Enhancement Of The Anti-Angiogenic Effects Of Delphinidin When Encapsulated Within Small Extracellular Vesicles Part 2

Please contact oscar.xiao@wecistanche.com for more information

3.2. Delphinidin Metabolites within sEVs

In addition to delphinidin (Figure 2A), peonidin-3-galactoside (Retention time=11.11 min, [M]' m/z=463.12404)(Figure 2B) and delphinidin 3-O-β-rutinoside (Retention time=12.34 min, [M] m/z=611.16121)(Figure 2C) were detected under these experimental conditions in sEVs. Traces of these metabolites were also detected in the standard solution of delphinidin. The analysis was based on the exact mass, though an authentic standard solution is necessary to confirm this observation.

Please click here to know more

3.3. Effects of Delphinidin and sEV-Loaded Delphinidin on HAoEC Proliferation

Delphinidin alone or loaded in sEVs did not modify the viability of HAoECs for 24h at concentrations of 1 to 10 μg/mL （Figure 3A）.

VEGF (20 ng/mL), used as a positive control, non-significantly increased endothelial cell proliferation (Figure 3B). Native sEVs (10 ug/mL)had no effect. Delphinidin inhibited endothelial cell proliferation in a concentration-dependent manner, with a maximal effect reached at 10ug/mL(Figure 3B).In the same manner, sEV-loaded delphinidin induced a concentration-dependent inhibition of endothelial cell proliferation. Interestingly, the maximal inhibition was obtained at a concentration of 5 ug/mL of sEV-loaded delphinidin, a concentration two times lower than that for delphinidin alone. These results suggest that sEV-loaded delphinidin was two times more potent than delphinidin alone.

3.4.Effects of Delphinidin and sEV-Loaded Delphinidin on NO Production

As shown on Figure 4， ATP（10μM）induced an increase in DAF-2 fluorescence illustrating NO production in endothelial cells. Native sEVs did not affect NO level. Delphinidin alone increased NO production in a concentration-dependent fashion, with the maximal effect being reached at 10ug/mL. Delphinidin-loaded sEVs also elicited a concentration-dependent augmentation of endothelial NO production. Interestingly, the maximal effect of sEV-loaded delphinidin was obtained at 1 ug/mL, while the free delphinidin reached this effect at 10 ug/mL. Thus, sEV-loaded delphinidin was 10 times more potent than delphinidin alone in increasing NO production.

3.5. Effects of Delphinidin and sEV-Loaded Delphinidin on Angiogenesis

HAoECs restructured and formed capillary-like structures (Figure 5A,B). Native sEVs had no effect on the formation of capillary-like structures. As previously described [6], delphinidin alone reduced the number of branchings of capillary-like structures in a concentration-dependent manner, with the maximal effect being reached at 10 ug/mL with a 40% reduction. Interestingly, delphinidin-loaded sEVs exerted a potent reduction of the number of capillary branchings. Delphinidin at 0.1 ug/mL loaded within sEVs already decreased the number of branchings by 60%, and this effect was greater than that obtained with 10 μg/mL of delphinidin alone. Thus，sEV-loaded delphinidin was more than 100 times more potent in inhibiting angiogenesis than delphinidin alone.

Figure 5. In vitro angiogenesis assay. (A)Representative phase-contrast micrographs of tubular structures in cultured HAoECexposed for 24 h to delphinidin or sEVs loaded with delphinidin at different concentrations.Magnification:40×. Four areas from each well were analyzed. (B) The bar graph illustrated the significant decrease in the percentage of branch points after treatment compared to PBS-treated (control).Data are shown as mean ±SEM of sixindependent experiments in comparison with control: a: p<0.0001 vs.sEV;b:p<0.0001 vs.Del 1;c:p<0.0001 vs. Del 5;d:p<0.001 vs. Del5;e:p<0.01vs.Del5;f:p<0.05vs.Del5;g: p<0.05 vs.sEV Del0.1;h:p<0.001 vs.sEVDel0.5 and sEVDel5;i:p<0.0001 vs.sEVDel 1.

4. Discussion

The current study shows that the delphinidin loaded within sEVs was obviously more potent than free delphinidin regarding its ability to release endothelial NO, to inhibit endothelial proliferation, and reduce capillary-like structures. In addition to delphinidin found into sEVs, the analysis of delphinidin metabolites within the sEVs showed the presence of two metabolites(i.e., delphinidin 3-O--rutinoside and peonidin-3-galactoside)present in delphinidin samples. Thus, delphinidin degraded into the same metabolites in its free form or when loaded into sEVs; however, these were more potent in acting on endothelial cells. Of importance, when encapsulated within sEVs, delphinidin (used as a generic term and encompassing natural metabolites delphinidin 3-O-ß-rutinoside and peonidin-3-galactoside) was 2-fold, 10-fold, and 100-fold more potent than free delphinidin regarding endothelial proliferation, endothelial NO production and capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on different steps leading to angiogenesis. These results indicate that sEVs may be considered as a promising delivery of delphinidin as an innovative approach to target diseases associated with increased angiogenesis, including cancer, atherosclerosis, and diabetic retinopathies.

Cistanche can improve immunity

The encapsulation of polyphenols to protect them from degradation is a natural phenomenon. Indeed, it has been shown that plants rich in polyphenols produce EVs carrying these molecules. For instance, the flavonoid glycoside naringin and its metabolite, naringenin, are found in grapefruit-derived EVs [12]. Moreover, nanoparticles derived from plants can be used as vectors for other molecules of interest. Indeed,it has been reported that grapefruit-derived nanoparticles loaded with a STAT3 inhibitor inactivate STAT3 in GL26 tumor cells and improve the survival rates of mice [19]. Another strategy for the encapsulation of polyphenols is to use vesicles derived from mammalian cells. A recent report evaluated the exosomal formulation of anthocyanidins against different types of cancer [19]. sEVs harvested from raw bovine milk loaded with a mixture of cyanidin, delphinidin, petunidin, peonidin and malvidin increase the anti-proliferative activity of anthocyanidins against six different types of cancer cells via the inhibition of TNFo-induced activation of NF-kB [20]. Indeed, the effects of sEVs loaded with anthocyanidins are more effective than those obtained by free anthocyanidins. This method has advantages; however, it remains risky. Indeed, the use of EVs from mammalian cells can cause immune reactions.sEVs de-rived from immature human dendritic cells did not induce any toxicity, and the immature nature of dendritic cells induced low immunogenicity [21,22]. To our knowledge, this is the first time that a loading efficiency for delphinidin within JAWS ⅡI sEVs has been described. Loading of delphinidin into the sEVs(9%) protects and probably limits its degradation into metabolites under the experimental conditions used. The mechanisms involved require further study. The metabolites found in the sEVs, such as delphinidin 3-O-β-rutinoside or peonidin-3-galactoside, are also found in in vivo experiments with delphinidin 14,15]. Pre-vious works have reported that degradation products of delphinidin have potent biological activities, including anti-cancer and anti-inflammatory activities [20]. Among phenolic acids, gallic acid is mostly formed by the degradation of delphinidin in culture media [23]. In the present study, we found that metabolites detected in sEV-loaded delphinidin were identical to those detected from free delphinidin [18]. Although the exact proportion of metabolites encapsulated in these sEVs was not determined, they were more effective on target cells than metabolites alone. Thus, delphinidin and its metabolites were probably more stable and protected from degradation.

We previously reported that, in bovine aortic endothelial cells, delphinidin stimulates NO release by increasing intracellular Ca2+ concentrations via the increase of superoxide anion formation. This was associated with increased tyrosine phosphorylation of several intracellular proteins, resulting in endothelium-dependent vasodilatation [24,25]. Delphinidin interacts directly with the activator site of ERα, leading to the activation of endothelial NO-synthase, NO production and endothelium-dependent vasorelaxation 4]In the present study, sEV-loaded delphinidin was 10 times more potent than free delphinidin; thus, it would probably be more effective in correcting the NO-endothelial dysfunction associated with cardiovascular diseases, including hypertension, stroke or metabolic diseases [3].

We previously reported that upregulation of the NO pathway is not responsible for the antiproliferative effect of delphinidin. Indeed, delphinidin inhibits endothelial cell proliferation by the activation of the ERK-1/-2 pathway, leading to cell cycle arrest and accumulation of cells in the G0/G1 phase via down-regulation of cyclin A and D1 expression and an upregulation of p27kip1 [6,7]. We also found that delphinidin reduces tumor growth of melanoma cells in vivo by acting specifically on endothelial cell proliferation via the inhibition of VEGFR2 signaling, MAPK, PI3K and at transcription level on CREB/ATF1 factors, and the inhibition of phosphodiesterase 2 [9]. In the present study, delphinidin-loaded sEVs were two-fold more potent than free delphinidin in inhibiting endothelial proliferation. Thus, these results indicate that delphinidin-loaded sEVs are a promising approach to pre-vent pathologies associated with excess endothelial proliferation and, therefore, generation of the vascular network such as plaque development and stability in atherosclerosis and tumor development in cancer.

In concordance with these findings, we show that delphinidin decreases capillary-like formation in an experimental model of angiogenesis. Interestingly, when encapsulated within sEVs(even at loading as low as 9%), delphinidin was 100-fold more potent than free delphinidin in decreasing capillary-like formation.

Limitation of the study: The anti-angiogenic potential exhibited by many natural compounds contained in many Mediterranean diet constituents, including delphinidin, makes this dietary pattern especially interesting as a source of chemopreventive agents, defined within the angioprevention strategy. This has been recently reviewed by Martinez-Podeva et al. [26]. Delphinidin appears to be as potent as other flavonoids in inducing anti-angiogenic properties. Although abundant in the diet, anthocyanins in general, and delphinidin in particular, are poorly absorbed. One consequence of the poor bioavailability of anthocyanins is that many effects observed in vitro(e.g., inhibition of COX-2)are unlikely to occur in vivo, which is not the case for delphinidin, based on our former studies [6-10]. However, additional studies using delphinidin encapsulated in sEV are needed to confirm the increase in the anti-angiogenic properties of this approach in vivo.

In summary, sEV-loaded delphinidin increased the efficacy of delphinidin 100-fold for proliferation, 10-fold for NO, and 2-fold for capillary-like formation. Thus, sEVs either protected delphinidin and its metabolites from degradation, or some unidentified delphinidin metabolites contained in the sEVs were more potent. The differential potency obtained for proliferation, NO production, and angiogenesis supports the hypothesis that delphinidin-loaded EVs exert effects on different steps leading to angiogenesis. Nevertheless, we provide evidence that we optimized delphinidin efficacy, probably by reducing its degradation and increasing its delivery when encapsulated in EVs. Thus, delphinidin-loaded sEVs represent a powerful delivery system to decrease angiogenesis in endothelial cells, with no unwanted side effects, knowing the low bioavailability of this compound. We underscore an innovative therapeutic strategy based on bio-engineered EVs as vectors of delphinidin in helping to increase its potential health benefit to target angiogenesis-related diseases, including cancer, which could eventually be extended to further diseases with excess vascularization.

This article is extracted from Nutrients 2021, 13, 4378. https://doi.org/10.3390/nu13124378 https://www.mdpi.com/journal/nutrients