Extraction and drug determination of exosomes
The SHRss micelles used for drug loading were synthesized using disulfide cross-linked stearyl-peptides containing arginine, histidine, and cysteine, according to our previous report . Further, DTX-loaded, and DTX/siPLK1 co-loaded SHRss micelles (Co-PMs) were prepared using the probe-based ultrasonication technique.
Before the addition of Co-PMs to Raw264.7 cells, the sensitivity of Raw264.7 cells to DTX and Co-PMs was evaluated by performing a 24 h cytotoxicity assay (Fig. 2). As expected, Raw264.7 cells were shown to be extremely resistant to DTX and Co-PMs within the 24 h period. A low percentage of cell death was observed at the concentration of 8000 ng/mL DTX, whereas the IC50 value of DTX in various tumor cells has been reported as 50–700 ng/mL [21-23]. Thus, it was suggested that DTX would not affect the viability of Raw264.7 cells at the administered dose of 2500 or 5000 ng/mL.
As already known, cell contents usually appear in the released exosomes , and since Co-PMs contain both DTX and chemically modified stable siPLK1, it was expected that Raw264.7 cells that have accumulated Co-PMs, could release DTX and siPLK1 with their exosomes. Accordingly, we confirmed the presence of DTX and siPLK1 in exosomes produced by Raw264.7 cells.
Fig. 3A shows the DTX-loaded in exosomes as detected by HPLC analysis. DTX loading in exosomes obtained from cells treated with Co-PMs increased significantly compared with exosomes from cells treated with DTX alone, whether at concentrations of 2500 or 5000 ng/mL. After linking DSPE-PEG-FA, the DTX content in the Co-Exo-FA slightly decreased, which might have been due to the release of DTX from the Co-Exo during the procedure. As confirmed in our previous studies 21, SHRss micelles have been demonstrated to be able to deliver more chemotherapy drugs into cells. Therefore, more DTX was delivered into macrophages by Co-PMs to result in the secretion of exosomes containing more DTX. On the other hand, Co-PMs encapsulate DTX within the micelles, reducing the intracellular free DTX and thereby reducing the total DTX excreted by the powerful P-gp efflux pathway of the macrophage [24, 25].
Moreover, the amount of siPLK1 in the exosomes secreted by Raw264.7 cells transfected with co-loaded SHRss micelles was determined by qRT-PCR analysis. As shown in Fig. 3B, the amount of siPLK1 increased with the increase in the transfection dose, as compared with controls. Since free siPLK1 cannot be easily absorbed by cells, exosomes produced by Raw264.7 cells treated with free siPLK1 contained only a small amount of siPLK1. In order to ensure the stability of siPLK1 during endocytosis and exosome secretion, we chose chemically modified siPLK1, which is not easily degraded by enzymes and can remain stable in cells for an extended period of time . This result demonstrated that exosomes secreted from Raw264.7 cells that had incorporated DTX/siPLK1 co-loaded SHRss micelles contained intact siPLK1.
Ultrastructural analysis of exosomes
Raw264.7 cells with internalized Co-PMs were incubated with serum-free medium for 24 h to trigger the release of exosomes. Exosomes were extracted from the conditioned medium using the differential centrifugation protocol. Dynamic light scattering (DLS) analysis indicated that the hydrodynamic diameter of Co-Exo-FA (153 nm) was a little longer than that of Co-Exo and blank Exo (Fig. 4A, 4B). The measured particle size was in good agreement with that obtained by transmission electron microscopy (TEM) imaging. Similarly, the zeta potential detected for Co-Exo-FA decreased from -11.2 mV to -16.3 mV compared to Co-Exo, and this change might be caused by the presence of the negatively charged PEG-FA.
Exosomes were also observed using TEM, and results indicated that exosomes regularly exhibited a spherical shape with a complete double-layer membrane structure (Fig. 4C). Likewise, the various Co-Exo exhibited no significant difference compared to blank Exo, whereas the edges of the Co-Exo-FA appeared to be more blurred. This indicated that PEG-FA was successfully distributed around the exosomes, and thus could help exosomes escape clearance by the reticuloendothelial system (RES), as well as facilitate better tumor targeting.
In addition, the marker proteins of the exosomes were detected by western blotting analysis. As shown in Fig. 4D, CD9 molecule (CD9), CD63, CD81, and tumor susceptibility gene 101 (TSG101), which are exosome-specific proteins (exosome markers), were clearly detected in exosomes as compared to the respective levels in Raw264.3 cells. Moreover, calnexin-1, an endoplasmic reticulum marker protein was only detected in the Raw264.3 cell debris.
Co-Exo-FA targeting of prostate cancer cells in vitro
To study the cellular uptake of exosomes, DU145 and PC-3 cells were incubated with exosomes labeled with Nile red and FAM-siPLK1 for 4 h before analysis using flow cytometry. As shown in Fig. 5, there was an enhanced uptake of Co-Exo-FA by PC-3 and DU145 cells compared with unmodified Exo after 4 h of incubation, indicating that the PEG-FA modification contributed to the cellular uptake of Co-Exo-FA.
After 4 h of incubation, the qualitative internalization of Co-Exo-FA and their intracellular localization in PC-3 cells were evaluated using confocal laser scanning microscopy (CLSM) (Fig. 6). Blue fluorescence revealed nuclei stained by DAPI, and red fluorescence was due to the intracellular Nile red dye, whereas green fluorescence marked the internalized FAM-siPLK1.
In accordance with our flow cytometry analysis, both red (Nile red) and green (FAM-siPLK1) fluorescence was stronger in the Co-Exo-FA treated group relative to other groups, demonstrating the excellent targeting properties of Co-Exo-FA. In the merged view, the overlay of red and green fluorescence formed a yellow fluorescence, indicating that Nile red and FAM-siPLK1 were effectively co-delivered into PC-3 cells. On the other hand, the presence of widespread red and green fluorescence showed that part of the contents dissociated from the Co-Exo-FA upon delivery and spread into the cells.
Good biocompatibility of the membrane surface is crucial for the potential application of nanoparticles. As expected, the internalization mechanisms of free DTX and drug-loaded exosomes were different. Internalization of DTX was driven via passive diffusion, whereas the mechanisms of cellular uptake of exosomes were clathrin-mediated endocytosis and macropinocytosis . Moreover, it is known that the surface of prostate cancer cells carries folate receptors, and folate modifications have been verified to lead to increased cellular uptake of nanoparticles . Based on the results obtained from both the flow cytometry analysis and CLSM, it was suggested that folate receptor-mediated endocytosis played a vital role in the enhanced internalization of Co-Exo-FA. Above all, the most important outcome was that both DTX and siPLK1 were effectively co-delivered by Co-Exo-FA in vitro.
Effects on cell viability and apoptosis
To investigate the capacity of exosomes to influence the viability of prostate cancer cells, PC-3 and DU145 cells were treated with different exosome subtypes, and their viability was measured at 48 h. As shown in Fig. 7, blank exosomes did not affect the viability of DU145 and PC-3 cell lines, suggesting high compatibility and low cytotoxicity. Nevertheless, the viability of PC-3 and DU145 cells was obviously inhibited by either drug-loaded exosomes or DTX alone, in a concentration-dependent manner. PC-3 and DU145 cells treated with DTX-Exo had a lower IC50 (38.5 ng/mL and 26.1 ng/mL, respectively) than cells treated with DTX alone (39.8 ng/mL and 31.6 ng/mL, respectively), which might be due to the different cellular uptake between chemotherapeutic drugs and drug-loaded nanoparticles and the sustained release of DTX from the exosomes. Interestingly, co-loading of siPLK1 achieved a synergistic effect, which enhanced the cytotoxicity of DTX-Exo (IC50: 33.2 ng/mL and 19.7 ng/mL, respectively) in both cell lines. Among them, Co-Exo-FA exhibited the sharpest viability decrease (IC50: 22.1 ng/mL and 17.1 ng/mL, respectively), indicating that the introduction of folate contributed to the reduction of cell viability by approximately 33.4% in PC-3 and 13.2% in DU145 cells.
Annexin V-FITC/Propidium Iodide (PI) staining and flow cytometry analysis were used to evaluate the effect of the various exosome subtypes on cell apoptosis (Fig. 8). Results showed the untreated cells and blank Exo-treated cells did not show a noticeable change in overall apoptosis and necrosis (no more than 10% in both groups) (Fig. 8). In contrast, extensive cell apoptosis was observed in drug-treated groups in PC-3 cells, and this was consistent with previous reports that DTX can induce cell apoptosis and death. Moreover, the combined loading of siPLK1 and DTX into exosomes increased cell apoptosis by 31.2% in PC-3 cells, compared with DTX-Exo alone. In addition, a 55.5% increase in cell apoptosis was observed in the Co-Exo-FA treated group, indicating that incorporation of the folate group in Co-Exo resulted in increased induction of cell apoptosis.
Downregulation of the target gene
PC-3 cells were treated with blank Exo, DTX, DTX-Exo, siPLK1 loaded Co-Exo, and Co-Exo-FA. Consecutively, the expression of PLK1 was analyzed by qRT-PCR. Fig. 9A shows that the PLK1 mRNA levels in cells treated with siPLK1 loaded Co-Exo, and Co-Exo-FA were significantly reduced compared to those in cells treated with blank Exo, DTX, and DTX-Exo. In particular, the expression of PLK1 mRNA was reduced by 33.3% and 69.7%, respectively. We believe that siPLK1 was loaded in Co-Exo in sufficient quantities to subsequently downregulate the expression of PLK1 mRNA and that the folate enhanced this gene regulation effect at the translational level.
For western blotting analysis, the GAPDH housekeeping gene was used as a control. We explored the potential regulation of the PLK1 gene by exosomes that harbor siRNA (siPLK1) against PLK1. The protein levels of PLK1 were also downregulated in the Co-Exo-FA treated PC-3 cells, compared to other groups (Fig. 9B). These results demonstrated that Co-Exo downregulated PLK1 at translational and post-transcriptional levels. This finding was consistent with the results showing that Co-exosomes could function as therapeutic vesicles.
Co-Exo-FA targeting of tumors in a murine xenograft model
The in vivo tumor targeting performance of Co-Exo-FA was investigated in a murine xenograft model established by subcutaneously inoculating PC-3 cells into the right flanks of BABL/C nude mice. Blank exosomes and Co-Exo-FA were labeled with DIR, which is usually used as an indicator for tracing nanoparticles in vivo . Next, using in vivo imaging systems, we observed the fluorescence of tumor-bearing mice at 1, 4, 8, and 24 h after intravenous administration of DIR, Co-Exo, and Co-Exo-FA. As shown in Fig. 10A, the strong fluorescence of DIR was visualized in all mice subjected to intravenous injection. More specifically, 1 h after the injection, a strong DIR fluorescence signal was observed at the tumor site in the Co-Exo-FA/DIR injected mice, and this signal was found to be strongest at 8 h post-administration. The signal at the tumor site was much stronger than that in the rest of the tissues. Once the mice were injected with Co-Exo-FA/DIR, a large amount of DIR appeared to be still accumulated in the tumor even 24 h post-administration. On the other hand, only faint DIR fluorescence was observed at the site of the tumor in the free DIR injected mice, and this fluorescence signal was mainly noted in the liver and kidneys. The intensity of the DIR signal in the tumors of the free DIR-treated mice did not change with time. Mice injected with Co-Exo/DIR exhibited a slight fluorescence accumulation in the tumor site after injection, which might have been a result of the EPR effect.
Tumors and organs were resected and scanned for visualization of their DIR fluorescence signal 24 h post-administration. As shown in Fig. 10B, Co-Exo-FA/DIR strongly accumulated in tumors, but exhibited very low accumulation in the other organs, including the heart, lung, and kidneys. However, strong fluorescence intensity was observed in the liver and kidneys of mice in the free DIR-treated group, which was much higher than that in the tumor site.
These data indicated that the Co-Exo-FA formulation significantly improved the efficiency of tumor accumulation. The PEG present on the surface of exosomes provided protection from elimination by the RES; therefore, Co-Exo-FA could make full use of the EPR effect. Meanwhile, the folate also contributed to the cellular uptake of Co-Exo-FA. Thus, we believe that, consistent with previous findings, the folate contributed to the efficiency of the tumor targeting .
Tumor growth inhibition in vivo
Next, we evaluated the anticancer effects of Co-Exo-FA in in vivo tumor growth experiments. PC-3 cells were subcutaneously engrafted on the back of male BALB/c nude mice. Subsequently, Co-Exo-FA was intravenously injected every 4 d. Tumor sizes and body weights in each group were monitored periodically.
As shown in Fig. 11A, intravenous injection of blank exosomes had no significant inhibiting effect in tumor growth compared with the phosphate-buffered saline (PBS)-injected group. However, DTX, DTX-Exo, Co-Exo, and Co-Exo-FA treated mice exhibited different levels of tumor growth inhibition. The most significant antitumor activity was observed in the Co-Exo-FA treated group. The average tumor volume at day 27 was 72.3 mm3 in the Co-Exo-FA group, which was 17.4-, 15.7-, 7.1-, 4.1-, and 2.8-fold smaller than that in groups treated with PBS, blank Exo, DTX, DTX-Exo, and Co-Exo, respectively. The data obtained from our tumor xenograft mouse model confirmed that co-loading of DTX and siPLK1 into exosomes resulted in a synergistic effect against prostate cancer in vivo. The isolated tumors at day 27 were significantly smaller in the Co-Exo-FA group than in other groups, consistent with the observed growth curve of tumor volume (Fig. 11B).
The body weight measurements were used as an indicator of systemic toxicity by Co-Exo-FA. Fig. 11C shows that during the experiment, no significant body weight loss was observed in any of the treated mice compared to control groups, except for mice that were systemically administered free DTX. Therefore, it was suggested that DTX-loaded in exosomes could remarkably reduce the systemic toxicity of free DTX in vivo.
Tumor tissues were further evaluated through hematoxylin and eosin (H&E) staining and IHC staining. As shown in Fig. 11A, both the blank Exo-treated and the control group exhibited a high density of cells displaying intact structures, and both the nuclei and the cellular outline remained clearly visible. In contrast, karyolysis, karyorrhexis, and cell disruption were observed in the tissues of tumors treated with DTX, DTX-Exo, Co-Exo, and Co-Exo-FA. In particular, cells treated with Co-Exo-FA lost their nuclei and cytoplasm, becoming an amorphous material, thus indicating the most evident and significant signs of necrosis and apoptosis. H&E staining was also used to evaluate the influence of the administered exosomes to essential organs, including the heart, liver, spleen, lung, and kidneys. Fig. 12A shows that there was little necrosis of vital organ tissues in all the Exo-treated groups. However, we observed necrosis in the liver tissue of mice in the free DTX group. Since DTX was loaded in exosomes, the majority of which localized in tumor tissues, only low-level systemic toxicity was expected.
To detect the expression of PLK1 protein in tumor tissues, we performed IHC staining. As shown in Fig. 12B, the expression of PLK1 was significantly reduced in the tumors treated with Co-Exo-FA compared with other groups. Meanwhile, the administration of free DTX or DTX-Exo showed no visible effect on the expression of PLK1.
By promoting tumor apoptosis and downregulating the expression of the PLK1 gene, this study revealed the synergistic antitumor effects of the Co-Exo-FA delivery system. These results confirmed that Co-Exo-FA could function as an efficient chemotherapeutic and siRNA delivery system.