Enhancement Of The Anti-Angiogenic Effects Of Delphinidin When Encapsulated Within Small Extracellular Vesicles Part 1

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Abstract:(1)Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (EV)properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells(HAoECs).(2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide(NO)production, and Matrigel assay were evaluated in HAoECs.(3)Results: Delphinidine, 3-O-β-rutinoside, and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs.sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation,10-fold for endothelial NO production, and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions:sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

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Keywords: delphinidin; endothelial cells; angiogenesis; small extracellular vesicles; cancer; cardiovascular diseases

1. Introduction

Polyphenols are found mainly in plant-derived foods and beverages and provide the taste and color of plant foods. Moreover, epidemiological studies have reported a greater reduction in cardiovascular risk and cancer associated with diets rich in polyphenols [1-3.

Delphinidin (2-(3,4,5-tri-hydroxyphenyl)chromenylium-3,5,7-triol) is an anthocyanin abundantly identified in pigmented vegetables and fruits, particularly berries and red grapes. We previously reported that delphinidin possesses the same pharmacological profile as a total extract of red wine polyphenolic compounds to promote the increase of intracellular calcium concentration and activation of tyrosine kinases [3], leading to endothelial nitric oxide (NO) production subsequent to estrogen receptor alpha (ERo)stimulation [4]. In addition, we reported that delphinidin via ERo acts as an immunomodulatory and anti-inflammatory molecule that can alter T lymphocyte proliferation and differentiation in patients with cardiovascular risk factors [5].

Finally, we demonstrated that delphinidin displays anti-angiogenic properties, both in in vitro and in vivo angiogenesis models, and reduces in vivo tumor growth of melanoma [6-10]. Indeed, delphinidin inhibits endothelial cell proliferation through the involvement of cyclin D1- and A-dependent pathways [6,7]. We also reported a possible association between inhibition of VEGF-induced mitochondrial biogenesis through the Akt pathway by delphinidin and its anti-angiogenic effect [8]. Moreover, delphinidin reduces tumor growth of melanoma tumor cells in vivo by acting specifically on endothelial cell proliferation. The mechanism implies an association between inhibition of VEGF-induced proliferation via VEGFR2 signaling, MAPK, PI3K, and at transcription level on CREB/ATF1 factors, and the inhibition of phopsphodiesterase2[9]. Most interestingly, high doses of delphinidin decreased neovascularization in an in vivo model of angiogenesis triggered by ischemia using a rat model of femoral artery ligature [10]. Together, these data show that delphinidin is a promising compound to prevent pathologies associated with cardiovascular disorders and tumorigenesis.

However, delphinidin is less potent to induce these beneficial effects compared to total red wine polyphenol extracts, especially in inducing endothelium-dependent NO-mediated vasodilatation [11]. Indeed, delphinidin is light-sensitive and stable only at pH<3; therefore, it degrades rapidly under physiological conditions. Moreover, delphinidin is poorly absorbed, and thus its modest bioavailability and stability reduce its effects both in vitro and in vivo. The measurement of delphinidin and its conjugated metabolites in plasma indicates its low bioavailability [12]. Hence, it is important to find new strategies to enhance delphinidin bioavailability and efficacy.

One strategy to overcome such problems is the use of extracellular vesicles (EVs)as a drug delivery system. We recently found that EVs, including large and small EVs (sEVs), are nanostructures originating from different subcellular compartment properties, overcoming the limitations of classical nano-formulations. sEVs decrease instability and immunogenicity, improve bioavailability and target selectivity [13]. Some reports under-score the protective effects of EVs released by cells treated with polyphenols. Indeed, sEVs enriched with miR-21 from cells treated with curcumin decreased tumor cell growth and angiogenesis, corrected endothelial permeability, and decreased the cell viability of different cancer cell lines [14]. In addition, miR-16-enriched sEVs from cells treated with epigallocatechin gallate suppressed tumor growth [15].

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In the present study, we took advantage of sEV properties to enhance both the stability and efficacy of delphinidin.sEV-loaded delphinidin induced angiogenesis inhibition using human aortic endothelial cells (HAoECs). Delphinidin content in terms of metabolites within these EVs was also determined.

2. Materials and Methods

2.1.Cell Culture

HAoECs(Promocell, Heidelberg, Germany)were cultured at 37°C and 5% CO2 in endothelial cell growth medium MV2 (Promocell)supplemented with 1%penicillin/streptomycin (Sigma-Aldrich, St. Quentin Fallavier, France). Cells were trypsinized at 70/80% confluence and were used between passages 3 and 6 for all experiments.

The JAWS II dendritic cell line was purchased from the American Type Culture Collection(CRL-1194; ATCC; Manassas, VA, USA). JAWS II cells were grown at 37°C and 5% CO, in a complete culture medium composed of alpha minimum essential medium (Lonza; Basel, Switzerland) containing ribonucleosides and deoxyribonucleosides and supplemented with 20% fetal bovine serum (FBS)(Gibco, Life Technologies; Grand Island, NY, USA),4 mM L-glutamine(Lonza),1 mM sodium pyruvate(Lonza), 1% penicillin/streptomycin(penicillin/-streptomycin, Sigma-Aldrich) and 5ng/mL murine GM-CSF (Miltenyi Biotec; San Diego, CA, USA). Cells were trypsinized at 70/80% confluence and were used between passages 8 and 16 for all experiments.

2.2.sEVIsolation

JAWS II cells were seeded at a density of 5×10° cells in a T175 cell culture flask in a complete growth medium, and they have starved in FBS before any isolation. Cell medium was centrifuged at 300×g and 2000×g for 10 min to remove cells and cell debris, respectively. The resultant supernatant was centrifuged at 20,000× g for 30 min to exclude large EVs. The supernatant was centrifuged at 200,000×g (Optima MAX-XP ultracentrifuge and MLA-50 rotor, Beckman Coulter, Villepinte, France) for 2h to pelletize sEVs. Then,sEVs were washed in phosphate-buffered saline (PBS)(NaCl 137 mM, KCl 2.7 mM, Na2HPO410 mM, KH2PO41.8 mM, pH=7.4) and recentrifuged at 200,000×g for 2 h. Finally, sEVpellets were resuspended in 1 mL of PBS and stored at 4°C until subsequent use. The amount of sEVs was determined using the method of Lowry, with bovine serum albumin (Sigma-Aldrich) as the standard.sEVs were used at 10 ug/mL.

2.3. Delphinidin Loading

Delphinidin was prepared in water at pH=2 with 0.1% DMSO in order to reach the concentration of 10 ug/mL. The sEVs were added (2 mg), and the solution was stirred and then vortexed for 10 min. After 2 h of ultracentrifugation at 200,000×g, the obtained pellet was reconstituted in 1 mL of 0.1%DMSO or PBS. Delphinidin absorbance was measured at 530 nm, and a standard curve with different concentrations(0.1 to 10 ug/mL) of free delphinidin was performed. The percentage of the efficacy of the loading of sEVs was 9%, independently of the concentration of delphinidin used (data not shown). Thus, the amount of delphinidin was adjusted to obtain the desired concentration (0.1 to 5 ug/mL)within 10 μg/mL sEVs. To remove free delphinidin， these vesicles were washed twice.

2.4. Nanoparticle Tracking Analysis (NTA)

sEV samples were diluted in sterile NaCl0.9%, and size distribution was analyzed using the NanoSight NS300 (Malvern Instruments Ltd., Malvern, UK). Videos were recorded. NTA software determined the size distribution using the Stokes-Enstein equation.

2.5. Transmission Electronic Microscopy

sEVs were first fixed overnight at 4°C with 2.5% glutaraldehyde (LFG Distribution, Lyon, France) in 0.1 M PBS. Then, sEVs were washed two times in PBS by 100,000×g centrifugation for 70 min.sEVs were deposited on copper grids for 2 min and negatively stained with 20 μL of uranyl acetate 5%（diluted in ethanol 50%）for 30 s. Grids were then observed with a Jeol JEM 1400 microscope(Jeol, Croissy sur Seine, France)operated at 120 keV.

2.6. Determination of Delphinidin Metabolites within sEVs

Sample preparation was as follows∶250 μL methanol （MeOH） was added to 10μg sEVs reconstituted in PBS, and samples were subjected to a 20 min ultrasonication. Two hundred uL of MeOH was further added, and samples were centrifuged(10,000× g, 10 min,4°C) and evaporated in a miVac duo concentrator(Genevac Ltd., Ipswich, UK). The dry extract was reconstituted with 200 μL LC-MS grade water containing 1% formic acid. The mixture was subjected to second centrifugation(10,000× g, 5 min, 4°C)prior to ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry(UHPLC-HRMS) analysis in order to analyze delphinidin metabolites with accurate mass measurements.

The chromatographic separation was achieved with a Kinetex@ 1.7 μm XB C18，150×2.1 mm column together with the corresponding SecurityGard C18 column (Phenomenex). Mobile phases consisted of HO in channel A and acetonitrile in channel B， both containing 0.1% formic acid. The elution gradient（A∶B， o/ø）was as follows∶ hold initial conditions 95:5 for 2 min, followed by a linear gradient from 95:5 to 0:100 over a 6 min period, hold at 0:100 for 3 min, return to initial conditions 95:5 and hold these conditions for 3.5 min. A constant flow rate of 0.300 mL/min was used; the injection volume was 10 μL.

Full scan and targeted SIM mass spectra were acquired in positive ionization mode, using resolution 70,000 Full Width at Half Maximum (FWHM) with automatic gain control (AGC) target of 3 × 10°ions and a maximum ion injection time (IT) of 200 ms. Data-dependent MS/MS experiments were acquired in'Top5's data-dependent mode.

Metabolites reported in the literature [16-18] were monitored:Delphinidin, alde-hyde, phloroglucinol aldehyde, gallic acid, chalcone, petunidin-3-galactoside, petunidin-3-arabinoside,petunidin 3-O-rutinoside, delphinidin-3-arabinoside, delphinidin-3-galactoside, delphinidin 3-O-(6-coumaroylglucoside),delphinidin 3-O-β-rutinoside, cyanidin-3-galactoside, cyanidin 3-O-β-rutinoside, Peonidin-3-galactoside and malvidin-3-galactoside.

Daily instrument calibration was performed by infusion of Pierce LTO Velos ESI positive/negative calibration kits as recommended by the manufacturer. Xcalibur 2.2 soft-ware (Thermo Fisher Scientific, San Jose, CA, USA) was used for data acquisition, and TraceFinder 3.0 software (Thermo Fisher Scientific)was employed for data processing.

2.7. Cell Viability Assay

1 ×104 HAoECs were seeded onto a 96-well plate and cultured for 24 h and treated with delphinidin （1 to 10 ug/mL）. Then， 5 μg/mL of 3-（4，5-dime-thylthiazol-2-yl）-5-（3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS reagent, Promega, WI, USA)was added into each well and incubated at 37°C for 120 min. The absorbance was measured on a CLARIOstar(BMG LABTECH, Ortenberg, Germany)spectrophotometer at 490 nm.

2.8. Proliferation Assay

Proliferation assays were conducted using CyQUANT Cell proliferation Assay kit (Invitrogen, Carlsbad, CA, USA)according to the manufacturer's recommendations. Briefly, 1.5 × 10* cells were seeded in a 96-well plate. Cells were serum-starved for 2 h and then treated with delphinidin, native sEVs or sEVs loaded with delphinidin at different concentrations. After 24 h of incubation, cells were washed with PBS, and a dye-binding solution was added. Cells were incubated at 37 °C for 30 min. A fluorescent microplate reader(CLARIOstar9, BMG LABTECH, Ortenberg, Germany)with filters for 485 nm excitation and 530 nm emission was used for fluorescence measurement.

2.9.NO Production Assaty

HAoECs were seeded on an 8-well slide （Ibidi， Gräfelfing， Germany） at a rate of 3×104 cells per well （i.e.，3×104 cells/cm²）in 300 μL of the medium. At70-80% confluence， cells were stimulated for 24 h with delphinidin, native sEVs or sEV-loaded delphinidin. Adenosine triphosphate（ATP）was used as a positive control （10 μM， Sigma-Aldrich） to stimulate the production of NO. After 24 h, the medium of each well was removed, and the diaminofluorescein diacetate （DAF-2 DA）probe was added （5 μM for 30 min， Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the wells were washed with PBS. Cells were fixed with paraformaldehyde(4%, 20 min). Fluorescence was read by confocal microscopy (Zeiss, Jena, Germany, LSM700). Four pictures were acquired, and ImageJ software was used for quantification.

2.10.Matrigel Assay

HAoECs were seeded in wells coated with Matrigel(gel of extracellular matrix of murine sarcoma of Engelbreth-Holm-Swarm， Sigma-Aldrich）. Briefly， 10 μL of liquid Matrigelwas placed in each well of a 15-well Ibidi μ-slide Angiogenesis plate （lbidi）and then incubated for 45 min at 37 °C to form a gel. HAoECs were then seeded and incubated at 37°C and 5%CO, for 45 min before treatments with either delphinidin, native sEVs or sEV-loaded delphinidin, followed by an incubation of 12 to 14h at 37 °C and 5%CO. The formation of"capillary-like structures" was observed with an optical microscope (Olympus CK40). Quantification was performed by measuring the number of capillary-like structures using Image Software.

2.11.Statistical Analysis

Results are expressed as mean ± SEM. The significance of the differences between groups was determined by analysis of variance(ANOVA), followed by Tukey's multiple compar-isons test. p-values of<0.05 were considered significant.

3. Results

3.1.sEV Characterization and Loading of Delphinidin

In agreement with the literature, delphinidin loaded within sEVs did not induce changes in the size of the vesicles, being118.7± 2.9 and 111.7± 1.8nm for empty sEVs and delphinidin-loaded sEVs, respectively, as determined by Nanoparticle Tracking Analysis (Figure 1A) and confirmed by electron microscopy analysis (Figure 1B). In addition, both types of sEVs, native and those loaded with delphinidin, expressed exosomal markers such as ALIX, CD63, and TSG101 at similar levels (Figure 1C), whereas they did not express ß-actin， a marker of large EVs.

Figure 1. Characterization of sEVs.(A) Size distributions of native sEVs and sEVs loaded with delphinidin based on NTA measurements. (B)Representative Transmission Electron Microscopy image of native sEVs and sEVs loaded with delphinidin. Scale bar=100 nm.(C) Western Blot analysis showing the expression of Alix， CD63, TSG101, and ß-Actin in sEVs and sEVs loaded with delphinidin.

This article is extracted from Nutrients 2021, 13, 4378. https://doi.org/10.3390/nu13124378