Resveratrol-Loaded Lipid Nanocarriers Are ... - ACS Publications

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Resveratrol-Loaded Lipid Nanocarriers Are Internalized By Endocytosis in Yeast Ceĺ ia Barbosa,†,# Cat́ ia Santos-Pereira,†,‡,# Ineŝ Soares,† Viviana Martins,†,§ Joana Terra-Matos,† Manuela Côrte-Real,† Marlene Lúcio,*,⊥ M. E. C. D. Real Oliveira,⊥ and Hernan̂ i Geroś *,†,‡,§

J. Nat. Prod. Downloaded from pubs.acs.org by NEWCASTLE UNIV on 04/09/19. For personal use only.

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Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal ‡ Centre of Biological Engineering (CEB), Department of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal § Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal ⊥ Centre of Physics (CFUM), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal S Supporting Information *

ABSTRACT: Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 μM. The partition coefficient (Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers’ suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (ΔH = 43.76 ± 5.68 kJ mol−1; ΔS = 0.20 ± 0.005 kJ mol−1; and ΔG = −18.46 ± 3.48 kJ mol−1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant (end3Δ) and with the endocytosis inhibitor methyl-β-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications. saturated fat.5−7 The polyphenol resveratrol (RSV) has been pointed out as the main contributor to cardiovascular protection.5,8 The beneficial cardiovascular effect of RSV can, in turn, be related with its proved capacity to act as a modulator of the metabolism of blood lipoproteins,5,9 inhibiting the oxidation of low-density lipoproteins (LDL) and preventing either platelet aggregation or proatherogenic eicosanoid production by human platelets and neutrophils.5 An RSV-rich diet has been associated with a higher abundance of high density lipoproteins (HDL), which take part in

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atural phenolic compounds are the major group of phytochemicals with antioxidative properties found in plants, particularly in fruits, seeds, and leaves, and their derivative foods and drinks, including chocolate, tea, and wine.1,2 These are a class of phenylalanine-derived chemical compounds with a reactive hydroxyl group (−OH) bound directly to an aromatic hydrocarbon ring. They are classified as simple phenols or polyphenols according to the number of phenol units.3,4 The consumption of red wine has been associated with the so-called “French paradox”, a term coined to describe the observation that the French population has a very low incidence of cardiovascular disease, despite a diet high in © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 27, 2018

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DOI: 10.1021/acs.jnatprod.8b01003 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Characterization of unloaded and RSV-loaded nanocarriers. (A) Size distribution (in intensity) of unloaded and RSV-loaded DODAB:MO (1:2) nanocarriers (bars) and their polydispersity (blue dots). (B) Cryo-SEM images of DODAB:MO (1:2) nanocarriers. (C) ζPotential values of DODAB:MO (1:2) unloaded and RSV-loaded nanocarriers. The dashed blue line represents the threshold of +30 mV, as a minimal value for nanocarrier stability. (D) Encapsulation efficiency (EE%) (bars) and loading efficiency (RL%) (blue dots) of increasing RSV concentrations (5, 20, 40, 60, 100, and 200 μM) to a 1000 μM lipid concentration. Values reported are the mean diameter ± standard deviation for at least three determinations.

(DODAB):monoolein (MO) lipid nanocarriers27−29 and on the development of lipid nanocarriers for RSV delivery30 by designing new strategies for improving RSV bioavailability and bioactivity using an efficient encapsulation nanosystem of RSV in DODAB:MO (1:2) liposomes. These nanosystems present interesting features that differ from conventional liposomes. At molar ratios where MO is in excess (e.g., DODAB:MO (1:2)) DODAB bilayers enclose MO-enriched reversed lyotropic nonlamellar liquid crystalline mesophases. This means that, instead of containing aqueous solutions, the core of these nanosystems is filled by honeycomb-like lipid assemblies, rich in MO, that offer the possibility to entrap higher cargos.31 Therefore, we chose a ratio of 1:2 to ensure a higher RSV loading in both the lipid bilayer and core. Furthermore, the lipid bilayer is rich in DODAB, which offers stability to the nanocarriers, while providing cationic surface charge to favor cellular internalization.27 To characterize the mechanism of internalization of RSVloaded nanocarriers by eukaryotic cells, we selected as a model system the fermentative yeast Saccharomyces cerevisiae. This idea emerged from the pioneering work of Howitz and colleagues,32 who identified RSV as a potent SIRT1 activator, regulating the longevity of this microorganism. The observation that an endocytic mechanism is involved in the internalization of RSV-loaded nanocarriers by yeast cells opens new avenues for future research on medical applications of RSV.

cholesterol removal from the atheroma, preventing the obstruction of arteries.10 In addition, RSV may protect diabetic patients from meal-induced oxidative stress,11 and several other pharmacological effects have been attributed to this compound, including antibiotic and chemopreventive properties.5,9,12−14 Different studies on RSV bioavailability after oral ingestion and clinical trials have been conducted in both rodents and humans.7,15 Results indicate that the increase of food nutritional value by RSV suplementation is limited by its low bioavailability, low water-solubility, and rapid degradation.7 The nanoencapsulation of resveratrol and other phenolic compounds has been reported to be an efficient approach to overcome these limitations (reviewed by refs 15−19). In fact, its incorporation in nanocarriers such as polymeric micelles,20 gelatin nanoparticles,21 and cyclodextrin-based nanosponges,22 among others, was shown to improve RSV solubility, stability, and availability. Liposomes are the most clinically established nanocarrier systems for drug delivery.23,24 They are composed by amphiphilic molecules, which spontaneously self-assemble into vesicles made of lipid bilayers enclosing aqueous solutions in their core. The control of the structure of the liposomes, namely, size and size distribution, morphology, and supramolecular organization, is important for the design of drug delivery systems.23−26 In turn, the lipid molecules forming the nanoaggregate strongly influence the structure and stability of liposomes in physiological conditions. In the present study, we extended our previous work on the characterization of dioctadecyldimethylammonium bromide B



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RESULTS AND DISCUSSION

RSV-Loaded Nanocarriers Gathered Biophysical Properties Adequate for Therapeutic Application. Due to the low water solubility and labile properties of RSV and its rapid metabolism and elimination in humans, in the present paper we exploited its encapsulation in DODAB:MO nanocarriers as a strategy to improve the preservation of its biological properties and to enhance its bioavailability. DODAB:MO (1:2) nanocarriers have an average hydrodynamic diameter size of 130 ± 4 nm determined by dynamic light scattering (DLS) and present a sphere-like smooth surface as observed by cryo-scanning electron microscopy (SEM) (Figure 1A and B). It is worthwhile to recall that in cryo-SEM lipid nanocarriers undergo a cryogenic procedure to remove water before imaging. This procedure is required for soft materials, but tends to impose some deformation on lipidbased nanosystems,33 whose diameter cannot be unambiguously compared with the solvated nanocarriers evaluated by DLS. Notwithstanding, the nanocarriers’ sizes by cryo-SEM were 164 ± 37 nm, which is not significantly different from the value of 130 ± 4 nm measured by DLS. RSV-loaded nanocarriers showed an RSV-dependent hydrodynamic size (measured by DLS) increase from 130 nm (empty liposomes) to 160 nm (100 μM RSV) and 180 nm (200 μM RSV) (Figure 1A). The small values of polydispersity obtained (≤0.18) suggest a fairly narrow and monomodal liposomal size distribution (Figure 1A). All lipid nanocarriers presented a highly positive ζ-potential owing to the presence of the positively charged lipid DODAB. Unloaded nanocarriers showed ζ-potential values (+49.7 ± 5.6 mV) higher than RSVloaded nanocarriers (Figure 1C), most likely because the neutral RSV molecule is located near the positive DODAB polar headgroups, which decreases the density of positive charges and reduces the surface charge of the nanocarrier. The encapsulation (EE%) and loading (RL%) efficiencies of RSV in the lipid nanocarriers are shown in Figure 1D. EE% was above 70% for incubation with 200 μM RSV. Furthermore, RL% increased with increasing RSV concentration, reaching 14% at 200 μM RSV. As it provides information about the amount of bioactive loaded per nanocarrier unit weight, RL% is actually the most important parameter for evaluation of nanocarrier encapsulation. The RL% showed a slow increase at lower RSV concentrations (0−60 μM) and a steeper increase at higher concentrations (100 and 200 μM). At concentrations below the solubilization limit (0−60 μM), RSV is still very soluble in water,19 and thus a small encapsulation in the lipid nanocarriers is achieved. However, for concentrations near or above the solubilization limit (100 and 200 μM),19 RL% increases because RSV is no longer soluble in water and the bilayer permeation is improved at saturation levels, further demonstrating the suitability of these lipid nanosystems as carriers for high RSV concentrations. The partition coefficient (Kp) between lipid/aqueous phases is a measure of the bioactive lipophilicity within the nanocarrier system, i.e., of its distribution in the hydrophobic and hydrophilic microenvironments.34,35 Kp was determined from 30 to 60 °C to calculate the thermodynamic parameters associated with the partition of RSV in a biphasic system DODAB:MO (1:2)/water. The nonlinear regression fittings of the third derivative values at different temperatures, obtained according to eq 3 of the Experimental Section, are presented in Figure 2A. A high Kp value was obtained (Llog Kp = 3.37 ±

Figure 2. Biophysical properties of RSV-loaded nanocarriers. (A) Normalized third derivative absorbance values at λ = 311 nm with increasing DODAB:MO (1:2) concentration for the determination of Kp of RSV in the lipid/water biphasic system at several temperatures: 30, 37, 50, 55, and 60 °C. Continuous lines are the best fitted curves according to eq 3. (B) Normalized average (AVG) count rate of DODAB:MO (1:2) lipid vesicles unloaded and RSV-loaded (200 μM) as a function of temperature. Continuous lines are the best fitted curves according to a modified Boltzmann regression as previously reported.37 Values reported are the mean ± standard deviation of three replicate samples.

0.10), suggesting that this bioactive compound has a lipophilic character,36 which facilitates its efficient encapsulation in the liposomal nanocarrier, in agreement with the observed high values of EE% and RL% (Figure 1D), indicative that the nanocarrier can incorporate high amounts of RSV. From the application of the van’t Hoff linear fitting to the experimental data, RSV partitioning at 37 °C is associated with ΔH > 0 (43.76 ± 5.68 kJ mol−1), ΔS > 0 (0.20 ± 0.005 kJ mol−1), and ΔG < 0 (−18.46 ± 3.48 kJ mol−1), reflecting the spontaneity of the partition process and the establishment of hydrophobic interactions between RSV and the liposomal nanocarrier. The thermotropic behavior of the lipid nanocarriers unloaded or loaded with RSV was evaluated by DLS, which measures the changes in the optical properties of lipid vesicles at increasing temperatures. Results are shown in Figure 2B, where AVG is the average number of photons scattered by the liposomes per second. Lipid nanocarriers in more ordered phases induce higher light scattering and, consequently, higher AVG count rate values than the fluid lipid systems, as previously reported.38 The data presented in Figure 2B were fitted by a modified Boltzmann regression as previously reported,37 to determine the values of the main phase transition temperature, Tm, and of the cooperativity of the transition, B (Table 1). The unloaded DODAB:MO (1:2) nanocarrier presented a high cooperativity, sugesting that all the lipid molecules transit to a different phase C



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Table 1. Biophysical Parameters (B and Tm) of DODAB:MO (1:2) Nanocarriers in the Absence and Presence of RSV (200 μM) DODAB:MO(1:2) DODAB:MO(1:2) + 200 μM RSV

B (cooperativity)

Tm (°C)

217 ± 58 169 ± 12

41.7 ± 1.0 36.7 ± 0.3

is also confirmed by the high log Kp obtained as well as by the high EE% and RL% of RSV. The stability of the nanocarrier was also evaluated by addition of increasing concentrations of liposomal nanocarriers to human serum albumin (HSA), used to mimic serum stability. As shown in Figure 3A, both unloaded and RSVloaded nanocarriers suffered an inversion of their ζ-potential in the presence of HSA, due to the progressive adsorption of HSA negatively charged residues to the positively charged surface of the nanocarriers. The aggregation of the uncharged nanocarriers occurs at the neutrality point of the ζ-potential, which is a sign of colloidal instability. In the unloaded nanocarriers, the neutrality point is observed immediately after the addition of 100 μM of DODAB:MO (1:2). Interestingly, lipid nanocarriers containing RSV present a different binding profile to HSA, which is evidenced by the slower inversion of the ζ-potential. This suggests that RSVloaded nanocarriers bind less extensively to HSA, which might be caused by the RSV location at the superficial part of the lipid headgroups, thereby reducing the positive surface charge density of the vesicles and hindering HSA adsorption to their surface. As shown in Figure 3B, a controlled biphasic release of RSV from the lipid nanocarriers occurs over a period of almost 50 h at pH 5.0 and 7.4. The best fitting to RSV release data was achieved by the Gallagher−Corrigan kinetic profile, and the corresponding parameters are presented in Table 2. While the first phase (until ∼8 h) is more pronounced and pHindependent, and likely corresponds to the release of RSV superficially located in the liposomal bilayer, the second one is more controlled at pH 5.0, in agreement with the counterion effect already observed for DODAB:MO (1:2) nanocarrier. During this phase there must occur the release of RSV encapsulated at the nanocarrier core.27 The shelf stability of the nanocarriers was evaluated during 84 days at 4.0 °C. As shown in Figure 3C, the ζ-potential decreased to values below the desired +30 mV, which assures

practically at the same time.39 However, RSV reduces the cooperativity of the transition. This supports that RSV was successfully encapsulated in the nanocarrier. Also, it indicates that the RSV molecules are not evenly distributed in the lipid nanocarrier, since RSV is affecting the cooperative unit of the lipid system by only influencing certain lipid molecules near its location. This is in agreement with previous studies that report the effects of drugs and proteins in the cooperative unit of the lipid nanosystems.39 The 5 °C decrease in the Tm when the nanocarrier was loaded with RSV is probably due to a weaker interaction between the lipid headgroup moieties combined with a lateral expansion of the interfacial region. This suggests that RSV molecules are located in the most organized portion of the lipid membrane system, within the DODAB polar headgroups and interacting at the C1−C8 level of the lipid acyl chains, as previously reported for another lipid/drug system.40 Indeed, despite being a lipophilic compound, the three hydroxyl (−OH) groups of RSV are embedded in the polar zone. The observed effect of RSV on the reduction of the cooperativity of the lipid phase transition corroborates this membrane location, since the cooperative unit that undergoes the transition is largely regulated by the carbons of the headgroup region.41 Therefore, it is possible to conclude that RSV decreases a lipid nanosystem’s microviscosity by insertion at the nanocarriers’ rigid portion. However, it should be noted that the microviscosity reduction does not compromise the nanocarrier integrity. The observed RSV effect on the biophysics of the nanocarrier are once more a proof of RSV high loading, which

Figure 3. Stability of RSV-loaded nanocarriers. (A) Serum stability evaluation by ζ-potential variation of unloaded and RSV-loaded (200 μM) DODAB:MO (1:2) nanocarriers with increasing lipid concentrations in the presence of human serum albumin (HSA). (B) pH stability evaluation by cumulative RSV release from DODAB:MO (1:2) nanocarriers in pH = 7.4 and pH = 5.0. (C) Shelf stability evaluation by ζ-potential measurements of unloaded and RSV-loaded (200 μM) DODAB:MO (1:2) nanocarriers. D



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Table 2. Values of k (h−1), tmax (h), and Fb for the Release of 200 μM RSV from DODAB:MO (1:2) Nanocarriers, Fitted to Eq 4 kinetic parameter

pH 7.4

pH 5.0

Fb Fmax k1 (h−1) k2 (h−1) tmax (h) R2

40.63 ± 8.76 88.88 ± 7.11 0.0444 ± 0.025 0.9241 ± 0.208 2.775 0.996

72.96 ± 13.42 94.68 ± 2.73 0.0845 ± 0.018 1.255 ± 0.769 2.488 0.997

release strongly suggest that the RSV-loaded nanocarriers developed herein are suitable for therapeutic purposes. An Endocytic Pathway Is Involved in the Internalization of RSV-Loaded Nanocarriers. After ensuring that the novel RSV-loaded nanocarriers gathered good characteristics for a potential therapeutic application, the nanocarriers were tested in yeast cells as a simple eukaryotic cellular model to characterize the mechanism of internalization. RSV-loaded nanocarriers were labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH)48,49 to evaluate by fluorescence microscopy how they are incorporated by yeast cells. As shown in Figure 4A, 30 min after incubation, yeast cells readily internalized nanocarriers, which appeared in lipid droplets as bright blue spots in the periphery of the central vacuole. Moreover, when yeast cells were incubated with DPH alone, no fluorescence was detected in the cytosol (not shown). Colocalization experiments with the lipophilic fluorescent probe N-(3-triethylammoniumpropyl)-4-(4(dibutylamino)styryl)pyridinium dibromide (FM1-43) showed that the green fluorescence of this endocytosis marker delimited the blue fluorescence of DPH, suggesting that nanocarriers are efficiently internalized through an endocytic pathway. Moreover, the percentage of internalization of unloaded and RSV-loaded nanocarriers was similar, showing that the presence of RSV does not affect the uptake of the nanocarriers by yeast cells (Figure 4B). Figure 5 shows a representative result of the internalization of RSV-loaded liposomes in wild-type yeast cells and in the endocytosis-defective mutant end3Δ. The analysis of the microscope images showed that the number of cells with internalized nanocarriers and the number of internalized nanocarriers per cell were consistently lower in the endocytosis-defective mutant end3Δ than in wild-type cells.

enough surface charge to avoid aggregation,42 only after the first month of storage. The results obtained in the present study together with previous ones27,28,43,44 supported that DODAB:MO nanocarriers are indeed adequate drug delivery systems. While DODAB provides structural rigidity to the lipid bilayer, required for the retention of the encapsulated compound, MO promotes inverted nonlamellar phases at the inner core of the vesicular structure, important for increasing the encapsulation efficiency. The MO content also provides the nanosystem with fusogenic properties,45 which might be relevant for a therapeutic application.46 The average diameter size of DODAB:MO (1:2) nanocarriers, either unloaded or loaded with RSV, falls in the optimal range of a nanosized agent (between 20 and 250 nm) to induce an efficient therapeutic effect, being above the kidney clearance and below the mononuclear phagocytic system thresholds, which allows the nanocarriers to circulate for prolonged periods of time.47 Furthermore, the high EE% and RL% of RSV, together with nanocarriers’ stability, and the pH-dependent RSV controlled

Figure 4. RSV-loaded nanocarriers are internalized in lipid vesicles by yeast cells. S. cerevisiae W303 cells were incubated for 30 min with DODAB:MO (1:2) nanocarriers unloaded or loaded with 200 μM RSV and labeled with 3 μM DPH (blue fluorescence). (A) RSV-loaded nanocarriers were further stained with the endocytosis fluorescent marker FM1-43 (green fluorescence) for 10 min before observation in the fluorescence microscope. DIC images clearly show internalized nanocarriers (head arrows), which are enclosed within lipid vesicles (arrows). (B) Percentage of yeast cells with internalized unloaded and RSV-loaded nanocarriers. At least 200 cells were counted per condition, and values are the mean of three independent experiments. E



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Figure 6. Internalization of RSV-loaded nanocarriers is delayed by the endocytosis inhibitor methyl-β-cyclodextrin. S. cerevisiae W303 cells pretreated for 30 min with 5 mg/mL methyl-β-cyclodextrin (+MβCD) or in the absence of the inhibitor (−MβCD) and then incubated for 30 min with DODAB:MO (1:2) nanocarriers loaded with 200 μM RSV and labeled with 3 μM DPH (blue fluorescence) before observation in the fluorescence microscope.

In summary, an important achievement of the present study regarded the clear-cut conclusion that an endocytic mechanism is involved in the internalization of RSV-loaded nanocarriers, supported by the following observations: (i) the green fluorescence of the endocytic marker FM1-43 colocalized with the blue fluorescence of the DPH-stained nanocarriers; (ii) the rate of internalization of RSV-loaded nanocarriers is delayed in the endocytosis-defective mutant end3Δ in comparison with the wild-type cells, and (iii) the incorporation of the nanocarriers is delayed by the endocytic inhibitor MβCD. Major questions regarding the physiology of the lipid droplets have not been solved yet;50 thus future studies of the role of this subcellular organelle in the storage/delivery of RSV-loaded nanocarriers will be of great scientific interest. Very few studies regarding the interaction of nanocarriers and yeast cells have been reported. In one of these studies, S. cerevisiae protoplasts were transfected with liposome-encapsulated plasmid DNA;51 however, the details of the mechanism involved remain obscure. Droplets of essential oils were efficiently encapsulated within yeast cells, but the resolution of the images did not allow further conclusions regarding the mechanisms of encapsulation.52 Regarding the uptake of free RSV, it was suggested that its loading process into yeast cells might occur by passive diffusion facilitated by the hydrophobic interactions and hydrogen bonds established between the RSV −OH groups and the −NH2, −OH, and −COOH groups of the polar headgroups of the phospholipids in the yeast membrane.53 In hepatic cells, the involvement of both passive diffusion and carrier-mediated processes in the uptake of free RSV was reported.54 Contrasting with these findings, a clathrin-independent endocytosis mechanism was proposed for free RSV uptake in cancer cells, namely, via lipid rafts. RSVinduced activation of downstream signaling pathways and caspase-dependent apoptosis were prevented by endocytosis inhibitors, lipid raft-disrupting molecules, and the integrin antagonist peptide arginine-glycine-aspartate.55 However, fewer studies report the uptake mechanism of encapsulated RSV by living systems. RSV-loaded polymeric micelles were shown to internalize in PC12 prostate cells and to localize at the cytoplasm, but the mechanism of uptake remained

Figure 5. Internalization of RSV-loaded nanocarriers is delayed in the endocytosis-defective mutant end3Δ. (A) S. cerevisiae cells were incubated for 30 min with DODAB:MO (1:2) nanocarriers loaded with 200 μM RSV and labeled with 3 μM DPH (blue fluorescence) and observed in the fluorescence microscope. (B) Three hundred minutes after incubation ca. 20% of wild-type cells exhibit DPH blue fluorescence in the vacuole.

When the cells were counted (at least 100 cells per condition; two independent experiments), the number of cells with internalized RSV-loaded nanocarriers was ca. 80% for the wildtype cells and only ca. 50% for end3Δ cells, 30 min after incubation. After a long-term incubation, the number of blue fluorescent cells was similar for both the wild-type and endocytosis-defective mutant end3Δ, but while the number of cells with vacuolar luminal blue fluorescence increased from 2% (t = 30 min) to 20% (t = 300 min) in wild-type cells, no vacuolar fluorescence was observed up to 300 min in the end3Δ mutant. The incubation of yeast cells with RSV-loaded nanocarriers labeled with DPH in the presence of the endocytic inhibitor methyl-β-cyclodextrin (MβCD) resulted in a very clear delay in the uptake of the nanocarriers. As shown in Figure 6, strong vacuolar peripheral blue fluorescent nanocarriers were observed in control cells, contrasting to weak fluorescence signals recorded in cells treated with the endocytic inhibitor. The number of cells containing internalized nanoparticles decreased in the presence of MβCD, as did the average number of nanocarriers incorporated per cell. The growth of S. cerevisiae W303 cells in batch cultures in YPD medium up to 28 h was not significantly affected by the presence of RSV-loaded nanocarriers (Figure S1A). Moreover, RSV-loaded nanocarriers did not affect cell viability as evaluated by flow cytometry with the fluorescent probe fluorescein diacetate (FDA) (Figure S1B). F



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elusive.20 Nevertheless, in HepG2 cells, different endocytic mechanisms were proposed for the uptake of RSV incorporated into stable dispersed liquid crystalline nanoparticles (cubosomes).56 Recently, it was also demonstrated that transferrin-targeted RSV-loaded liposomes are internalized in human glioblastoma cells by transferrin receptor-mediated endocytosis.57 The present study opens good perspectives for future research on the exploitation of the biological activities of RSVloaded nanocarriers, including antioxidant activity (or a prooxidant effect), transcriptional reprogramming, and changes in yeast cell longevity, among others. Free RSV inhibited the proliferation of the yeast Schizosaccharomyces pombe in a dosedependent manner (11−88 μM), modulating the transcriptional landscape and reprogramming the intracellular metabolome, consequently unbalancing the intracellular pool sizes of several classes of amino acids, nucleosides, sugars, and lipids.58 More recently, the effects of free RSV were shown to be dependent on the cellular energy status in S. cerevisiae and linked to respiration.59 Altogether, this study contributes to the medium term exploitation of RSV-loaded nanocarriers as a therapeutic approach in humans given the well-reported beneficial effects of RSV on the inhibition of cancer cell growth,60−63 as well as in other human diseases.64

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determined by DLS in a Zetasizer Nano ZS laser scattering device (Malvern Instruments Ltd., Malvern, UK) at a backscattering angle of 173°. ζ-Potential of the liposomal nanocarriers was evaluated by electrophoretic light scattering (ELS) in a Zetasizer Nano ZS (Malvern Instruments Ltd.). The size and ζ-potential results reported are the mean ± SD of three independent determinations. Scanning Electron Microscopy Analysis of Nanocarriers. The samples were mounted on conductive carbon adhesive attached to aluminum stubs and coated with platinum under vacuum in a sputter coater, after which they were visualized with a FEI Quanta 400 FEG scanning electron microscope at Materials Centre of the University of Porto (CEMUP) facilities. Determination of RSV Encapsulation Efficiency and RSV Loading Content. The liposomal nanocarriers were centrifuged (Jouan BR4i, multifunction centrifuge) through centrifugal filter units (Amicon Ultra-4, PLGC Ultracel-PL membrane, 10 kDa, Milipore) at 3000 rpm and 25 °C for 10 min or until complete separation between the lipid and the aqueous phase. RSV was quantified in both phases by UV/vis spectroscopy (PerkinElmer Lambda 45 UV/vis spectrometer). In the case of RSV quantification in the lipid phase, the derivative spectroscopy method was used to eliminate the light scattering caused by the presence of lipids.34 Spectra were taken in the range of 220−500 nm. Encapsulation efficiency was calculated by eq 1: EE (%) =

[resveratrol]loaded × 100 [resveratrol]total

(1)

The RSV loading content was also calculated by the following eq 2:

EXPERIMENTAL SECTION

RL (%) =

General Experimental Procedures. DODAB (purity >98.0%), MO (purity >99.0%), RSV (purity >99.0%), HSA (purity ≥98.0%), and MβCD were purchased from Sigma-Aldrich and used without further purification. FM1-43, FDA, and DPH dyes were purchased from Molecular Probes. Preparation of Unloaded and Resveratrol-Loaded Nanocarriers. Liposomal nanocarriers were prepared in a 1:2 molar fraction ratio of DODAB:MO by the lipid film hydration method followed by extrusion.65 Briefly, defined volumes of DODAB and MO (20 mM stock solution in ethanol) were added to a round glass tube, and the solvent was evaporated under a N2 stream until the lipid film was completely dried. Afterward, the lipid film was hydrated with 10 mL of ultrapure water above the lipids’ main phase transition temperature, Tm (60 °C), producing multilamellar vesicles (MLVs). MLVs were then repeatedly passed through a Nuclepore TrackEtched polycarbonate membrane filter with different pore diameters (400, 200, and 100 nm) in a Lipex extruder to obtain homogeneous large unilamellar vesicles (LUVs). The final concentration of DODAB:MO (1:2) was 1000 μM. The lipid nanocarriers obtained were then stored at 4 °C. DODAB:MO (1:2) liposomal nanocarriers were loaded with RSV to obtain final RSV concentrations of 100 or 200 μM and a final nanocarrier concentration of 1000 μM. Briefly, an appropriate amount of an ethanolic solution of RSV was added to a glass tube and the solvent evaporated under a N2 stream. Unloaded LUVs were added to the flask containing the dried film of RSV. Finally, the mixture of lipid nanocarrier and RSV was vortexed and incubated above Tm (60 °C). RSV-loaded nanocarriers were then separated from unloaded free RSV. Preparation of DPH-Labeled Nanocarriers. Labeled liposomal nanocarriers were prepared by evaporation to dryness of the lipids’ ethanolic solutions of DODAB and MO (1:2), mixed with the ethanolic solution of the fluorescent probe DPH. The lipid:probe ratio was kept at 100:1 to prevent changes in the structure of the nanocarrier. Labeled liposomal suspensions were protected from light in every step of their use. The resultant dried lipid film was dispersed with ultrapure water, and the subsequent steps followed the lipid film hydration method already described.66 Determination of the Size and ζ-Potential of the Nanocarriers. Liposomal nanocarriers’ size and distribution were

[resveratrol]loaded × 100 [lipid nanocarrier]

(2)

where [lipid nanocarrier] is the concentration of DODAB:MO (1:2) nanocarrier. Both EE (%) and RL (%) were calculated for different initial concentrations of RSV: 5, 40, 60, 100, and 200 μM. Determination of RSV Partition Coefficient. The partition coefficient (Kp) of RSV between lipid and aqueous phase was determined by derivative spectroscopy by a method previously published.67 Briefly, samples of LUVs of DODAB:MO (1:2) with increasing concentrations (0 to 2000 μM) and a fixed concentration of RSV (40 μM) were prepared. Samples were incubated at 37 °C for 30 min, after which UV/vis spectra were analyzed in the range of 220 to 500 nm (SpectraMax Plus 383 ultraviolet−visible plate reader (Molecular Devices)). The references’ absorption spectra of unloaded nanocarriers were subtracted from the samples’ absorption spectra of RSV-loaded nanocarriers. Derivative spectra were calculated using the Savitzky−Golay method, in which a second-order polynomial convolution of 13 points was employed to eliminate the light scattering caused by the presence of lipid nanocarriers. Applying eq 3) to the experimental data (D versus [L]) using a nonlinear leastsquares regression method, the adjustable parameter, Kp was obtained. In this equation, D is the derivative value obtained from the total absorbance, DM is the derivative of the absorbance of RSV distributed on the lipid membrane phase, and DW is the derivative of the absorbance of RSV distributed in the aqueous phase. [L] represents the lipid concentration (in M), and VΦ is the lipid molar volume (M−1). D = DW +

(DM − D W )K p[L]Vθ 1 + K p[L]Vθ

where D =

∂ N ABS ∂ N%

(3)

Kp of RSV in the biphasic system lipid nanocarrier/aqueous media was evaluated at different temperatures (21−41 °C). The van’t Hoff analysis of Kp’s temperature dependence was used to calculate the thermodynamic parameters involved in RSV partition.68 Effect of RSV on the Nanocarrier’s Main Phase Transition. With the purpose of understanding how RSV impacts membrane biophysical properties such as Tm and phase transition cooperativity (B), a 1000 μM liposomal nanocarrier of DODAB:MO (1:2) G



Article

unloaded and loaded with RSV (200 μM) was prepared as previously described. The intensity of the scattered light was analyzed by DLS in a trending mode with temperature variation between 25 and 60 °C with 1 °C temperature intervals. Data were collected as average count rate versus temperature (T) and adjusted by a modified Boltzmann regression as previously reported.37 Effect of Blood Serum on the Stability of Nanocarriers. Unloaded and RSV-loaded (200 μM) liposomal nanocarriers with increasing lipid concentrations (10−1500 μM) were incubated with 9 μM HSA at 37 °C. ζ-Potential of the resultant lipid−HSA aggregates was measured by ELS (Zetasizer Nano ZS) with an equilibration time of 60 s. The ζ-potential results reported are the mean ± SD of three independent determinations. Effect of pH on the Stability of Nanocarriers and Shelf Stability. DODAB:MO (1:2) liposomal nanocarriers (1000 μM) loaded with RSV (200 μM) were maintained in a 50 mL release media with either HEPES buffer, pH 7.4, or acetate buffer, pH 5.0, at 37 °C. At selected time points up to 46 h the RSV concentration was quantified in the release media by UV/vis spectroscopy (PerkinElmer Lambda 45 UV/vis spectrometer). The release profiles were obtained by plotting the cumulative percentage of RSV released against time. In order to clarify the possible mechanisms involved in the RSV release process, the release data were fitted with different mathematical models: zero-order, first-order, Higuchi, Weibull, Korsemeyer− Peppas, and Gallagher−Corrigan equations.69,70 From these the best fit and the one presented was that obtained with the Gallagher− Corrigan according to eq 4): ij e−k 2t − k 2tmax yz zz Ftot = Fb(1 − e−k1t ) + (Fmax − Fb)jjj j 1 + e−k 2t − k 2tmax zz k {

DODAB:MO (1:2) nanocarriers. After adjusting both cell suspensions to an OD640nm = 0.1, growth was recorded every 2 h up to 28 h. Effect of RSV-Loaded Nanocarriers on Yeast Cell Viability. W303-1A cells were grown in YEPD at 30 °C until an OD640nm = 0.5 and then incubated for 30 min with RSV (200 μM)-loaded DODAB:MO (1:2) nanocarriers. Samples were centrifuged at 3500g for 2 min, and the resulting pellet was resuspended in 1 mL of PBS buffer (pH 7.4, NaCl 137 mM, KCl 2.7 mM, phosphate 10 mM). The resulting suspension was stained with 4 μg/mL of the viability probe FDA for 20 min in the dark at room temperature. Autofluorescence was recorded from nonstained cells. Flow cytometry analysis was performed with an Epics XLTM (Beckman Coulter) flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic, and a 525 nm band-pass filter. For each sample, 20 000 events were evaluated. Data were analyzed using the Flowing software (version 2.5).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01003. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (M Lúcio): mlucio@fisica.uminho.pt. *E-mail (H Gerós): [email protected].

(4)

where Ftot is the total fraction of RSV released at time t, Fb is the fraction of RSV available for direct surface release, and k1, k2, and tmax are the rate constants and time to maximum drug release rate, respectively. With the purpose of assessing the unloaded and RSV-loaded lipid nanocarriers in terms of shelf stability, ζ-potential was measured from the moment of preparation up to 84 days. Study of the Uptake of RSV-Loaded Nanocarriers in Saccharomyces cerevisiae by Fluorescence Microscopy. A S. cerevisiae wild-type W303-1A strain and a mutant strain lacking the END3 gene (end3Δ), known to be endocytosis defective,71 were used. Yeast cells were grown in YEPD medium (1% yeast extract, 1% peptone, and 2% glucose) up to the midexponential growth phase (OD640 nm = 0.5) and mixed (1:1) with RSV (200 μM)-loaded or unloaded DODAB:MO (1:2) nanocarriers labeled with the fluorescent dye DPH, which is used for structural and dynamic studies of hydrophobic regions in biological membranes.48,49 The resulting mixture was incubated at 30 °C under shaking (200 rpm) for 30 min and then stained with the endocytosis fluorescent marker FM1-43 (5 μM) for 10 min before observation under a fluorescence microscope. The sequestration of the blue fluorescence of DPH by the vacuole was studied 300 min after mixing the cells with the nanocarriers. In the wild-type W303-1A cells, uptake experiments were also performed in the presence of the endocytosis inhibitor MβCD. Cells grown under the same conditions as aforementioned were pretreated for 30 min with 5 mg/mL of MβCD and then incubated for 30 min with RSV (200 μM)-loaded DODAB:MO (1:2) nanocarriers labeled with 3 μM DPH. Fluorescence microscopy images were acquired with a Leica Microsystems DM-5000B epifluorescence microscope with appropriate filter configurations for DPH and/or FM1-43. The images were obtained with a Leica DCF350FX digital camera and processed with LAS Leica Microsystems software. All images were acquired using the same fluorescence intensity settings, and the images depicted in Figures 4−6 are representative of three independent experiments. Effect of RSV-Loaded Nanocarriers on Yeast Cell Growth. W303-1A cells were grown in YEPD medium at 30 °C under shaking (200 rpm) in the presence or absence of RSV (200 μM)-loaded

ORCID

Hernâni Gerós: 0000-0002-3040-4095 Author Contributions #

C. Barbosa and C. Santos-Pereira contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the strategic funding UID/BIA/04050/2019, UID/FIS/04650/ 2019 and UID/AGR/04033/2019; and by the projects PTDCBIA-FBT/28165/2017 (POCI-01-0145-FEDER-028165), PTDC/BIA-FBT/30341/2017 (POCI-01-0145-FEDER030341) and PTDC/NAN-MAT/32651/2017 (POCI-010145-FEDER-032651). C.S.P. is a recipient of the PD/BD/ 128032/2016 fellowship funded by FCT under the scope of the Doctoral Program on Applied and Environmental Micrô biology (DP_AEM). We thank J. Demaitre (Faculty of Pharmaceutical Sciences, University of Ghent) for her contribution to HSA binding assays.

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REFERENCES

(1) Tsao, R. Nutrients 2010, 2, 1231−1246. (2) Ding, Y.; Yao, H.; Yao, Y.; Fai, L.; Zhang, Z. Nutrients 2013, 5, 2173−2191. (3) Signorelli, P.; Ghidoni, R. J. Nutr. Biochem. 2005, 16, 449−66. (4) Khoddami, A.; Wilkes, M.; Roberts, T. Molecules 2013, 18, 2328−2375. (5) Lu, Z.; Zhang, Y.; Liu, H.; Yuan, J.; Zheng, Z.; Zou, G. J. Fluoresc. 2007, 17, 580−7. (6) Moran, B. W.; Anderson, F. P.; Devery, A.; Cloonan, S.; Butler, W. E.; Varughese, S.; Draper, S. M.; Kenny, P. T. M. Bioorg. Med. Chem. 2009, 17, 4510−22. H



Article

(7) Timmers, S.; Auwerx, J.; Schrauwen, P. Aging 2012, 4, 146−58. (8) Zunino, S. J.; Storms, D. H. Cancer Lett. 2006, 240, 123−34. (9) García-García, J.; Micol, V.; de Godos, A.; Gómez-Fernández, J. C. Arch. Biochem. Biophys. 1999, 372, 382−8. (10) Galfi, P.; Jakus, J.; Molnar, T.; Neogrady, S.; Csordas, A. J. Steroid Biochem. Mol. Biol. 2005, 94, 39−47. (11) Losa, G. A. Eur. J. Clin. Invest. 2003, 33, 818−23. (12) Brisdelli, F.; D’Andrea, G.; Bozzi, A. Curr. Drug Metab. 2009, 10, 530−46. (13) Luther, D. J.; Ohanyan, V.; Shamhart, P. E.; Hodnichak, C. M.; Sisakian, H.; Booth, T. D.; Meszaros, J. G.; Bishayee, A. Invest. New Drugs 2011, 29, 380−391. (14) Roy, P.; Kalra, N.; Prasad, S.; George, J.; Shukla, Y. Pharm. Res. 2009, 26, 211−7. (15) Neves, A. R.; Lucio, M.; Lima, J. L. C.; Reis, S. Curr. Med. Chem. 2012, 19, 1663−81. (16) Rezaei, A.; Fathi, M.; Jafari, S. M. Food Hydrocolloids 2019, 88, 146−162. (17) Esfanjani, A. F.; Jafari, S. M. Colloids Surf., B 2016, 146, 532− 543. (18) Esfanjani, A. F.; Assadpour, E.; Jafari, S. M. Trends Food Sci. Technol. 2018, 76, 56−66. (19) Amri, A.; Chaumeil, J. C.; Sfar, S.; Charrueau, C. J. Controlled Release 2012, 158, 182−193. (20) Lu, X.; Ji, C.; Xud, H.; Li, X.; Dinga, H.; Yef, M.; Zhu, Z.; Ji, C.; Jiang, X.; Lu, X.; Guo, X.; Ding, D.; Ye, M.; Xu, H.; Li, X.; Ding, H.; Ding, X. Int. J. Pharm. 2009, 375, 89−96. (21) Karthikeyan, S.; Prasad, R. R.; Ganamani, A.; Balamurugan, E. Biomed. Prev. Nutr. 2013, 3, 64−73. (22) Ansari, K. A.; Vavia, P. R.; Trotta, F.; Cavalli, R. AAPS PharmSciTech 2011, 12, 279−286. (23) Bamrungsap, S.; Zhao, Z.; Chen, T.; Wang, L.; Li, C.; Fu, T.; Tan, W. Nanomedicine 2012, 7, 1253−1271. (24) Deshpande, P. P.; Biswas, S.; Torchilin, V. P. Nanomedicine 2013, 8, 1509−1528. (25) Jesorka, A.; Orwar, O. Annu. Rev. Anal. Chem. 2008, 1, 801−32. (26) Elizondo, E.; Moreno, E.; Cabrera, I.; Córdoba, A.; Sala, S.; Veciana, J.; Ventosa, N. Prog. Mol. Biol. Transl. Sci. 2011, 104, 1−52. (27) Oliveira, A. C. N.; Nogueira, S. S.; Gonçalves, O.; Cerqueira, M. F.; Alpuim, P.; Tovar, J.; Rodriguez-Abreu, C.; Brezesinski, G.; Gomes, A. C.; Lúcio, M.; Real Oliveira, M. E. C. D. R. RSC Adv. 2016, 6, 47730−47740. (28) Silva, J. P. N.; Oliveira, I. M. S. C.; Oliveira, A. C. N.; Lúcio, M.; Gomes, A. C.; Coutinho, P. J. G.; Real Oliveira, M. E. C. D. Biochim. Biophys. Acta, Biomembr. 2014, 1838 (1838), 2555−67. (29) Oliveira, I. M. S. C.; Silva, J. P. N.; Feitosa, E.; Marques, E. F.; Castanheira, E. M. S.; Real Oliveira, M. E. C. D. J. Colloid Interface Sci. 2012, 374, 206−17. (30) Neves, A. R.; Lúcio, M.; Martins, S.; Lima, J. L. C.; Reis, S. Int. J. Nanomed. 2013, 8, 177−87. (31) Barkat, M., Harshita, A., Beg, S., Ahmad, F. J. In Multifunctional Nanocarriers for Contemporary Healthcare Applications; Hershey, P. I. G. Ed.; IGI Global, 2018; DOI: 10.4018/978-1-5225-4781-5. (32) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L.-L.; Scherer, B.; Sinclair, D. A. Nature 2003, 425, 191−6. (33) Laouini, A.; Jaafar-Maalej, C.; Limayem-Blouza, I.; Sfar, S.; Charcosset, C.; Fessi, H. J. Colloid Sci. Biotechnol. 2012, 1, 147−168. (34) Fernandes, E.; Soares, T. B.; Gonçalves, H.; Lúcio, M. Front. Chem. 2018, 6, 1−20. (35) Avdeef, A. Curr. Top. Med. Chem. 2001, 1, 277−351. (36) Di, L.; Kerns, E. H. Curr. Opin. Chem. Biol. 2003, 7, 402−408. (37) Pereira-Leite, C.; Nunes, C.; Lima, J. L. F. C.; Reis, S.; Lúcio, M. J. Phys. Chem. B 2012, 116, 13608−13617. (38) Fernandes, E.; Soares, T. B.; Gonçalves, H.; Bernstorff, S.; Real Oliveira, M. E. C. D.; Lopes, C. M.; Lúcio, M. Int. J. Mol. Sci. 2018, 19, 1−26. (39) Seydel, J.; Wiese, M. Drug-Membrane Interactions: Analysis, Drug Distribution, Modeling; Methods and Principles in Medicinal

Chemistry (Book 15); Wiley-VCH Verlag, 2003; Vol. 15, DOI: 10.1002/3527600639. (40) Engelk, M.; Bojarski, P.; Bloss, R.; Diehl, H. Biophys. Chem. 2001, 90, 157−73. (41) Jain, M.; Wu, N. J. Membr. Biol. 1977, 34, 157−201. (42) Xu, R. Particle Characterization: Light Scattering Methods; Springer Science & Business Media, 2001; DOI: 10.1007/0-30647124-8. (43) Silva, J. P. N.; Oliveira, A. C. N.; Lúcio, M.; Gomes, A. C.; Coutinho, P. J. G.; Real Oliveira, M. E. C. D. Colloids Surf., B 2014, 121, 371−9. (44) Oliveira, A. C. N.; Raemdonck, K.; Martens, T.; Rombouts, K.; Simón-Vázquez, R.; Botelho, C.; Lopes, I.; Lúcio, M.; GonzálezFernández, Á .; Real Oliveira, M. E. C. D.; Gomes, A. C.; Braeckmans, K. Acta Biomater. 2015, 25, 216−229. (45) Oliveira, A. C. N.; Martens, T. F.; Raemdonck, K.; Adati, R. D.; Feitosa, E.; Botelho, C.; Gomes, A. C.; Braeckmans, K.; Real Oliveira, M. E. C. D. ACS Appl. Mater. Interfaces 2014, 6, 6977−89. (46) Csiszár, A.; Csiszar, A.; Pinto, J. T.; Gautam, T.; Kleusch, C.; Hoffmann, B.; Tucsek, Z.; Toth, P.; Sonntag, W. E.; Ungvari, Z. J. Gerontol., Ser. A 2015, 70, 303−13. (47) Kobayashi, H.; Watanabe, R.; Choyke, P. L. Theranostics 2014, 4, 81−9. (48) Fini, L.; Hotchkiss, E.; Fogliano, V.; Graziani, G.; Romano, M.; De Vol, E. B.; Qin, H.; Selgrad, M.; Boland, C. R.; Ricciardiello, L. Carcinogenesis 2008, 29, 139−46. (49) van Erk, M. J.; Roepman, P.; van der Lende, T. R.; Stierum, R. H.; Aarts, J. M. M. J. G.; van Bladeren, P. J.; van Ommen, B. Eur. J. Nutr. 2005, 44, 143−56. (50) Radulovic, M.; Knittelfelder, O.; Cristobal-Sarramian, A.; Kolb, D.; Wolinski, H.; Kohlwein, S. D. Curr. Genet. 2013, 59, 231−242. (51) Mochizuki, K.; Nakanishi, K.; Yasui, T. Agric. Biol. Chem. 1986, 50, 399−407. (52) Bishop, J. R.; Nelson, G.; Lamb, J. J. Microencapsulation 1998, 15, 761−73. (53) Shi, G.; Rao, L.; Yu, H.; Xiang, H.; Yang, H.; Ji, R. Int. J. Pharm. 2008, 349, 83−93. (54) Lançon, A.; Delma, D.; Osman, H.; Thénot, J. P.; Jannin, B.; Latruffe, N. Biochem. Biophys. Res. Commun. 2004, 316, 1132−1137. (55) Colin, D.; Limagne, E.; Jeanningros, S.; Jacquel, A.; Lizard, G.; Athias, A.; Gambert, P.; Hichami, A.; Latruffe, N.; Solary, E.; Delmas, D. Cancer Prev. Res. 2011, 4, 1095−106. (56) Abdel-Bar, H. M.; El Basset Sanad, R. A. Biomed. Pharmacother. 2017, 93, 561−569. (57) Jhaveri, A.; Deshpande, P.; Pattni, B.; Torchilin, V. J. Controlled Release 2018, 277, 89−101. (58) Wang, Z.; Gu, Z.; Shen, Y.; Wang, Y.; Li, J.; Lv, H.; Huo, K. PLoS One 2016, 11, No. e0150156. (59) Madrigal-Perez, L. A.; Canizal-Garcia, M.; GonzálezHernández, J. C.; Reynoso-Camacho, R.; Nava, G. M.; RamosGomez, M. Yeast 2016, 33, 227−34. (60) Sprouse, A. A.; Herbert, B.-S. Anticancer Res. 2014, 34, 5363− 74. (61) Schuster, S.; Penke, M.; Gorski, T.; Petzold-Quinque, S.; Damm, G.; Gebhardt, R.; Kiess, W.; Garten, A. PLoS One 2014, 9, No. e91045. (62) Vanamala, J.; Reddivari, L.; Radhakrishnan, S.; Tarver, C. BMC Cancer 2010, 10, 238. (63) Wang, X.; Wang, D.; Zhao, Y. Cell Biochem. Biophys. 2015, 73, 527−531. (64) Taylor, E. J. M.; Yu, Y.; Champer, J.; Kim, J. Dermatol. Ther. (Heidelb). 2014, 4, 249−57. (65) Weissig, V. Liposomes. In Methods and Protocols, Vol. 1: Pharmaceutical Nanocarriers; Springer, 2010. (66) Brittes, J.; Lúcio, M.; Nunes, C.; Lima, J. L. F. C.; Reis, S. Chem. Phys. Lipids 2010, 163, 747−54. (67) Magalhães, L. M.; Nunes, C.; Lúcio, M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C. Nat. Protoc. 2010, 5, 1823−30. I



Article

(68) Avila, C. M.; Martínez, F. Chem. Pharm. Bull. 2003, 51, 237− 40. (69) Gallagher, K. M.; Corrigan, O. I. J. Controlled Release 2000, 69, 261−72. (70) Costa, P.; Sousa Lobo, J. M. Eur. J. Pharm. Sci. 2001, 13, 123− 33. (71) Raths, S.; Rohrer, J.; Crausaz, F.; Riezman, H. J. Cell Biol. 1993, 120, 55−65.

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