Resveratrol encapsulated in novel fusogenic liposomes activates Nrf2 and attenuates oxidative stress in cerebromicrovascular endothelial cells from aged rats

Abstract

Resveratrol (3,4',5-trihydroxystilbene) is a plant-derived polyphenolic trans-stilbenoid, which exerts multifaceted antiaging effects. Here, we propose a novel delivery system for resveratrol, which significantly increases its cellular uptake into aged cells. Combination of resveratrol with a positively charged lipid component to "conventional" liposomes converts these lipid vesicles to a robust fusogenic system. To study their cellular uptake and cellular effects, we treated primary cerebromicrovascular endothelial cells isolated from aged F344xBN rats with resveratrol encapsulated in fusogenic liposomes (FL-RSV). To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dye, DiR, and resveratrol in cerebromicrovascular endothelial cells was confirmed using flow cytometry and confocal microscopy and high-performance liquid chromatography electrochemical detection. Treatment of aged cerebromicrovascular endothelial cells with FL-RSV activated Nrf2 (assessed with a reporter gene assay), significantly decreased cellular production of reactive oxygen species (assessed by a flow cytometry-based H2DCFDA fluorescence method), and inhibited apoptosis. Taken together, encapsulation of resveratrol into novel fusogenic liposomes significantly enhances the delivery of resveratrol into aged cells, which subsequently results in rapid activation of cellular Nrf2-driven antioxidant defense mechanisms. Our studies provide proof-of-concept for the development of a novel, translationally relevant interventional strategy for prevention and/or control of oxidative stress-related pathophysiological conditions in aging.

Keywords: Endothelial; Fusogenic liposomes; Oxidative stress; Polyphenol..

Figure 1.

Proposed model for enhanced membrane fusion between resveratrol-containing fusogenic liposomes and aged CMVECs. The synergistic interaction of the three components, the neutral lipid DOPE, the positively charged lipid DOTAP, and the aromatic resveratrol, results in an effective fusogenic mixture. Earlier studies of Brittes and coworkers (83) demonstrated that resveratrol is deeply located in the lipid bilayer, and only 10% of resveratrol molecules is found in the aqueous phase. The deep membrane location of resveratrol can be explained by its high lipophilicity, which is in turn related with the high partition coefficients determined for this compound. The positively charged lipids can polarize the delocalized π electrons of the highly aromatic structure of resveratrol inducing temporal dipoles in the liposome bilayers. These dipoles likely promote local instabilities in the molecular lipid arrangements (arrow), which will lead to subsequent fusion of the liposome and the opposing cell membrane. The model predicts that even short-term treatment of cells with resveratrol encapsulated in fusogenic liposomes will (i) deliver resveratrol in cells in sufficient quantities to activate Nrf2, an important cytosolic target of resveratrol, which will (ii) confer antioxidant effects, significantly attenuating age-related oxidative stress in CMVECs. CMVEC = cerebromicrovascular endothelial cell; DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammonium-propane.

Figure 2.

Effective resveratrol delivery to aged CMVECs using fusogenic liposomes (incubation time: 15 minutes). The effective concentration of resveratrol to induce membrane fusion was determined by flow cytometry by analyzing the cellular uptake of the fluorescent tracer DiR. (A) The percentage of DiR-loaded aged CMVECs as a function of applied resveratrol concentration (FL-RSV). Resveratrol delivered by conventional liposomes (PC-RSV) did not increase incorporation of DiR in cells. *p < .05 vs PC-RSV. (B) Liposomal delivery in CMVECs was visualized by detecting the fluorescent tracer DiR (red) using laser scanning microscopy. Resveratrol incorporated in positively charged liposomes (c_RSV = 100 µmol/L) induced homogenous DiR signal distribution in the plasma membrane of CMVECs (FL-RSV; upper panel). Resveratrol incorporated in conventional liposomes (PC-RSV; lower panel) was less effective (c_RSV = 100 µmol/L). Arrowheads: fusogenic liposomes attached to the cell surface. Arrows: incorporation of DiR in the cell membrane. *Debris. Scale bar: 100 µm. (C) Confocal images showing the effective uptake of resveratrol-containing fusogenic liposomes in endothelial cells of isolated vessel segments (incubation time: 15 minutes). Arrows: incorporation of DiR in endothelial cells (EC). Scale bar: 150 µm. CMVEC = cerebromicrovascular endothelial cell; DiR = 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide.

Figure 3.

(A) Trans-resveratrol (arrow) content, determined using high-performance liquid chromatography electrochemical detection, in aged CMVECs loaded with FL-RSV (upper panel) or PC-RSV (middle panel). The 5 nmol/L resveratrol standard is shown as reference (lower panel). (B) Normalized cellular resveratrol content upon loading aged CMVECs with FL-RSV or PC-RSV. *p < .05 vs untreated control. ^# p < .05 vs FL-RSV. Data are mean ± standard error of the mean. CMVEC = cerebromicrovascular endothelial cell.

Figure 4.

Reporter gene assay showing the effects of short-term incubation with resveratrol encapsulated in fusogenic liposomes or conventional liposomes (FL-RSV and PC-RSV, respectively; for 15 minutes, followed by washout) on Nrf2/ARE reporter activity in cultured primary CMVECs derived from aged rats. Cells were transiently cotransfected with ARE-driven firefly luciferase and cytomegalovirus-driven renilla luciferase constructs followed by liposomal resveratrol treatment. After a 2-hour period, the cells were then lysed and subjected to luciferase activity assay. After normalization, relative luciferase activity was obtained from four to six independent transfections. Data are mean ± standard error of the mean. *p < .05. ARE = antioxidant response element; CMVEC = cerebromicrovascular endothelial cell.

Figure 5.

(A) Short-term incubation with resveratrol encapsulated in fusogenic liposomes (FL-RSV; for 15 minutes, followed by washout) attenuates cellular production of free radicals in aged CMVECs (CM-H₂DCFDA staining, flow cytometry). The effect of short-term pretreatment with FL-RSV was comparable to the effect of chronic (for 24 hours) treatment with resveratrol encapsulated in conventional liposomes (PC-RSV). Short-term treatment of CMVECs with PC-RSV was ineffective. Data are mean ± standard error of the mean. The concentration-dependent decline in ROS production was significant (p < .05) for RSV, FL-RSV, and PC-RSV as well. (B) Time-dependent decreases in H₂O₂ production in aged CMVECs after treatment with FL-RSV. Changes in H₂O₂ production reached statistical significance (*p < .05) at 60 minutes posttreatment and continued to decline up to 24 hours. Cellular H₂O₂ production was assessed by the Amplex Red/horseradish peroxidase method. Dashed line indicates level of H₂O₂ production in CMVECs derived from young animals. CMVEC = cerebromicrovascular endothelial cell; ROS = reactive oxygen species.

Figure 6.

Short-term treatment of aged CMVECs with resveratrol encapsulated in fusogenic liposomes (FL-RSV) significantly inhibits apoptosis both under basal conditions and after H₂O₂ treatment. Shown are decreases in caspase 3/7 activity (a marker of apoptosis) in aged CMVECs posttreatment with FL-RSV. Compared with young CMVECs, aged CMVECs exhibited significantly increased caspase 3/7 activity (*p < .05). ^# p < .05 vs untreated aged, ^$ p < .05 vs aged + H₂O₂, and ^& p < .05 vs untreated young. Data are mean ± standard error of the mean (n.s. = nonsignificant). CMVEC = cerebromicrovascular endothelial cell.