Isolation, characterization, and stability of GENs
To isolate exosome-like nanoparticles from Panax ginseng, raw fresh ginseng was thoroughly ground in 1X PBS. After sequential low-velocity centrifugation steps, large debris and impurities were removed from the ginseng juice. The sucrose cushion method was applied to prevent the aggregation and degradation of the exosomes, achieved by increasing centrifugal force (Fig. 1a, b). GENs were observed through sucrose gradient fractionation at 1.13–1.19 g/mL (Fig. 1c) and identified based on the average size of 151.6 nm with a low polydispersity index (PDI) ranges. The zeta potential of GENs indicated that GENs held a negative charge of –17.9 mV (Fig. 1d). The size distribution and particle concentration were measured by nanoparticle tracking analysis (NTA). Transmission electron microscopy (TEM) analysis indicated that the GENs were mostly spherical and uniform in shape, without aggregation or degradation. The quantification of GENs was performed by measuring the protein concentration using a Bradford assay kit, which indicated that GENs from ginseng were produced abundantly (approximately 1.68 mg per 10 g of ginseng tissue, based on the protein concentration). Those results suggest that homogenous GENs can be obtained on a large scale using a simple and eco-friendly method (Additional file 1: Fig. S1e).
To characterize the profile of GENs, we extracted total RNA, encapsulated lipids, and protein and performed RNA sequencing analysis, total lipid profiling, and proteomic analysis (Fig. 1e, Additional file 1: Table S2, S3). The results indicated that GENs mainly consisted of phospholipids enriched in phosphatidylcholine (PC; 28.8%), triglycerides (TG; 16.8%), and ceramides (Cer; 12.9%). A total of 98 miRNAs targeting various genes in C6 glioma cells and 86 proteins similar to those found in Panax ginseng were identified. We hypothesized that the abundant membrane lipids in GENs contribute to their stability and may facilitate preferential uptake by the BBB and brain tumor tissues.
In addition, the ginsenosides were extracted from GENs and analyzed through high performance liquid chromatography (HPLC). The extraction of ginsenosides from GENs was carried out under optimized extraction conditions. The main active components of ginsenosides, including Rg1, Re, Rg3, and Rb1, were detected in GENs at varying concentrations, which potentially contributes to its effectiveness in the treatment (Fig. 1f).
To assess the stability of GENs, their size and zeta potential were measured for 30 days. GENs were aliquoted and stored at –80 ℃. The size and zeta potential of GENs were measured to stay intact around 150 nm with a low PDI value, while the zeta potential of GENs maintained a negative charge of approximately –20 mV (Additional file 1: Fig. S1a, b). Typical freeze-drying of GENs was also performed to guarantee long-term stability. The morphology of GENs remained in the same uniform spherical shape after the freeze-drying as observed by TEM. The size and zeta potential of freeze-dried GENs showed slight increases compared to the fresh GENs (Additional file 1: Fig. S2a, b). Hence, the freeze-dried GENs were used in the consecutive experiments. To further analyze the stability and profile of GENs, the concentrations of total RNA and protein were measured. The integrity of total RNA extracted from ginseng tissue and GENs was analyzed by 10% denaturing polyacrylamide gel electrophoresis. It was observed that the total RNA of GENs was enriched, and the integrity and length of RNA fragments in GENs were slightly distinct from those extracted from ginseng tissues. As a result, the RNA fragments isolated from GENs consisted of various RNA fragments ranging in size from 50 to 500 base pairs (bp), while the RNA fragments isolated from ginseng tissues were 80–1000 bp in size.
In addition, the samples of total protein extracted from ginseng tissue and GENs were separated by 15% SDS-PAGE gel. The proteins isolated from ginseng tissues and those isolated from GENs revealed similar protein bands, indicating the reliable consistency of the protein components (Additional file 1: Fig. S2c–e).
Effect of GENs on glioma cell apoptosis in vitro
To quantify the ratio of C6 glioma apoptosis, apoptosis induced by GENs was analyzed by an Annexin V/PI assay. As the concentration of GENs increased, the C6 glioma cells showed higher rates of early apoptosis (FITC + /PI−) and late apoptosis/necrosis (FITC + /PI +) compared with the negative control group (Fig. 2a, b). To study the effect of GENs on C6 glioma cell viability, the cells were treated with GENs at concentrations ranging from 1 to 62.5 μg/mL for 24 h. As a result, C6 glioma apoptosis was initiated by treatment of GENs at a concentration of 15.625 μg/mL and the total apoptosis was significantly increased at a concentration of 62.5 μg/mL of GENs, showing 81 ± 1.4% of apoptotic cell death. Collectively, flow cytometry analysis indicated that GENs could mainly affect C6 glioma cell apoptosis when administered at the highest concentration.
Cellular uptake and internalization of GENs and in vitro BBB penetration
C6 glioma cells were treated with DiD-labeled GENs and analyzed by confocal microscopy to determine the internalization of GENs. As shown in Fig. 2c, after 6 h of incubation, the distribution of DiD-labeled GENs in cells significantly increased compared with DiD-labeled liposomes, indicating the selective uptake of GENs by C6 glioma cells. Also, the uptake of GENs continued to increase over a 24 h period (Fig. 2c–e). To stimulate the brain environment, an in vitro transwell BBB model was used (Additional file 1: Fig. S3). By utilizing a co-culture transwell membrane with a pore size of 0.4 μm, we aimed to assess the internalization of GENs through the BBB. Brain capillary endothelial cells (BCECs) were seeded on the upper compartment and incubated for 16 days until the transepithelial/endothelial electrical resistance (TEER) level reached above 200 Ω cm2 (Additional file 1: Fig. S3b). Prior to the 24 h incubation of GENs, C6 glioma cells were seeded in the lower compartment. Then, DiD-labeled GENs were placed in the upper compartment and further incubated for 12 h. C6 glioma cells in the lower compartment were collected and the efficient cellular uptake of GENs was observed. As a result, the DiD-labeled GENs could pass through and be taken up by C6 glioma cells (Additional file 1: Fig. S3c). Thus, GENs can be internalized into the tumor by penetrating through the mimicked BBB and have the potential for drug delivery to brain endothelial cells.
To evaluate the uptake mechanism of GENs into C6 glioma cells through the BBB, BCECs seeded on a transwell membrane were treated for 6 h with a series of inhibitors including chlorpromazine (an inhibitor of clathrin-mediated endocytosis), cytochalasin D (a phagocytosis inhibitor), methyl-β-cyclodextrin (lipid raft disruptor), colchicine (pinocytosis and phagocytosis inhibitor), and antibodies for occludin and claudin-5 (paracellular transport inhibitors) (Additional file 1: Fig. S4). Additionally, to evaluate the internalization of DiD-labeled GENs at low temperatures, BCECs on the transwell were incubated at 4 ℃ for 1 h, and then C6 glioma cells incubated with DiD-labeled GENs for 12 h in the upper compartment were collected, and the fluorescence intensity was measured (Additional file 1: Fig. S4). Compared with the control group, a significant reduction (60.1% and 60.2%) in cellular uptake was observed at 4 ℃ and with colchicin, a microtubule-disrupting drug. In contrast, chlorpromazine, methyl-β-cyclodextrin, and cytochalasin-D either had no affect or slightly disturbed the endocytosis of GENs by C6 glioma cells. Samples treated with occludin and claudin-5 antibodies, which block the transmembrane protein of tight junctions involved in paracellular transport, exhibited approximately 98.2% and 91.2% cellular uptake compared to the controls. The internalization of DiD-labeled GENs significantly decreased at low temperatures and upon the addition of colchicine. It can be concluded that low-temperature conditions can hinder the internalization of GENs, and that the cellular uptake mechanism of GENs in BCEC is mediated by phagocytosis.
To evaluate the cytotoxicity of GENs, an MTT assay was performed to assess the growth of C6 glioma cells after treatment with various concentrations of GENs (ranging from 0.97 to 31.25 μg/mL) (Additional file 1: Fig. S5). The proliferation of C6 cells was not decreased and remained at about 100% after treatment with 31.25 μg/mL GENs for 24 h, and the inhibitory concentration 50 (IC50) was determined to be 53 μg/mL (data not shown). These results indicated that GENs are safe and do not compromise cell viability. Furthermore, GENs could provide a safe and effective platform for anticancer therapy.
Expression of apoptosis-related genes induced by GENs
To evaluate the effect of GENs on apoptosis in tumor cells, the induction of apoptosis was measured using western blotting and RT-qPCR. C6 glioma cells were incubated in the presence of GENs ranging from 0.24 to 62.5 μg/mL. Protein expression levels of BAX family genes were analyzed. As a result, the levels of the apoptosis-related gene BAX increased, whereas those of the anti-apoptotic genes such as BCL-2 and Survivin significantly decreased (Additional file 1: Fig. S6a). These observations indicate that GENs can activate BAX and downregulate BCL-2 and Survivin in C6 glioma cells in a dose-dependent manner (Additional file 1: Fig. S6b–g).
The results of RT-qPCR analysis showed consistent results with the western blotting findings. After 48 h of treatment with GENs, an upregulation of BAX mRNA was observed, whereas mRNA levels of BCL-2, BCL-xL, and Survivin were reduced by 29%, 61%, and 74%, respectively, compared with the control group (Fig. 2f). Overall, GENs appear to induce apoptosis in brain tumor cells (Fig. 2f).
GENs suppress cellular growth in glioma cells, fibroblasts, and endothelial cells
As shown in Fig. 3, we performed multiplex cytokine analysis to further assess the regulation of CAFs by GENs. We evaluated the mRNA expression levels of α-SMA, a marker of CAFs, and those of cell growth factors including c-MYC, TGF-β1/2/3, and integrin αVβ1/3/6 by RT-qPCR. After treatment with 10 μL of 1 mg/mL GENs for 48 h, we observed a significant decrease in the mRNA levels of c-MYC, TGF-β1/2/3, integrin αVβ1/3/6, and α-SMA. Similar trends were observed in cell growth factors in 3T3 cells and BCECs. However, the expression levels of some cytokines, such as TGF-β3 and integrin αVβ1 in 3T3 cells and TGF-β1 and α-SMA (a marker of CAFs) in BCECs, did not differ significantly.
Moreover, we co-cultured 3T3 and C6 at a 1:1 ratio to understand the role of GENs in mimicking TMEs. After treating the co-cultured cells with GENs at the same concentration as in the previous experiment, most cytokine genes, except those encoding integrin αV and integrin β6, were significantly downregulated (Fig. 3d). Collectively, GENs generally effectively suppressed cell growth factors, CAFs, and a marker of CAFs in vitro.
GENs suppress M2 macrophage polarization
Macrophage phenotype transition is considered to be one of the major factors in developing an effective treatment option for tumors. To understand the effect of GENs on macrophages, we treated BMMs with GENs in vitro and assessed M2 macrophage polarization. Figure 3e shows the morphological differences in BMMs treated with various stimuli, including GS-CSF and IL4. Unstimulated macrophages, MФs, showed a relatively smaller and round morphology, while M2-treated macrophages showed a flat, elongated, and branching morphology. After treating the BMMs with serially diluted GENs ranging from 0.1 to 5 μg/mL, we evaluated the gene expression levels of CD206 and IL10, indicators of M2 macrophage polarization. As shown in Fig. 3f, compared with the control group, the mRNA expression levels of CD206 and IL10 in the treatment groups decreased with increasing concentrations of GENs. These results indicate that GENs effectively down-regulate M2 macrophage polarization and the expression of pro-tumoral cytokine without causing cell damage.
Targeting effect of GENs in vivo
To evaluate the targeting ability of GENs in the brain tumor, we prepared 6–8-week-old Balb/C mice (18–20 g). DiD-labeled GENs were intravenously injected, and the fluorescence intensity of DiD-labeled GENs was measured using an in vivo imaging system (IVIS) at different time points over a time period of 24 h (Fig. 4a). Prior to each sample injection, we confirmed the comparable fluorescence intensity of conventional liposomes based on egg-yolks and GENs to ensure consistent experimental conditions (not shown). Compared with the conventional liposomes, GENs showed a rapidly increasing accumulation in the brain at all timepoints. In particular, GENs efficiently crossed the BBB and reached the tumor within 1 h after the administration. In addition, to determine the targeting effect and bio-distribution of DiD-labeled GENs, we dissected and imaged several organs including the heart, liver, pancreas, lung, kidney, and brain after 24 h of the injection of DiD-labeled conventional liposomes and GENs (Fig. 4a–c). GENs demonstrated a preferential targeting of brain tumors, showing a higher fluorescence intensity than conventional liposomes. By measuring the relative fluorescence intensities of the conventional liposomes and GENs in the brain by region of interest (ROI), we found that GENs resulted in almost 2.26-fold higher fluorescence intensity compared with the control group (Fig. 4c). We also observed a greater accumulation of GENs in the liver, spleen, lung, and partly in the heart compared to the conventional liposomes. These findings can be attributed to the physiochemical properties of GENs.
To further evaluate the selective and specific penetration ability of GENs in the steric tumor environment, we generated a mimic tumor model using cellular aggregates through 3D non-scaffold-based cell cultures (Fig. 4d). 10 days after seeding C6 glioma cells, the diameter of the spheroids was measured to be approximately 400 μm. DiD-labeled GENs and conventional liposomes were incubated with the tumor spheroids for 12 h. DiD-labeled GENs showed a much deeper penetration (118 ± 16.805 μm) compared with conventional liposomes (60 ± 13.445 μm) (Fig. 4d). We hypothesize that GENs have an advantage in infiltrating tumors through macropinocytosis and due to the overexpression of glucose transporters 1 (GLUT-1) in C6 glioma cells [1, 16, 17]. In support of this idea, when C6 glioma cells were pre-treated with Nocodazole and a GLUT-1 inhibitor, the cellular uptake of GENs was significantly inhibited (Additional file 1: Fig. S7). This suggested that macropinocytosis and the GLUT-1 pathway are the major pathways involved in the endocytosis of GENs in C6 glioma cells. Therefore, GENs demonstrate suitable physiological and chemical properties that enable enhanced intratumoral accumulation and fast penetration of the BBB.
Antitumor effect induced by GENs in vivo
To evaluate the anticancer effect of GENs, tumor-bearing Balb/C mice were treated with 5 μL of 2 mg/mL GENs (based on the protein concentration) through intracranial (IC) injection and 200 μL of 2 mg/mL GENs (based on the protein concentration) through intravenous (IV) injection every other day for 14 days after orthotopic tumor implantation (Fig. 5a). The progression of tumor growth was evaluated using an IVIS from day 0 to 8, and the luminescence intensities of tumors in the experimental groups were measured after the first injection of GENs (Fig. 5b). Figure 5b displayed the luminescence intensity of the tumor in a Balb/c mice, showing a higher luminescence intensity in the control group, indicating tumor growth, compared with the GENs’ treatment group. In mice receiving both IV and IC injections of GENs, the luminescence intensities of C6-Luc decreased markedly as the treatment continued. Notably, a significant decrease in the luminescence intensity of C6-Luc in the two experimental groups, especially in mice receiving IC injection within day 4 (Fig. 5b). The administration route appeared influence the efficacy of GENs, as they exhibited faster tumor reduction. The median survival time of the mice in the IV injection group was 105 days, significantly longer than that in the control group, with a median survival rate of 62.5 days (Fig. 5c).
To visualize changes in the tumor mass within the brain, we utilized the Wistar rat, and measured the tumor volume of C6 glioma before and after treatment with GENs. Orthotopic implantation of C6 glioma cells was performed in 8-week-old Wistar rats (200–250 g). 14 days after the injection of GENs, tumor growth was monitored using magnetic resonance imaging (MRI) with T2-weighted images 14 days after the injection of GENs (Fig. 6a). As shown in Fig. 6b, tumor volume in the treatment group decreased significantly from 64.16 ± 34.62 mm3 to 24.46 ± 13.73 mm3, displaying a marked contrast with the control group where tumor volume increased from 55.37 ± 75.46 mm3 to 67.02 ± 73.8 mm3.
In addition, three mice from each group (control, IV, and IC) were sacrificed to collect brain tissues for analysis. The brain tissues were stained with hematoxylin and eosin (H&E) and cut in the coronal plane. The results revealed that a significant decreased in tumor size in the treatment groups, whereas an increase in tumor size was observed in the control group (Fig. 6d, e). The H&E-stained brain tumor biopsy treated by GENs showed a reduced and small population of tumor cells, while the control group displayed significant histologic atypical changes and a large population of tumor cell, as indicated by strong nuclear staining.
Evaluation of liver toxicity of GENs in vivo
We further analyzed the cytotoxicity of GENs in vivo. (Additional file 1: Fig. S8) After 7 times of intravenous injections of GENs at the same concentration of treatment, the blood plasma was collected and the blood chemistry analysis including the blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), urea, creatine, and uric acid was performed. As a result, those indicators of liver function were not elevated, indicating that the GENs were not harmful and did not induce toxicity in the liver.
The regulation of CAFs leads to tumor-associated macrophages (TAMs)
Regulating the tumor microenvironment (TME) is considered to be an important approach for effective therapy. The TME consists of a heterogeneous population of cells, including infiltrating cancer cells, epithelial cells, endothelial cells, mesenchymal macrophages, and fibroblasts. Cytokines and chemokines are excessively secreted from various cells, and uncontrolled cell signaling induces oncogenesis and results in tumor progression. Thus, when evaluating the therapeutic effects against tumors, it is essential to consider various aspects. To validate the immunomodulatory effect of GENs, we first examined the gene expression levels of CAF in the TME in the mice. We assessed the mRNA expression levels of growth factors, angiogenic factors, and chemokines. Compared with the control group, we noted decreased mRNA levels of inducible nitric oxide synthase (iNOS), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), signal transducer and activator of transcription-3 (STAT-3), and chemokines including CCL2, CCL3, CXCL12, CXCL14, and the transforming growth factor beta (TGF-β) family in the treatment groups (Fig. 7a–c). Decreased iNOS expression is responsible for lower macrophage toxicity and can therefore induce an immune response to treat cancer and suppress tumor growth . However, there was no significant difference in the mRNA expression levels of iNOS between the treatment groups. Interestingly, the expression levels of STAT3, which can potentially control cell growth and act as an oncogenic driver by regulating several apoptosis-related pathways, were significantly decreased in the treatment groups .
Additionally, the mRNA levels of chemokines CCL2, CCL3, CXCL12, and CXCL14 were also obviously reduced in the treatment groups. All things considered, GENs might not only be a regulator of macrophage proliferation but also contribute to the downstream signaling pathway of apoptosis-related signal molecules in TAMs. Thus, GENs have the potential to become a great adjuvant therapeutic strategy by reducing the expression of cytokines and promoting tumor suppression.
To investigate the role of GENs in CAFs, the in vivo expression of α-SMA in the brain tissue was analyzed by immunohistochemical staining (Fig. 7d, e). Immunohistochemical staining of the brain tissue treated with GENs showed that the expression of α-SMA was significantly reduced compared to the brain tissue treated with PBS. The ratio of α-SMA-positive area was 31.6 ± 2.2% in the control group, whereas it was reduced to 12.3 ± 1.2%, and 8.9 ± 1%, in the GENs IV injection and GENs IC injection group, respectively. These results confirmed that treatment with GENs effectively suppresses CAFs in the brain tumor-bearing mice, suggesting that GENs can serve as a promising therapeutic approach to enhance the effectiveness of cancer treatment and improve prognosis.
We had a particular interest in understanding the regulation of macrophages by GENs in the TME. To gain further insights into immune regulation, we investigated the effects of GENs on T cells and regulatory T cells (Tregs) (Fig. 8). T cells plays a crucial role in defending against infections and cancer, while Tregs regulate the immune system. There is a close interaction between T cells and Tregs through several mechanisms. For example, Tregs can inhibit T cell function by secreting suppressive cytokines such as TGF-β, IL-10, and IL-35. In addition, Tregs can suppress anticancer immunity and impede immune surveillance, thereby promoting tumor growth and progression . Consequently, targeting Tregs holds promise as an approach for cancer therapy. As shown in Fig. 8, treatment with GENs, led to a significant increase in CD8 + T cells, known as cytotoxic T cells, within the TME compared with the control group, whereas CD4 + T cells, known as helper T cells, were decreased (Fig. 8a). The abundance of Tregs (FoxP3 + and CD25 +) was notably reduced in the treatment groups (Fig. 8b). These findings support the previously described regulatory mechanism between Tregs and T cells. Molecular analysis of macrophages displayed in Fig. 8c–e led us to expect greater therapeutic effects on T cells and Tregs in response to GENs treatment through IC injection. However, the groups receiving GENs via IV and IC injections showed similar abundances of T cells and Tregs. We speculated that, assessing the proliferation of T cells and Tregs would require, more than one week after the treatment to observe a discernible difference in activation. The results may also be attributed to the attenuation of exponential regulation of accumulation after treatment. Immune cells were stimulated with appropriately concentrated GENs, and the immune cell analysis results were consistent with their maximal response to antigens. Previous studies have demonstrated that the expression of CD8 + and CD4 + in T cells is not significantly altered by different administration routes when a high concentration of vaccine is administered [43, 44]. Therefore, although different administration routes were employed, GENs were administered at a high concentration to all tumor treatment groups, likely resulting in the immune cells reaching a plateau in terms of their final expression. This hypothesis is supported by previous reports [43, 45].
To assess the impact of GENs on TAMs and their cytokine release, we conducted molecular analysis using M1 markers (including CD86), M2 markers (including CD206), and a pan macrophage marker (F4/80). M1-like macrophages are known to promote an antitumor response in TAMs by supporting a type 1 T helper cell (Th1)-mediated anticancer response and facilitating the recruitment of cytotoxic T cells. Based on the M1/M2 ratio, the treatment groups exhibited a general increase in M1-macrophages compared with the control groups, indicating an adjuvant antitumor effect on the glioma (Fig. 8c–e). The mRNA expression levels of M2-macrophage markers, CD206 and IL10, were significantly decreased in the treatment groups (Fig. 8e). M1 macrophages were induced with RAW 264.7 cells by adding 100 ng/mL lipopolysaccharide and 50 ng/mL interferon gamma (IFNγ). Subsequently these M1 macrophages were treated with GENs in vitro. The results showed a significant increase in CD86 expression, a marker of M1 macrophages, at all concentrations of GENs (Additional file 1: Fig. S9). Overall, in the GENs treatment groups, tumor suppression was observed through the promotion of macrophage proliferation and modulation of the immune system, including T cells and Tregs, which are involved in angiogenesis regulation.
Gene silencing effect of ptc-miR396f on c-MYC
Previous studies reported [46, 47] the presence of bioactive compounds, including nucleic acids, in vegetables, which have shown potential in the treatment. Consequently, PENs containing genes could serve as a therapeutic RNA delivery agents themselves, benefiting from their inherent abundance of genes and biocompatibility. Furthermore, several specific genes including siRNAs and miRNAs derived from plants or vegetables have been functionally validated and offer new prospects for nucleic acid-based therapeutics targeting cancer and inflammation . In addition, Teng et al.  reported that ginger-derived exosome-like nanoparticles that are preferentially taken up by gut biota, exerting regulatory actions on bacterial mRNA through their miRNAs. It is confirmed that the miRNAs within these nanoparticles can induce the production of IL-22, which is associated with the improvement of intestinal barrier function. However, it is not clear whether the miRNAs in GENs play a role in suppressing tumor growth and inducing antitumor immunity in cells and the TMEs.
Hypothesizing that miRNA might be a regulatory factor, we analyzed the miRNA profile of GENs. Subsequently, we utilized the miRNA databases (TargetScan and miRanda) to identify miRNAs in GENs and predicted their potential targets based on sequence-based miRNA target prediction (Fig. 9a). miRNA targets were sorted by cumulative weighted context + + score. Among the miRNA targets, we selected the target gene and potential oncogene and further examined the target site accessibility of miRNAs in the coding transcripts [48, 49]. Then, a potential miRNA-target interaction was identified. c-MYC is a predominant oncogene and the c-MYC signaling pathway is one of the well-known pleiotropic gene-regulating signal pathways, including BCL2 and TGF-β (Fig. 9b) [50, 51]. In C6 glioma cells, a parallel reduction was observed in gene expression levels of c-MYC and BCL2 after transfection with the miRNAs named vvi-miR396b, ptc-miR396f, and ptc-miR396f, compared to non-targeting controls (Fig. 9c; Additional file 1: Tables S1–S5). The highest gene silencing effect was seen with ptc-miR396f in both target genes, with 58% and 76% reductions in c-MYC and BCL2. In addition, to make the result of RNA sequencing data more helpful and clear, a pie chart of the miRNA abundance by miRNA family and the ten most abundant miRNAs in GENs by the read count were presented in Additional file 1: Fig. S10 and Fig. S11.
Given that the lipid composition of GENs primarily consists of phospholipids (28.8% of PC), we anticipated that we could demonstrate the efficient delivery of miRNAs using the lipid component of GENs as a delivery vector . To examine whether the suppression of gene expression was induced as expected, the miRNAs were delivered via GENs. We extracted lipids from GENs using the Bligh and Dyer lipid extraction protocol and used the lipid content of GENs as a transfection reagent in conjunction with the miRNAs (Additional file 1: Fig. S12a). The delivery of ptc-miR396f via the extracted lipids exhibited the highest gene silencing effect on both c-MYC and BCL2 (Additional file 1: Fig. S12b).
In this study, we evaluated the anticancer activity and the regulatory effects of GENs on glioma. Various miRNAs involved in diverse signaling pathways related to apoptosis or cell proliferation were examined. The findings of this study confirmed that GENs are capable of suppressing tumor growth by inducing apoptosis in tumors, regulating macrophages and TAMs, and exerting gene silencing effects through exosomes. The results highlight the promising potential of GENs for the treatment of glioma.
Gene Ontology (GO) classification
A total of 7,072,614 genes in GENs were determined by GO enrichment analysis. The results indicated that these genes were involved in biological processes (BP), cellular components (CC), and molecular functions (MF) (Additional file 1: Fig. S14). In the categories, the most abundant subcategories were cellular component (5427 genes) and organic substance metabolic process (5408 genes). The top 5 most prominent enrichment subcategories involved in the BP category were organic substance metabolic process (5408 genes), primary metabolic process (5204 genes), cellular metabolic process (5033 genes), DNA metabolic process (1039 genes), and organic acid metabolic process (803 genes).
Scatter plot of differential gene Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
To further understand the mechanism of the differential expression of genes (DEGs) from the GENs described above, we implemented KEGG pathway enrichment testing. The degree of KEGG enrichment was evaluated by the qvalue, the rich factor, and the number of genes enriched in the pathway . The enrichment represents the ratio of the proportion of DEGs involved in the pathway to all genes. The higher rich factor is, the more significant the enrichment level of the DEGs in the pathway. The top 20 pathways with the most significant enrichment after screening are presented in Additional file 1: Fig. S15. These genes were enriched in the porphyrin and chlorophyll metabolism, propanoate metabolism, ABC transporters, alanine, aspartate, glutamate metabolism, and pyrimidine metabolism pathways.