 . The relationship between the ECM1 molecule and mitochondrial dynamics, followed by apoptosis, is yet to be understood. Extracellular vesicles (EVs) are nanometric lipid bilayer spherical-shaped that biologically originate from final endosomes created by the concave spherical protruding multivesicular body (MVB) membrane enclosing intraluminal vesicles (ILVs). These vesicles either combine with lysosomes for decay or merge with the plasma membrane on the cytoplasmic side with inside-out membrane orientation and are released into the extracellular space 12 . Their size range of 30–200 nm in diameter is released from the cell membrane as a significant monitoring tool for cellular transmission. They have applications in the medical field to combat a broad spectrum of illnesses 13 . Chinese hamster ovary (CHO) cells constitute an epithelial cell line that is isolated from the ovary of a Chinese hamster and is cultured either as adherent cells or in suspension. These cells are grown extensively for antibody production and recombinant proteins 14 . CHO cells can be reconstructed for the post-translational modifications of human cells and minimize the risk of immunogenic reactions 15 . In this study, we employed CHO cell-derived extracellular vesicles (CE) as a delivery system to ensure the precise transport of therapeutic agents into cells for ovarian cancer treatment. The Flavonoid family comprises diverse secondary metabolites that are efficient for cancer therapy. Flavonoids are organic phytochemicals found in fresh fruits, green vegetables, tea, and specific medicinal plants. Myricetin (MYR) is a phenolic compound that is chiefly segregated from the bark of Myrica nagi, and is found in the subtropical Himalayas and North-West Provinces of Kumaon, Uttarakhand, India 16 . Myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one) is a dietary flavonoid also found in fruits (such as grapes, oranges, and berries), vegetables (such as sweet potatoes, parsley, and broad beans), tea, and wine 17 . Its structure is composed of an aromatic ring with hydroxyl groups at the 3, 5, 7, 3', 4', and 5’ positions, which is considered efficient in purifying free radicals generated by both enzymatic and nonenzymatic mechanisms 18 . MYR can function as an antioxidant in cancer prevention by regulating the cell cycle, curbing cell proliferation, and producing an anti-inflammatory response 19 . MYR promotes an anti-inflammatory response by suppressing the MAPK and AKT-mTOR pathways, and lowering the levels of TNFɑ, IL-6, and cytokines 20 . MYR presents a therapeutic challenge as its hydrophobic nature results in lower absorption in cells. It is a yellow-colored, shiny, needle-shaped substance poorly soluble in water; however, it is soluble in dimethyl sulfoxide 21 . Therefore, there is a need for an efficient delivery vehicle that protects the compound while maintaining its stability and conveys it proficiently to the target cells. This work emphasized the efficiency of Myricetin-loaded CHO cell-derived extracellular vesicles (CE + MYR) in combating tumor growth, focusing on its impact on mitochondrial dynamics and apoptosis via the ECM1 pathway.

Supplementary Information 1. Supplementary Information 2. Supplementary Information 3. Supplementary Information 4. Supplementary Information 5. Supplementary Information 1. Supplementary Information 2. Supplementary Information 3. Supplementary Information 4. Supplementary Information 5.