Liposomes as carriers of the lipid soluble antioxidant resveratrol: Evaluation of amelioration of oxidative stress by additional antioxidant vitamin

Life Sciences 93 (2013) 917–923

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Liposomes as carriers of the lipid soluble antioxidant resveratrol: Evaluation of amelioration of oxidative stress by additional antioxidant vitamin K. Vanaja a,b, M.A. Wahl c, L. Bukarica d, H. Heinle a,⁎ a

Institute of Physiology, Eberhard Karls Universität Tübingen, Gmelinstr. 5, Tuebingen D-72076, Germany Visveswarapura Institute of Pharmaceutical Sciences, Bangalore, India c Pharmazeutische Technologie, Eberhard Karls Universität Tübingen, Tuebingen D-72076, Germany d Department of Pharmacology, University of Belgrade, Serbia b

a r t i c l e

i n f o

Article history: Received 13 May 2013 Accepted 15 October 2013 Keywords: Resveratrol Vitamin C Antioxidant activity Liposomes Chemiluminescence Flow cytometry

a b s t r a c t Aim: Resveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems. Main methods: Liposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H2DCF-DA dye. Key findings: Particle size varied from 134.2 ± 0.265 nm to 103.3 ± 1.687 nm; PDI ≤0.3; zeta potential values were greater than −30 mV and PDE ≥80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H2O2 further intensified the antioxidative effects of pure RES and liposomal formulations. Significance: The present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS. © 2013 Elsevier Inc. All rights reserved.

Introduction Oxidative stress is a problem of aerobic life and is considered to be involved in many diseases like atherosclerosis, cancer, neurodegenerative diseases and ageing (Halliwell, 2000, 2006). Cellular mechanisms involved in the production of reactive oxygen species (ROS) include the inflammatory response, free radical leak from mitochondria, auto-oxidation of catecholamines, activation of xanthine oxidase, pro-oxidant activities of toxins or even remedies and exposure to ionising radiations. On the molecular level, ROS may attack and modify proteins, unsaturated lipids and nucleic acid leading to altered functions or even destruction causing cell death (Chanvitayapongs et al., 1997). Therefore antioxidants (AO) have received a great deal of attention, ⁎ Corresponding author at: Institute of Physiology, University of Tuebingen, Tübingen 72076, Germany. Tel.: +49 7071 2973420; fax: +49 7071 293073. E-mail address: [email protected] (H. Heinle). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.10.019

for their prophylactic and therapeutic potential for diseases in which the physiological cell defence against ROS is compromised (Ratman et al., 2006). Dietary nutritional components are the primary source of antioxidants providing substances like polyphenols, flavonoids, carotenoids, vitamin E or vitamin C to the body. Yet, although these compounds very often reveal strong antioxidant activity during in vitro investigations, their in vivo effect is sometimes limited due to the hydrophobic properties, limited uptake in intestine or rapid biodegradation by the liver (Tesoriere et al., 2009). In the last years, many studies focussed on the polyphenolic substance resveratrol (RES) which is described to provide pharmacological protection in many diseases by virtue of its antioxidant activity (Lee et al., 2012; Bonechi et al., 2012; Kristl et al., 2009). However, despite the unquestionable protective activity of resveratrol in several in vitro models, clinical studies demonstrated only minimal effect in humans which is mainly related to pharmacokinetics of resveratrol (Lu et al., 2009; Das et al., 2008). Trans-resveratrol in plasma is very sparse with short

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half-life (8–14 min) because of its poor water-solubility (b 1 μM) and instability, as it converts to cis form (a less active form). To circumvent the solubility limitations, loading polyphenols into water soluble carriers such as liposomes would offer chemical and biological protection (Bonechi et al., 2012; Cadena et al., 2013; Kristl et al., 2009; Caddeo et al., 2008). The use of antioxidant liposomes referring to liposomes containing lipid-soluble antioxidants, water-soluble antioxidants, chemical antioxidants, enzymatic antioxidants or combination of these various antioxidants have been thoroughly reviewed (Stone and Smith, 2004). Combination of resveratrol with antioxidant vitamins was found to be more effective in protecting the cells from oxidative stress rather than any of these antioxidants alone (Chanvitayapongs et al., 1997). Effect of resveratrol in combination of known antioxidant such as vitamin C in ameliorating oxidative stress has been investigated earlier using pure resveratrol only (Chanvitayapongs et al., 1997), whereas the additive antioxidative effect of vitamin C with resveratrol loaded into liposomes has not been tested earlier. Hence, the present study aims to examine the antioxidative capacity of liposomal resveratrol utilising chemiluminescence test systems and cellular systems with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) dye. Hence, the goal of the present work was to prepare resveratrol loaded liposomes with and without vitamin C to investigate the ameliorative antioxidant effect using chemical and cellular test systems. Materials and methods Materials AAPH [2,2′-azobis(2-amidinopropane)dihydrochloride] was obtained from Polyscience, Warrington, USA. Resveratrol (RES), cholesterol (CH), dihexadecyl phosphate (DP), Triton-X 100, 2′,7′dichlorodihydrofluorescein diacetate (H2DCF-DA), and 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich, Steinheim. Zymosan A was obtained from Sigma, Deisenhofen. Vitamin C (VC) was purchased from Caesar & Loretz GmbH, Hilden. Phospholipon 90H (PL) was a gift sample from Lipoid AG, Cologne. Luminol, methanol, ethanol, chloroform, and DMSO were obtained from Merck, Darmstadt. HPLC estimation of vitamin C was commercially analysed by SGS Institut Fresenius GmbH, Berlin. Buffy coat cells were provided by Institute of Transfusion Medicine, University of Tuebingen. Human umbilical vein endothelial cells (HUVECs) were provided by Institute for Pathology, University of Tuebingen. Preparation of liposomes Liposomes loaded RES with and without VC were prepared as per the composition shown in Table 1 using thin film hydration method (Vanaja et al., 2008). Briefly, lipid phase comprised of PL, CH, and DP dissolved in CHCl3:MeOH (2:1 v/v) mixture in a dry round bottom flask. Organic solvents were removed by a vacuum evaporator (RotavaporR, W. Büchi, Flawil Schweiz) above the lipid transition temperature (51 °C) to obtain a uniform, thin lipid film on the wall of the flask. The deposited lipid film was hydrated with appropriate volume of water (double distilled) by rotation for 1 h at 51 °C. Small unilamellar (SUV) liposomes were obtained by subjecting the dispersions to probe sonication (Ultrosonic Processor, UP200S, Hielscher Ultrasound Technology) for 2 to 6 min using cooling pads. Finally the liposomal dispersions prepared were stored at room temperature for 2 h to anneal any structural defects. RES was added to the lipid phase during lipid film formation; VC was added in the aqueous phase during the lipid film hydration (L-RES — resveratrol loaded liposomes; L-RESVC — resveratrol loaded liposomes with vitamin C). Blank liposomes were prepared similarly without resveratrol and vitamin C (L-BLANK). The antioxidative capacity of the liposomal formulations was compared

with pure resveratrol. Pure drug stock solution of 200 μM of RES (PD-RES) was prepared in methanol and stored at − 20 °C. Liposome characterisation Percentage entrapment efficiency (% EE) was determined after separation of free and entrapped drug. Five millilitres of liposome suspension was subjected to ultracentrifugation (Beckman L7 ultracentrifuge, Beckmann Coulter GmbH) for 50 min at 40,000 RPM at 6 °C. Resveratrol content was determined directly from the liposomal pellet. Methanol was added on the pellet, vortexed, filtered through 0.45 μm pore size filter and analysed using HPLC as reported earlier (Atanackovic et al., 2011). Percentage EE of vitamin C was determined indirectly from the free concentration in the supernatant after ultracentrifugation using reversed phase HPLC as reported earlier (Kall and Andersen, 1999). %EE ¼

Amount of drug entrapped  100 Total amount of drug

ð1Þ

Particle size (Z-average mean size and polydispersity index) and ζ-potential were determined by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE), respectively, using a nanosizer (Nano-ZS, Nanoseries, Malvern Instruments, UK) after appropriate dilution of liposome preparation (Kristl et al., 2009; Anabousi et al., 2005). Six independent measurements were performed in each case. Mass distribution of particle size with polydispersity index as well as the electrophoretic mobility was obtained. Sizes are reported as the Z-average mean diameter for the hydrodynamic diameter (nm) along with polydispersity index. Zeta potential (mV) was calculated from electrophoretic mobility in the electric field. Microscopical examinations of the liposome preparations were carried out using freeze fracture electron microscopy (Schubert et al., 1986). Vesicle suspensions were drawn up with a micropipet, mounted directly into the central hole (1 mm in diameter) of a pair of gold specimen holders, shock frozen in nitrogen slush (−210 °C), and transferred to a freeze-fracturing BAF 400 D apparatus (Balzers) equipped with an oscillating quartz (QSG 301). The specimens were fractured at about 5 × 10−6 mbar and −150 °C and then shadowed with platinum/carbon (45 °C, 2 nm) and carbon (20 nm) for replica stabilisation. To avoid any condensation at the fracture faces, shadowing was started before fracturing. Replicas were directly cleaned several times in distilled water and mounted on pioloform-coated copper grids and observed in Siemens Elmiscop 102 electron microscope. Determination of anti-oxidative capacity Inhibition of AAPH induced luminol enhanced chemiluminescence The experimental procedure carried out was similar to the previous report (Germann et al., 2006). Briefly, the reaction mixture consisted of 500 μl Tyrode solution including AAPH (10 mM), luminol (0.3 mM) and DMSO (0.5%) thermostated at 37 °C; after 20 min, the liposomal preparations (50 μl) were added and the reaction was followed for another 20 min. Chemiluminescence detecting free radicals formed by spontaneous decay of AAPH was measured throughout the time and is given either as actual time dependent intensity or as integrated values of photons detected in a luminometer (Berthold 9600, Wildbad, Germany). Relative radical scavenging activity was calculated as follows: Radical scavenging activity ¼

AUCAAPH –AUCSAMPLE AUCAAPH

ð2Þ

with AUCAAPH: CL0–20 min; AUCSAMPLE: CL20–40 min, values ranging from 0 (no scavenging) to 1 (maximum scavenging).

K. Vanaja et al. / Life Sciences 93 (2013) 917–923

Measurement of intracellular oxygen-derived radicals

Table 1 Composition of liposomes loaded with resveratrol and addition of vitamin C. Molar ratio

Resveratrol (μM)

Vitamin C (μM)

100 150 200 100 150 200

100 150 200

PL:CH:DP L-RES 1 L-RES 2 L-RES 3 L-RESVC 1 L-RESVC 2 L-RESVC 3

2:0.4:0.6 3.4:0.4:0.6 4:0.8:1.2 2:0.4:0.6 3.4:0.4:0.6 4:0.8:1.2

Cellular ROS production was detected using the dye 2′-7′dichlorodihydrofluorescein diacetate (H2DCF-DA), a cell permeable non-fluorescent probe which is de-esterified intracellularly by means of esterases (H2DCF) and turns to highly fluorescent 2′-7′dichlorofluorescein (DCF) on oxidation (Rothe and Valet, 1990; Rotte et al., 2012). Blood leukocytes and human umbilical vein endothelial cells were used for this method. Using isolated blood leukocytes

L-RES: liposomes loaded resveratrol without additional vitamin C. L-RESVC: liposomes loaded resveratrol with additional vitamin C.

Inhibition of ROS production using isolated blood leukocytes As a second assay, the inhibiting effect on zymosan-induced oxidative burst reaction of blood leukocytes was determined (Germann et al., 2006; Heinle and El-Dessouki, 1995). For each experiment, buffy coat cells (50 μl) from one healthy donor (cell count of approximately 2.5 × 105) were incubated with 500 μl Tyrode solution and 0.11 mM luminol (in 1% DMSO). Chemiluminescence (Berthold 9600, Wildbad, Germany) was measured for a period of 60 min: 0 to 10 min without further addition, 10–20 min with addition of liposome preparations (50 μl), and finally after the stimulation of phagocytosis by zymosan (20–60 min). Basal chemiluminescence was related to the standard cell count; the effects of liposomal preparations on the basal radical production and on the degree of activation by zymosan were evaluated. Results obtained are reported in relative to control.

Inhibition ¼

919

Integral control−Integral sample Integral control

ð3Þ

Inhibition values range from 0 (no inhibition) to 1 (maximum inhibition).

Briefly, to buffy coat cells, phosphate buffered saline (PBS) and 50 μl of sample preparations (liposome loaded resveratrol with and without vitamin C, blank liposomes, pure resveratrol) were added; the mixture was incubated in the dark at 37 °C for 60 min. Thereafter, H2DCF-DA was added (final concentration of 10 μM); cells were washed with PBS, followed by incubation at 37 °C for 30 min. After a washing step, emission of trapped, oxidised DCF in cells was analysed with flow cytometry; FACS-calibur from Becton Dickinson; Heidelberg, Germany (Sarkar et al., 2006). Cells incubated without antioxidants or liposomal preparations were defined as control. Stock solution of 200 μM of RES (PD-RES) was prepared in methanol and stored at −20 °C. Using human umbilical vein endothelial cells (HUVECs) HUVECs cultured to confluence were pre-incubated at 37 °C, 5% CO2 + 95% O2 with H2DCF-DA dye (final concentration 10 μM) and 500 μl of sample preparations (liposome loaded resveratrol with and without vitamin C, blank liposomes, pure resveratrol) for 1 h. Then, the cells were washed with PBS, and incubated in fresh medium containing H2O2 (50 μM) for 1 h at 37 °C. Cells incubated without antioxidants or liposomal preparations were defined as control. Thereafter, the cells were trypsinized, washed, and centrifuged at 1000 RPM; the pellet was resuspended with PBS and stored on ice for immediate FACS analysis (FACS-calibur from Becton Dickinson; Heidelberg, Germany). Fluorescence intensity of DCF was measured in FL-1 with an excitation wavelength of 488 nm and emission wavelength of 530 nm.

Cytotoxicity assay Statistics Cytotoxicity of formulations was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Briefly, buffy coats (5 × 105 cells/well) and HUVECs (1 × 104cells/well) were plated into 96-well culture plate and incubated at 37 °C in a humidified 5% CO2 incubator. The test samples (L-RES 3 and L-RES VC 3 and pure resveratrol) were added to the wells and incubated for 24 h at 37 °C (5% CO2). After 24 h of incubation, 100 μl of fresh media and 10 μl MTT (5 mg/ml; Sigma) were added to each assay well and incubated for 4 h, protected from the light. The formazan crystals produced were dissolved by resuspension in 200 μl of DMSO and the absorbance at 570 nm was measured by using Versamax plate reader (Molecular Devices VERSAmax Tunable Microplate Reader).

Data is expressed as mean ± SEM; n represents the number of independent experiments. Differences were tested for significance using one way ANOVA-Tukey's multiple comparison test and Student t-test. P b 0.05 was considered to be significant. Results Preparation of liposomes loaded resveratrol with and without vitamin C Various liposomal formulations of resveratrol with and without vitamin C were prepared as per the compositions in Table 1. Resveratrol

Table 2 Characterisation of liposomes loaded with resveratrol and addition of vitamin C. Particle size d. (nm)

L-RES 1 L-RES 2 L-RES 3 L-RESVC 1 L-RESVC 2 L-RESVC 3

133.0 131.8 134.2 125.2 127.1 103.3

± ± ± ± ± ±

1.21 0.98 0.13 0.45 0.35 0.84

Data expressed as mean ± SEM, n = 6.

Poly dispersity index

0.265 0.249 0.321 0.251 0.315 0.191

± ± ± ± ± ±

0.01 0.01 0.01 0.00 0.02 0.01

Zeta potential (mV)

−37.26 −48.77 −49.28 −60.70 −60.60 −54.33

± ± ± ± ± ±

1.16 0.44 0.03 0.06 0.52 1.47

% Encapsulation efficiency Resveratrol

Vitamin C

78.53 80.11 85.19 78.56 83.72 79.93

55.37 ± 0.36 56.90 ± 1.17 60.7 ± 0.47

± ± ± ± ± ±

1.63 1.15 0.96 0.54 0.69 0.31

920

K. Vanaja et al. / Life Sciences 93 (2013) 917–923

a)

b)

0.2 µm

0.2 µm

Fig. 1. Freeze fracture electron micrographs of liposomes loaded with resveratrol and addition of vitamin C. a) L-RES and b) L-RES VC. Bar indicates 0.2 μm.

being imparted due to the presence of dicetyl phosphate in the formulations (Caddeo et al., 2008). Vesicle morphology evaluated by freeze fracture electron microscopy further confirmed that the formulations were unilamellar vesicles (Fig. 1) within the electrophoretically determined particle size.

was stably incorporated in liposomes with entrapment efficiency greater than 80% (Table 2). The size of the liposomes as determined by dynamic light scattering method varied from 134.2 nm to 103.3 nm. Polydispersity index (PDI) effectively accounts for particle distribution and homogeneity of colloidal suspension. Results obtained showed that L-RES and L-RESVC formulations displayed PDI between 0.191 ± 0.01 and 0.265 ± 0.01 indicating narrow and homogenous particle size distributions. Zeta potential (ZP) which is a good index of magnitude of repulsive interaction between colloidal particles assesses the electrostatic stability of vesicular and colloidal suspensions. All the liposome formulations exhibited ZP values in the range of −37.26 ± 1.16 to −60.70 ± 1.22 mV, with the negativity

Effects on AAPH-induced luminol enhanced chemiluminescence The AAPH reaction which is based on its thermic decomposition to form free radicals was used to test antioxidant activity of the liposomal formulations (Fig. 2). The comparison with the control shows that resveratrol does not lose its antioxidant properties after encapsulation

a) Chemiluminescence (cpm)

016E+08 014E+08 012E+08 010E+08 008E+08 006E+08 004E+08 002E+08 0,000E+00 0

10

20

30

40

50

60

Time (min) Control

PD-RES

L-RES VC 3

L-RES 3

% Radical scavenging activity

b) #*

100 90 80 70 60 50 40 30 20 10 0

*

#* *

*

* *

CONTROL L-BLANK

L-RES 1

L-RES 2

L-RES 3 L-RESVC 1 L-RESVC 2 L-RESVC 3 PD-RES

Fig. 2. Anti-oxidative effects of liposomes loaded with resveratrol and addition of vitamin C in AAPH reaction. a) Original tracing of the luminol-enhanced chemiluminescence reaction of AAPH is shown. The antioxidants were added at 20 min. b) Arithmetic mean ± SEM (n = 6 independent measurements). Control was performed with methanol alone. Final concentration of RES in L-RES 1 and L-RESVC 1 (10 μM); L-RES2 and L-RESVC2 (15 μM); L-RES3 and L-RESVC3 (20 μM) and PD-RES (20 μM). ⁎(P b 0.001) indicates significant difference from control. #(P 0.01) indicates significant difference of L-RES3 and L-RES VC3 vs. PD-RES.

K. Vanaja et al. / Life Sciences 93 (2013) 917–923

921

a) Chemiluminescence (cpm)

030E+08 025E+08 020E+08 015E+08 010E+08 005E+08 0,000E+00 0

10

20

30

40

50

60

Time (min) Control

L-RES 3

L-RES VC 3

PD-RES

b) 100

%Luminescence inhibition (relative to control)

90 80

# *

70

# *

*

*

60 50

*

*

40 30 20 10 0 L-BLANK

L-RES 1

L-RES 2

L-RES 3

L-RESVC 1 L-RESVC 2 L-RESVC 3

PD-RES

Fig. 3. Antioxidative effects of liposomes loaded with resveratrol and addition of vitamin C on free radical production of zymosan stimulated buffy coat cells. a) Original tracing of free radical production of zymosan stimulated buffy coat cells and inhibition by different formulations is shown. The reaction was followed by measurement of luminol-enhanced chemiluminescence. b) Arithmetic mean ± SEM (n = 6 independent measurements). Relative inhibition of chemiluminescence by liposomal formulations (100% means complete inhibition). Final concentration of RES in L-RES 1 and L-RESVC 1 (10 μM), L-RES2 and L-RESVC2 (15 μM), L-RES3 and L-RESVC3 (20 μM), and PD-RES (20 μM). ⁎(P b 0.05) indicates significant difference of L-RES vs. L-RES VC of respective concentrations. #(P b 0.05) indicates significant difference of L-RES3 and L-RESVC3 vs. PD-RES.

into liposomes; in contrast, the antioxidant capacity at least in this test system was improved be approximately 20%. As seen in Fig. 2a and b, the different formulations revealed an increased anti-oxidative capacity with increasing content of resveratrol loaded in liposomes, yet the addition of vitamin C did not improve this property correspondingly.

110

*

Cell Viability (%)

100

#

#

*

90

#

*

80 70

Cytotoxicity study Cytotoxicity potential of pure resveratrol and liposomes loaded with resveratrol was evaluated in buffy coat cells and HUVECS using MTT Tetrazolium assay. The yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of hydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan was solubilized and quantified by spectrophotometric means. Resveratrol pure and loaded into liposomes did not show significant reduction or improvement in cell viability of buffy coat cells and HUVECS at the tested concentration of 20 μM, which confirmed the elimination of cytotoxicity of resveratrol (Fig. 4).

60 50

Effects on ROS production in isolated blood leukocytes

40 30 20 10 0 Control

L-RES 3 Buffy coats

L-RESVC 3

PD-RES

HUVECs

Fig. 4. Cytotoxic effect of free resveratrol and liposomes loaded with resveratrol and addition of vitamin C on buffy coat cells and HUVECS. Arithmetic mean ± SEM (n = 4 independent measurements). Final concentration of RES in L-RES 3, L-RES VC3 and PD-RES was 20 μM. Cells incubated in the absence of antioxidants served as control. ⁎(P b 0.01) indicates significant differences from PD-RES in buffy coat cells. #(P b 0.001) indicates significant difference from PD-RES in HUVECS.

The results of these measurements showed again the dose dependency effect of liposomal resveratrol in the absence and presence of vitamin C (Fig. 3a and b). Yet, the presence of vitamin C did not show statistically significant additive effect. Increase in ROS levels is indicated as a shift in histograms showing frequency distribution of values of DCF fluorescence versus number of events or cells. Flow cytometric histograms showed a unimodal distribution of DCF fluorescence with decrease in the fluorescence as evident from the shift in the mean fluorescence intensity (MFI). Resveratrol loaded liposomes quenched the ROS produced by activated phagocytes in comparison with pure methanolic solution of resveratrol.

K. Vanaja et al. / Life Sciences 93 (2013) 917–923

a) Counts

CONTROL L-RES 3

PD-RES L-RESVC3

100

101

102

103

104

FL1-Height

% ROS positive cells

b)

90 80 70 60 50 40 30 20 10 0

* # *

CONTROL

L-Blank

L-RES 3

#*

L-RESVC 3

PD-RES

Fig. 5. Anti-oxidative effects of liposomes loaded with resveratrol and addition of vitamin C on spontaneous free radical production of buffy coat cells. a) FACs histograms depicting ROS dependent DCF fluorescence. b) Arithmetic mean ± SEM (n = 3 independent measurements). Final concentration of RES in L-RES 3, L-RES VC3 and PD-RES was 20 μM. Cells incubated in the absence of antioxidants served as control. ⁎(P b 0.05) indicates significant differences from control. #(P b 0.05) indicates significant difference from PD-RES.

Measurement of intracellular oxygen-derived radicals The results of the FACS analysis of buffy coat cells treated with liposomes are shown in Fig. 5. ROS dependent DCF fluorescence is shown in Fig. 5a; the results of which were quantitatively analysed as seen in Fig. 5b. It is obvious that in this case the combination of vitamin C with resveratrol is more active than the singly loaded liposomes and the pure resveratrol as well. However, concerning inhibition of spontaneous free radical production in buffy coat cells, there is no significant difference between L-RES 3 and L-RES VC 3. Results from the second cellular system with HUVECs for the antioxidant assay showed a similar property as observed with the buffy coat cells (Fig. 6). Hence, resveratrol loaded into liposomes could effectively protect the oxidative damage induced by H2O2 as well as oxidative burst in buffy coat cells. Data obtained from flow cytometric analysis reveals that resveratrol when encapsulated into liposomes could effectively quench the intracellular accumulation of ROS but a synergistic effect was not seen with additional vitamin C.

cytotoxicity, and its long-term stability (Kristl et al., 2009; Caddeo et al., 2008; Bonechi et al., 2012). The anti-oxidative action of RES in free and liposomal form was also evaluated on HEK293 cell line; the metabolic activity showed no cytotoxicity stimulated cellular metabolic and antioxidant activity levels to eliminate the harmful effect of the stress (Kristl et al., 2009). Peroxidation initiated thermally by the azo initiator AAPH demonstrated that resveratrol and some analogues were effective AO against linoleic acid peroxidation in sodium dodecyl sulphate (SDS) and cetyl trimethylammonium (CTAB) micelles (Fang et al., 2002). Although, the anti-oxidative property of RES is reported earlier, for the first time we studied the anti-oxidative actions of RES–vitamin C combination after delivery as liposomes using different test systems to examine whether ameliorative antioxidant effect exists. By application of film hydration procedure, liposomes incorporating RES with and without VC were prepared to get good encapsulation efficiency up to the range of 80%. Encapsulation efficiency of vitamin C was less prominent, which could mean that a substantial amount of VC was present in the aqueous solution. Furthermore, the polydispersity index less than 0.3 indicated a narrow size distribution and homogenous distribution of colloidal particles, hence a good quality for all the formulations. Also a large negative zeta potential further enhanced the stability of vesicular suspension by simple electrostatic stabilisation with negativity being imparted by dicetyl phosphate. These observations were similar to previously reported studies (Caddeo et al., 2008). Further, freeze-fracture electron microscope confirmed that L-RES and L-RESVC were small unilamellar vesicles (Fig. 1). Presence of VC in liposomes loaded with RES did not influence the physicochemical properties when compared with liposomes loaded with RES alone. The results of measurement of reduction in oxidative stress in various model systems show, firstly, that the anti-oxidative effect of RES was not impaired by liposome carrier system and its encapsulation within the lipid layers. Secondly, it is evident that under the experimental conditions of the test systems (cellular and cell free systems — chemical systems) used RES loaded into liposomes exhibited generally a small but significant increased activity in comparison with the same concentration of free RES. This could be due to the limited solubility of free RES in water,

a)

Control PD-RES

Counts

922

L-RES 3 L-RES VC 3

100

Discussion

102

103

104

FL1-Height

b) % ROS Positive cells

Previous studies have shown that resveratrol can prevent or slow the progression of a wide variety of illness including cancer and cardiovascular diseases, enhance stress resistance and extend the lifespan of various organisms from yeast to vertebrates (Baur and Sinclair, 2006). In vivo, resveratrol has reported to increase plasma antioxidant capacity and decrease lipid peroxidation; in vitro RES prevents LDL oxidation as well as scavenge free radicals. Studies conducted in the past indicate that RES can suppress pathological increases in the peroxidation of lipids and other macromolecules in vivo, but the mechanism being not clear (Baur and Sinclair, 2006). However, results from the pharmacokinetic studies report that RES is rapidly metabolised and has a short initial half-life, due to low solubility (Baur and Sinclair, 2006). To overcome the limitations of low bioavailability and solubility of RES, liposomes as appropriate delivery system for RES have been investigated by several authors. Localization of RES within liposomal bilayer was found to be crucial for stimulation of cell-defence system, prevention of resveratrol

101

56 48 40

* #*

#*

32 24 16 8 0 CONTROL

L-BLANK

L-RES 3

L-RESVC 3

PD-RES

Fig. 6. Measurement of intracellular oxygen-derived radicals-using HUVECS in presence of liposomes loaded with resveratrol and addition of vitamin C. a) FACs histograms depicting ROS dependent DCF fluorescence. b) Arithmetic mean ± SEM (n = 3 independent measurements). Final concentration of RES in L-RES 3, L-RES VC3 and PD-RES was 20 μM. Cells incubated in the absence of antioxidants served as control. ⁎(P b 0.001) indicates significant difference from control; #(P b 0.01) indicates significant difference from PD-RES.

K. Vanaja et al. / Life Sciences 93 (2013) 917–923

respectively, within the cellular systems, but with an improved uptake of liposomal RES into the cells. The cellular systems, i.e., buffy coat cells and HUVECs provided evidence that the liposomal preparations are able to scavenge ROS in the extracellular as well as the intracellular space: zymosan stimulated oxidative burst reaction leads to an extracellular secretion of oxygen free radicals which was effectively inhibited by liposomal RES. Similarly decrease in ROS production as indicated by decrease in DCF fluorescence is clearly an intracellular event. Further, in vitro cytotoxicity data obtained from MTT assay confirmed the elimination of resveratrol cytotoxicity (at 20 μM concentration) in the cellular systems, which suggests that liposomal formulations loaded with antioxidants would improve cellular drug uptake and retention. Previous studies performed with pure form of RES using FACS analysis with DCF-DA showed that pure resveratrol could strongly protect HT22 cells from glutamateinduced oxidative cytotoxicity by removing intracellular ROS (Fukui et al., 2010) and oxidative stress and isosmotic cell shrinkage were significantly blunted in the presence of pure resveratrol (Qadri et al., 2009). In the present study, resveratrol delivered via liposomes further enhanced the reduction in intracellular ROS. Another aspect is that addition of vitamin C did not affect the antioxidant activity of RES by a remarkable extent. This means that the vitamin C is less effective in the liposomal system, probably because it is mainly present in the aqueous phase (Wen-Lei and Jun-Min, 2008; Porter, 1993). The fact that vitamin C does not significantly enhance the antioxidative behaviour of pure RES was also described by Chanvitayapongs et al., 1997. These authors used t-BuOOH treatment of PC12 cells as a cellular model in studying oxidative stress and observed that combination of vitamin C and resveratrol did not significantly increase the protection over that afforded by resveratrol alone. However, the present study suggests that L-RES is more active when delivered as liposomes than pure RES as it could significantly improve the protection during oxidative stress, whereas presence of vitamin C did not synergise this protection to a great extent. Conclusion Protection of oxidative injury, also within the intracellular compartments as seen from FACs analysis, was more pronounced following delivery of resveratrol as small unilamellar liposomes in comparison to methanolic solution of resveratrol. However, a synergistic anti-oxidative effect was not assured in presence of vitamin C; hence other known antioxidants should be considered to study the ameliorative effect. Conflict of interest statement The authors have declared no conflict of interest.

Acknowledgement We would like to thank, Prof. Dr. Hartwig Wolburg and Dr. Petra Fallier-Becker, for helping us with the TEM analysis and cell culture studies. Thanks to Mr. Klaus Weyhing (Institute of Pharmacy) for the help rendered in characterisation studies. Thanks to Shefalee Bhavsar, Soumya Chatterjee (Institute of Physiology) for helping with FACS analysis and Helena, Cvejic, (Novisad, Serbia) for the help rendered in HPLC analysis. Finally, we would like to thank DAAD (Deutscher Akademischer Austausch Dienst) for providing fellowship to conduct this work. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2013.10.019.

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