Lipid-Encapsuled Grape Tannins Prevent Oxidative-Stress-Induced Neuronal Cell Death, Intracellular ROS Accumulation and Inflammation

The central nervous system (CNS) is particularly vulnerable to oxidative stress and inflammation, which affect neuronal function and survival. Nowadays, there is great interest in the development of antioxidant and anti-inflammatory compounds extracted from natural products, as potential strategies to reduce the oxidative/inflammatory environment within the CNS and then preserve neuronal integrity and brain function. However, an important limitation of natural antioxidant formulations (mainly polyphenols) is their reduced in vivo bioavailability. The biological compatible delivery system containing polyphenols may serve as a novel compound for these antioxidant formulations. Accordingly, in the present study, we used liposomes as carriers for grape tannins, and we tested their ability to prevent neuronal oxidative stress and inflammation. Cultured catecholaminergic neurons (CAD) were used to establish the potential of lipid-encapsulated grape tannins (TLS) to prevent neuronal oxidative stress and inflammation following an oxidative insult. TLS rescued cell survival after H2O2 treatment (59.4 ± 8.8% vs. 90.4 ± 5.6% H2O2 vs. TLS+ H2O2; p < 0.05) and reduced intracellular ROS levels by ~38% (p < 0.05), despite displaying negligible antioxidant activity in solution. Additionally, TLS treatment dramatically reduced proinflammatory cytokines’ mRNA expression after H2O2 treatment (TNF-α: 400.3 ± 1.7 vs. 7.9 ± 1.9-fold; IL-1β: 423.4 ± 1.3 vs. 12.7 ± 2.6-fold; p < 0.05; H2O2 vs. TLS+ H2O2, respectively), without affecting pro/antioxidant biomarker expression, suggesting that liposomes efficiently delivered tannins inside neurons and promoted cell survival. In conclusion, we propose that lipid-encapsulated grape tannins could be an efficient tool to promote antioxidant/inflammatory cell defense.


Introduction
During life, the central nervous system (CNS) is constantly exposed to reactive oxygen species (ROS) and other free radicals emerging from many sources, including exposure to oxidizing agents (smoke, ionizing radiations, toxic agents) and body-borne oxidants, tissues [32]. Liposomes are self-organizing colloidal particles that constitute of one or more lipid bilayer membranes, which surround an aqueous compartment that can be utilized to encapsulate both hydrophilic, hydrophobic and amphiphilic compounds [33]. The use of liposomes provides several advantages such as low toxicity, biocompatibility, lower clearance rates, the ability to target specific tissues and the controlled release of drugs [32,34]. Importantly, liposome structure can also serve to deliver drugs into the brain since they are able to cross the blood-brain barrier (BBB), suggesting a strong potential of liposomes for the delivery of polyphenolic mixtures to the brain, which then act as neuroprotectors. Indeed, previous studies have demonstrated that tannic-acid-charged liposomes can reduce Tau aggregation in human neuroblastoma cells, and have shown to significantly reduce intracellular ROS production upon oxidative stress induction [35,36]. In addition, recent reports have shown that orally administered tannins are capable to reduce brain neuroinflammation [29], supporting a high potential of grape tannins as novel protective agents against neuroinflammation. However, limited information is available regarding the effects of lipid-encapsulated grape tannins on in vitro and in vivo models of neuroinflammation. In this study, we used liposomes to encapsulate/package grape polyphenols to tackle their poor bioavailability and low stability. We thus tested the neuroprotective capacity of lipid-encapsulated grape tannins formulation against oxidative stress and neuroinflammation in a catecholaminergic neuronal cell line (CAD) [37], by evaluating their effects on cell viability under oxidative stress conditions, intracellular ROS production and oxidative/inflammatory gene expression.

Liposome Preparation and Tannins Encapsulation
Tannin-containing liposomal nanosuspensions (TLS) were prepared using the heating/homogenization method with lamellarity and size reduction by ultrasound cycles [38]. To prepare 100 mL of TLS [1 mg/mL], 0.1 g of condensed tannins obtained from green grape seeds (Sauvignon blanc variety) were dissolved in 20 mL of a solution (50:50% v/v) of ethanol-citrate buffer (0.1 M at pH 3) at 70 ± 1 • C and 700 rpm, respectively. Once dissolved, 1 g of phosphatidylcholine (PC) was incorporated and stirred at 700 rpm without temperature for 5 min. The suspension was then heated at 80 • C for 1 h in a thermoregulated bath. After this time, 0.76 g of glycerol was added as a lipoprotectant [39] dissolved in 40 mL of citrate buffer (0.1 M at pH 3.0), and heated again at 80 • C for 1 h. After this second heating period, the remaining 40 mL of buffer was added to complete the volume and 5 vortex cycles, and 20 ultrasound cycles were applied, respectively, using an ultrasonic cell disruptor (HIELSCHER UP100H, Teltow, Germany, max. 100 W) with a sonotrode. MS7 Micro tip 7 (7 mm diameter, 120 mm length, 130 W/cm 2 acoustic power density) working at 90% amplitude and 22.5 Hz. Finally, TLS were purified by centrifugation at 2500× g for 15 min at 15 • C and redispersed in MilliQ water. PC was obtained by purification of raw food-grade soy lecithin following the procedure described by López-Polo et al. to obtain saturated lipids [40]. Crude soy lecithin (10 g) was dissolved in 50 mL of ethyl acetate at 20 • C. Distilled water (2 mL) was then slowly added with manual stirring, resulting in the formation of two phases. The lower phase was separated and dispersed in 30 mL of acetone, forming clusters that were crushed with a glass rod. Then, the acetone was separated by decantation and a new aliquot (30 mL) of acetone was added, repeating the trituration process. The precipitate was vacuum filtered and dried in a desiccator at 20 • C for 48 h to finally obtain soy phospholipids (dipalmitoyl lecithin (1,2-dipalmitoyl-snglycerol-3-phosphocholine, DPPC, M.W.:734.05 g/mol, purity ≥ 99%). In total, 1 mg/mL of condensed tannin was encapsulated; this concentration was preliminarily determined with a multilevel factorial statistical design, which contemplated concentrations from 0.5 to 5 mg/mL of tannins. For determination of mean degree of polymerization, a suspension of condensed tannins (TS) was used as control. Tannic acid (hydrolysable) was used as a standard (Sigma, St. Louis, MI, USA; 403040) for those monomers that have remained free after vesicle formation with the ultrasonic disruptor, to determine encapsulation efficiency and loading capacity, by interpolation by spectrophotometry using tannic acid as standard at 280 nm absorbance.
Encapsulation efficiency and drug loading efficiency were calculated using the formulas: Encapsulation efficiency (%) = weight of tan nins into nanoparticle initial weight of tan nins Loading With C = dosage concentration, V = volume of administration, C = drug concentration in the separation solution after drug loading, V = total volume of liposome suspension, m = weight of tannins entrapped into liposomes, and M = weight of product (liposome + loaded tannins). C was determined by spectrophotometry by using standard curves of tannic acid standard at 280 nm absorbance (y = 0.021x − 0.114, r 2 = 0.955, p < 0.001). M was determined by weighting the dehydrated nanoparticles after freezedrying known aliquots of the dispersion 2.2. Mean Particle Size (MPS), Polydispersity Index (PDI), and z-Potential (ξ) The MPS, PDI and ξ analyses for each sample were performed using dynamic light scattering (DLS) with a measurement angle of 173 • Backscater and a mean and phospholipid refractive index of 1.330 and 1.334, respectively. (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK).

Proximal Composition
Proximal composition (see Supplemental Table S1) of tannins (TS) and nanoliposomes that encapsulated grape seed tannins (TLS) were carried out under the standards 925.45 for moisture; 923.03 for ashes; 990.03 for proteins; 996.06 for lipids of the A.O.A.C., 2016.

Quantification of Total Phenols
For the quantification of total phenols, expressed as gallic acid [µg/mL], the methodology described by Cano et al. (2020) was used with some modifications [41]. Briefly, 1 mL of 1 mg/mL TS or TLS (redispersed after centrifugation) was added to a 15 mL conical tube. A total amount of 200 µL of Folin-Ciocalteu reagent was added and allowed to stand for 3 min. After this time, 1.6 mL of 7.5% sodium carbonate solution was added and it was left to react for 1 h in the absence of light. Absorbance data were recorded at 760 nm for later quantification.

HPLC-UV
The identification of the compounds present in TS and TLS were carried out by means of depolymerization of tannins in acidic methanol and in the presence of toluene-α-thiol or cysteamine hydrochloride (acid thiolysis) and subsequent HPLC-UV analysis using a wavelength of 280 nm [42]. To extract the condensed tannins from the nanoliposomes, they were centrifuged at 9000 rpm for 1 h at 20 • C. In total, 200 µL of the supernatant was transferred to a microcentrifuge tube (2 mL) and mixed with 200 µL of thiolysis medium (Cysteine-Hydrochloric Acid-Methanol). Subsequently, the microcentrifuge tube was heated in a water bath at 65 • C for 1 h to carry out thiolysis. During thiolysis, the monomeric units of flavan-3-ol are released from the condensed tannins in their native state (terminal units) as well as their respective thioethers (extending units). After the thiolysis time, 1 mL of deionized water was added to stop the reaction [42]. In total, 25 µL of the sample (thiolyzed extract, non-thiolyzed extract, calibration standard) was injected onto the HPLC-UV column at 280 nm. Prior to injection, samples were conditioned to room temperature (20 • C). The mobile phase gradient described by Bianchi et al. [42] was applied at a flow equal to 1 mL/min. The composition of Phase A was 0.1% trifluoroacetic acid (TFA) in deionized water and Phase B was 0.08% TFA in a 4/1 ratio of acetonitrile/deionized water.

Trolox Equivalent Antioxidant Capacity Assay
The Antioxidant Assay Kit (Cayman Chemical, Ann Arbor, MI, USA, 709001) was used to measure antioxidant capacity of TLS and TS preparations (redispersed to 1 mg/mL after centrifugation), according to the method described by Compaoré et al., 2016 [43], based on the capacity of the sample to prevent the oxidation of ABTS (2,2 -azino-di-[3ethylbenzthiazoline sulphonate]) to ABTS•+. ABTS assay was performed according to the manufacturer instructions with minimal modifications. Briefly, 10 µL of suspension or Trolox standard was added to 200 µL of diluted ABTS solution, with incubation in the dark for 5 min. The absorbance was read at 620 nm, with a microplate reader (BioTek Instruments, New York, NY, USA). Trolox was used to generate the standard curve (y = −0.552x + 0.307, r 2 = 0.998, p < 0.001), and the results were expressed in micromole of Trolox equivalents (TE) per mL of the initial preparation (µmol TE/mL).

Cell Viability Assay
In total, 40,000 CAD cells were seeded in 6-well plates and differentiated for 7 days. After differentiation, cells were incubated with charged TLS, empty LS (1 mg/mL) and TS (0,1 mg/mL) for 6 h at 37 • C y 5% CO 2 . After the incubation, the toxicity of H 2 O 2 on cultured CAD cells was assessed by the trypan blue exclusion method ( Figure 1A). After exposure to 200 µM H 2 O 2 or SFM (control plates) for 24 h at 37 • C 5% CO 2 , cells were immediately stained with 1.5% Trypan blue for 10 min at room temperature. Cells were then examined by light microscopy (20x), counted in quadruplicate in the Neubauer chamber for determining the viability.

Intracellular ROS Assay
In total, 2500 CAD cells were plated in 35 mm dishes, differentiated for 7 days and first incubated with TS (0,1 mg/mL), TLS or LS (1 mg/mL) for 1 h at 37 • and 5% CO 2 . CellROX ® Deep Red Reagent ® (Thermo Fisher Scientific, Waltham, MA, USA, C10422) was then added in each well at 5µM for 30 min in the same experimental conditions. Then, 10 mM H 2 O 2 was added, and a time lapse (30 min, 2 Hz) was acquired by confocal microscopy (Zeiss 710, 10X objective, Jena, Germany) at 644/665 nm Ex/Em, respectively ( Figure 2A). Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 16   In total, 2500 CAD cells were plated in 35 mm dishes, differentiated for 7 days and first incubated with TS (0,1 mg/mL), TLS or LS (1 mg/mL) for 1 h at 37° and 5% CO2. CellROX ® Deep Red Reagent ® (Thermo Fisher Scientific, C10422) was then added in each well at 5µ M for 30 min in the same experimental conditions. Then, 10 mM H202 was added, and a time lapse (30 min, 2 Hz) was acquired by confocal microscopy (Zeiss 710, 10X objective) at 644/665 nm Ex/Em, respectively (Figure 2A).

RNA Isolation, cDNA Synthesis and RT-qPCR Analysis of Neuroinflammatory Biomarkers
In total, 100 mm culture dishes containing 100,000 differentiated CAD cells were used for molecular biology experiments. Briefly, cells were treated with TS, LS, and TLS with or without 200 µM H 2 O 2 for 24 h ( Figure 3A). Immediately after, cells were washed twice with ice-cold 1X PBS for stopping stimuli and RNA isolation was immediately performed using TRIzol reagent TM (Invitrogen 15596026) according to the manufacturer instructions. RNA samples were stored at −80 • C until quantification and cDNA synthesis by reverse transcription. RNA was quantified in Take3 TM plates in Epoch microplate reader (BioTek Instruments, New York, NY, USA). RNA purity (260/280 ratio) was 1.84 ± 0.06. cDNA synthesis was performed immediately after using the iSCRIPT kit (Bio-Rad, Hercules, CA, USA, 1708891) from 1 µg of RNA per reaction, according to the manufacturer instructions using the next thermal profile (1 cycle and 60 • C for 30 s (annealing/extension/acquisition), followed by a melting curve from 72 to 95 • C (0.3 • C/read). qPCR reactions were performed in duplicate for each sample, using the 2 ∆∆CT method for quantifications as described previously [44,45], using the geometric mean of CT values of control samples as internal calibrators for each target. We also tested the constitutive expression of 18S among all the experimental groups by using 2 −∆CT comparisons between calibrator and treated samples (Supplementary Table S2). CT values over 35 were considered insignificant and were excluded for RT-qPCR quantifications.

Statistical Analysis
The data in the tables are presented as mean ± standard deviation (S.D.), and for violin plots as median and quartiles. Normal distribution of the data was assessed using a Shapiro-Wilk test. Comparisons were performed through Student's t-test, one-way ANOVA test followed by Bonferroni's post hoc test, and two-way ANOVA with repeated measurements, according to the data structure. The level of significance was defined as p < 0.05. All the statistical analysis was performed with GraphPad Prism 9.0 software (Dotmatics, Boston, MA, USA). Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 16

Statistical Analysis
The data in the tables are presented as mean ± standard deviation (S. D.), and for violin plots as median and quartiles. Normal distribution of the data was assessed using

Lipid-Encapsulated Tannins Characterization
TS and TLS samples presented low protein content 0.08 [g/100 g], attributed to the interaction of tannins with proteins. Regarding lipids content, TLS reported a high value of 0.18 [g/100 g], which is mainly attributed to the addition of soy phosphatidylcholine. The energy contribution of TS (11.39) and TLS (12.04 kcal/100 g) was lower than the value reported by Czaplicka et al. (2016) [46] for fresh grapes 33 kcal/100 g ( Table 1). The encapsulation efficiency was determined by spectrophotometry using tannic acid as standard at 280 nm absorbance. The encapsulation efficiency of TLS was 91.0 ± 5.1 %, and the loading efficiency was 53.0 ± 3.0%. ABTS assay revealed that TLS showed negligible antioxidant activity compared to TS (Table 1, 9.10 ± 6.75 vs. 2017.51 ± 238.57 µM TEAC, p < 0.05; Table 1). In fact, in three of five independent assays, ABTS antioxidant activity was equal to 0 µM TEAC. As expected, total polyphenols content (TPC) was significantly higher in TS compared to TLS (78.34 ± 0.12 vs. 30.23 ± 0.14 GAE µg/mL, TS vs. TLS, p < 0.05) per mL (Table 1). These results could be attributable to the w/w tannins ratio present in TS and TLS formulations, since both TPC and ABTS assays were performed using 1 mg/mL of redispersed particles after centrifugation. Considering that TS consists of free tannin suspension, whereas TLS loading efficiency was 53%, lower antioxidant activity of TLS was expectable, since tannic acid concentrations per mL of TLS are two-fold lower compared with TS. Supplementary Table S1 shows the concentrations (mg/L) obtained by HPLC-UV at 280 nm of the different monomeric units of flavan-3-ol of the condensed tannins in their native state (terminal units) as well as their respective thioethers (units of extension) such as gallic acid, catechin, catechin-cysteine, epicatechin, epicatechin-cys, catechin gallate, epicatechin gallate, epitecatechin gallate cys and procyanidin B1(cis-trans) in the samples of suspended tannins (TS) and encapsulating nanoliposomes grape seed tannins (TLS), with catechin being the most abundant component in both samples, followed by epicatechin and gallic acid (Supplementary Table S1).

Tannins and Charged Liposomes Prevent Hydrogen Peroxide-Induced Cell Death
We analysed the effects of empty lyposomes (LS), TS and TLS on cell viability in differentiated CAD cells treated with or without hydrogen peroxide, in order to explore the capacity of nanoliposomes and free tannin suspensions to prevent ROS-induced neuronal cell death. For this, CAD cells were preincubated with 0. + TS, respectively; p < 0.05), despite TLS showed negligible antioxidant activity by the ABTS method and significantly lower polyphenols content expresed as gallic acid equivalents (Table 1). These data suggest that TLS efficiently delivered tannins inside the cells before H 2 O 2 exposure and then prevented hydrogen-peroxide-induced neuronal cell death since empty liposomes were unable to prevent cell death after H 2 O 2 treatment when compared with charged liposomes (48.72 ± 21.14 vs. 90.38 ± 11.17% cell survival, H 2 O 2 + LS vs. H 2 O 2 + TLS, respectively; p < 0.05 Figure 1). Neither LS, TLS or TS alone affected cell survival (100.00 ± 7.01 vs. 98.02 ± 3.84 vs. 96.58 ± 3.97 vs. 96.15 ± 9.42; Control vs. LS vs. TLS vs. TS, respectively).

Lipid-Encapsulated Grape Tannins Protects Neurons against ROS-Induced Neuroinflammation
We analysed the capacity of TLS (0.1 mg/mL concentration in plate) to prevent oxidative stress-induced neuroinflammation as well as to induce the expression of antioxidant gene defense in neurons. For that, we treated CAD cells with 200 µM H 2 O 2 with or without TLS for 24 h and we then analysed pro/anti-inflammatory cytokines expression and antioxidant enzymes by RT-qPCR. As shown in Figure 3B,C, H 2 O 2 resulted in marked increases in proinflammatory cytokines expression and reductions in antioxidant enzyme expression. Importantly, TLS treatment dramatically reduced the mRNA expression of two major proinflammatory cytokines after H 2 O 2 treatment (tumor necrosis factor alpha, TNF-α: 400.3 ± 1.7 vs. 7.9 ± 1.9-fold; and interleukin-1 beta, IL-1β: 423.4 ± 1.3 vs. 12.7 ± 2.6-fold; p < 0.05; H 2 O 2 vs. TLS+ H 2 O 2 , respectively), suggesting a neuroprotective role of TLS by reducing neuronal inflammation. H 2 O 2 treatment resulted in reduced expression of two main antioxidant enzymes, soluble superoxide dismutase (CuZn-SOD, 1.03 ± 0.12 vs. 0.28 ± 0.08-fold, Control vs. H 2 O 2 , p < 0.05) and mitochondrial superoxide dismutase (Mn-SOD, 1.59 ± 0.21 vs. 0.10 ± 0.06-fold, Control vs. H 2 O 2 , p < 0.05), and, surprisingly, neither TLS nor TS were able to restore their levels to control condition (CuZn-SOD, 0.28 ± 0.08 vs. 0.12 ± 0.04 and 0. 26 Figure S1). Neither H 2 O 2 nor TLS affected neuronal nitric oxide synthase expression ( Figure 3B). In addition, we found that transforming growth factor beta-1 (TGF-β1) was also increased by H 2 O 2 treatment and this was prevented by TLS treatment (1.03 ± 0.10 vs. 16 Figure S1). Regarding to free grape seeds tannins suspensions, TStreated (0.1 mg/mL) CAD cells displayed a non-significant trend to restore pro-oxidant and pro-inflammatory biomarker mRNA expression in response to H 2 O 2 , with the exception of IL-1β, on which H 2 O 2 + TS showed a significantly higher anti-inflammatory activity compared with H 2 O 2 + TLS (2.1 ± 0.3 vs. 12.7 ± 2.6-fold expression vs. control, respectively; p < 0.05; Supplementary Figure 1). Therefore, lipid-encapsulated grape seed tannin treatment reduced H 2 O 2 -induced proinflammatory gene expression without affecting main antioxidant genes. Considering that TLS prevented neuronal cell death (Figure 1), intracellular ROS formation ( Figure 2) and classic proinflammatory cytokine mRNA expression ( Figure 3) in response to H 2 O 2 , our results suggest that TLS may exerted neuroprotection by these mechanisms.

Discussion
The main findings of our work demonstrate that both grape tannins suspension (TS) as well as lipid-encapsulated grape tannins (TLS) exerted neuroprotective effects, preventing ROS-induced neuronal cell death, reducing intracellular ROS and diminishing proinflammatory gene expression, with no effect on main antioxidant enzyme expression, suggesting that the lipid encapsulation of tannins resulted in the effective delivery of grape seed polyphenols to neurons.
Tannic acid is one main polyphenol present in grape seeds and skin [27,28] and exerts strong antioxidant properties [48]. Therefore, its utilization as a natural source of antioxidants is a potential alternative for the development of novel neuroprotective drugs. However, the limited bioavailability of dietary polyphenols such as tannic acid mine its potential as a neuroprotective agent [31]. However, lipid encapsulation has been proposed as a feasible solution to cope with low bioavailability in vivo [32]. Indeed, our results showing that TLS antioxidant activity was negligible compared with TS (Table 1), and that TLS significantly (i) reduced intracellular ROS levels and (ii) reduced pro-inflammatory cytokines expression in CAD cells, strongly support the finding that lipid encapsulation is an efficient way to deliver grape tannins to neurons, acting as neuroprotectors by directly scavenging ROS as well as reducing neuronal inflammation. Indeed, empty liposome (LS) completely failed to protect cells against H 2 O 2 -induced cell mortality and intracellular ROS. Remarkably, we found that TLS mimicked the neuroprotective effects of free tannins suspension (TS). Considering that loading efficiency was~50% and encapsulation efficiency~90%, approximate tannins concentration in 0.1 mg/mL of TLS is~0.05 mg/mL, TLS showed 2-fold more in vivo antioxidant capacity compared to TS (0.1 mg/mL). Therefore, given that TLS mimicked TS neuroprotective effects (even at lower theoretical concentrations than free tannins suspensions) and that LS failed to protect neurons against oxidative stress, we believe that our results strongly suggest that observed effects of grape-tannin-charged liposomes are attributable to their cargo.
Neuroinflammation is a complex orchestrated process that comprises all CNS cells, including astrocytes, microglia, neurons and infiltrated leukocytes [4,49]. Previous reports showed that catecholaminergic neurons respond to proinflammatory stimuli by producing TNF-α, IL-1β, IL-6, among other inflammatory biomarkers [50,51]. The later support the notion that neurons actively participate in the neuroinflammatory response. In the present study, we found that ROS accumulation elicit an inflammatory response in cultured neurons in vitro and that lipid-encapsulated grape tannins are an effective mean to reduced ROS-induced neuroinflammation. Since we did not perform experiments in whole animals, caution is needed when trying to translate our findings into animal physiology. Further experiments are needed to fully determine the translational potential of lipid-encapsulated grape tannins on CNS inflammation in animals. Due to the nature of our study, we did not investigate the precise mechanism(s) by which TLS exerts neuroprotection. However, it has been shown that both NF-κB and AP-1 transcription factors are involved in the cellular responses following hydrogen peroxide-induced neuroinflammation in both physiological and pathological conditions [2,[11][12][13][14][15]. Despite the fact that we did not directly assess these signaling pathways, TNF-α and IL-1β (which we found to be elevated after H 2 O 2 treatment and reduced by TLS) are classic downstream targets of the NF-κB/AP-1 signaling pathway in neurons and glial cells [4,23,29,52], and there is evidence showing that prooxidant stimuli activates NF-κB and AP-1 in catecholaminergic neurons being the outcome of an increased expression of TNF-α and IL-1β [37]. Therefore, it is highly likely that the beneficial effects associated with TLS are partly linked to inhibition of the NF-κB/AP-1 signaling pathway in CAD neurons. Future studies are needed to fully determine the exact molecular mechanism(s) by which TLS offers neuroprotection.
Our results showed that lipid encapsulation of tannins could be an alternative strategy to prevent some of the pathological features of neurodegenerative diseases (i.e., AD, HD, PD, ALS) since they effectively reduced neuronal oxidative stress and inflammation, two main common hallmarks of these pathological states [6][7][8][9][10]. A recent report showed that periodic administration of high doses of tannic acid (not condensed) prevents cognitive impairment in an AD-like model induced by lipopolysaccharide intraperitoneal injections (the major activator of the proinflammatory TLR4 signaling pathway) [29]. This study confirms a major role of inflammation and oxidative stress in the genesis and progression of neurodegenerative disease, as well as the potential of dietary polyphenols to mitigate neuroinflammation and symptoms of neurodegenerative disease [29]. Interestingly, in the same study, authors were able to find that high doses of tannic acid (60 mg/kg) were effective in reducing LPS-induced neuroinflammation and cognitive impairment [29]. However, this high dosage of tannic acid is unlikely to be translated into human studies due to the well-known low tolerability of high dose tannic acid. Then, a carrier-based strategy may result in lower dosages without comprising tannins therapeutic potential. Given their capacity to easily cross the BBB and the chemical protection of their cargo, liposomesencapsulated tannins rise as a promising tool to afford neuroprotection in vivo [32,34].

Conclusions
In summary, our study provided the first evidence that lipid-encapsulated grape tannins are an effective means to control oxidative stress and inflammation in neurons following an oxidative insult. Then, lipid-encapsulated grape tannins could offer a novel strategy to confer neuroprotection.