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Zein-Based Nanoparticles Improve the Oral Bioavailability of Resveratrol and Its Anti-inflammatory Effects in a Mouse Model of Endotoxic Shock Rebeca Penalva, Irene Esparza, Eneko Larraneta, Carlos J. Gonzalez-Navarro, Carlos Gamazo, and Juan M. Irache J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505694e • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 6, 2015

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Journal of Agricultural and Food Chemistry

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Zein-Based Nanoparticles Improve the Oral Bioavailability of Resveratrol

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and Its Anti-inflammatory Effects in a Mouse Model of Endotoxic Shock

3 4

Authors

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Rebeca Penalva a, Irene Esparza a, Eneko Larraneta a, Carlos J. González-Navarro c,

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Carlos Gamazo b, Juan M. Irache a

7

8

Affiliation

9

a

Department of Pharmacy and Pharmaceutical Technology, University of Navarra,

10

31008 - Pamplona, Spain.

11

b

Department of Microbiology. University of Navarra, 31008 - Pamplona, Spain.

12

c

Centre for Nutrition Research, University of Navarra, 31080 - Pamplona, Spain.

13

Corresponding author:

14 15 16 17 18 19 20 21 22 23 24

Prof. Juan M. Irache Dep. Pharmacy and Pharmaceutical Technology University of Navarra C/ Irunlarrea, 1 31080 – Pamplona Spain Phone: +34948425600 Fax: +34948425619 E-mail: [email protected]

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Running title: Zein nanoparticles improve bioavailability and anti-inflammatory

26

effect of resveratrol

1 ACS Paragon Plus Environment


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Abstract

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Resveratrol offers pleiotropic health benefits including a reported ability to inhibit

29

lipopolysaccharide (LPS)-induced cytokine production. The aim of this work was to

30

prepare, characterize and evaluate a resveratrol nanoparticulate formulation based on

31

zein. For this purpose, the oral bioavailability of the encapsulated polyphenol as well as

32

its anti-inflammatory effects in a mouse model of endotoxic shock was studied.

33

The resveratrol-loaded nanoparticles displayed a mean size of 307±3 nm, with a

34

negative zeta potential (-51.1±1.55 mV), and a polyphenol loading of 80.2±3.26 µg/mg.

35

In vitro, the release of resveratrol from the nanoparticles was found to be pH

36

independent and adjusted well to the Peppas-Sahlin kinetic model, suggesting a

37

mechanism based on the combination of diffusion and erosion of the nanoparticle

38

matrix. Pharmacokinetic studies demonstrated that zein-based nanoparticles provided

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high and prolonged plasma levels of the polyphenol for at least 48 h. The oral

40

bioavailability of resveratrol when administered in these nanoparticles increased up to

41

50% (19.2-fold higher than for the control solution of the polyphenol). Furthermore,

42

nanoparticles administered daily for 7 days at 15 mg/kg, were able to diminish the

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endotoxic symptoms induced in mice by the intraperitoneal administration of LPS (i.e.,

44

hypothermia, piloerection and stillness). In addition, serum TNF-α levels were slightly

45

lower (approximately 15%) than those observed in the control.

46 47

Key words

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Resveratrol, zein, nanoparticles, bioavailability, anti-inflammatory.

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50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

Abbreviations Rsv: resveratrol SIRT1: sirtuin 1 LPS: lipopolysaccharide from Salmonella enterica serovar. Minnesota Rsv-NP-Z: resveratrol-loaded zein nanoparticles NP-Z: empty zein nanoparticles Rsv-sol: resveratrol solution in a PEG 400: water mixture Rsv-susp: suspension of resveratrol in purified water PCS: photon correlation spectroscopy SEM: Scanning electron microscopy EE: encapsulation efficiency iv: intravenous Cmax: maximal serum concentration Tmax: time in which Cmax is reached AUC: area under the concentration-time curve from time 0 to last time MRT: mean residence time Cl: clearance V: volume of distribution t1/2: half-life in the terminal phase Fr: relative bioavailability FRD: fraction of resveratrol dissolved FRA: fraction of resveratrol absorbed ip: intraperitoneal PGE2: prostaglandin E2 PDI: polydispersity index MRP: multidrug resistance proteins BCRP: breast cancer resistance protein



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Introduction

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Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a polyphenol molecule that was

79

identified in the dried roots of Polygonum cuspidatum, a plant used in traditional

80

Chinese and Japanese medicine

81

phytoalexin, as it is synthesized in spermatophytes in response to injury, UV irradiation

82

and fungal attack 2. This molecule is naturally found in a wide variety of plant species,

83

vegetables, fruits and food products, such as peanuts, grape skins, plums and red wine

84

2

85

effects

86

activities 6.

87

Over the last few years, the preventive effect of resveratrol against diabetes has been

88

demonstrated.

89

hyperinsulinemia, it reduces blood insulin 7. Similarly, resveratrol was reported to

90

reduce body weight and adiposity in obese recipients 8. These actions involve the

91

activation of sirtuin 1 (SIRT1), which inhibits inflammatory pathways in macrophages

92

and modulates insulin sensitivity 9. Furthermore, different studies have shown that

93

resveratrol is capable of inhibiting lipopolysaccharide (LPS)-induced cytokine

94

production

95

and gene expression of IL-1 and TNF-α, which are important endogenous pyrogens 11.

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Despite these potential health benefits, the use of resveratrol is limited due to its high

97

lipophilicity, short biological half-life, and chemical instability

98

resveratrol is orally administered, only very small amounts of the unchanged

99

polyphenol can be detected in the plasma

1

. Resveratrol (Rsv) has been classified as a

. Resveratrol offers pleiotropic health benefits, including antioxidant and anti-aging 3

and cardioprotective 4, anticancer 1, neuroprotective

Resveratrol

normalizes

hyperglycemia,

5

and

and anti-HIV/AIDS

in

animals

with

10

. This effect, exerted via modulation of NF-κB, decreases the production

12

1,2

. In addition, when

. This low bioavailability is due to

100

polyphenol biotransformation by UDP-glucuronosyltransferase and sulfotransferases,

101

which produce resveratrol-3’-glucuronide and the sulfate derivative, respectively 2. In

102

rats, the main metabolite of resveratrol is the glucuronide conjugate

103

humans, both the glucuronide and the sulfate derivatives have been described


13

, whereas in 14

.

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104

Renal excretion is the major route of elimination of the polyphenol and its derivatives

105

2,15

106

To address these drawbacks, different strategies have been pursued, including

107

encapsulation in different oral delivery systems, such as self-nanoemulsifying drug

108

delivery systems

109

others.

110

An alternative approach might be the use of zein nanoparticles. Zein is the major

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storage protein in maize and comprises approximately 45-50% of the total protein

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content in corn

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mixture of different peptides than can be divided into four main fractions: i) α-zein (75-

114

85% of total protein), with its two main MWs being 21-25 kDa and 10 kDa; ii) β-zein

115

(10-15%), with an MW of 17-18 kDa; iii) δ-zein, with a minor fraction of 10 kDa; and iv)

116

γ-zein (5-10%), with an MW of 27 kDa

117

high percentages of hydrophobic amino acids such as leucine (20%), proline (10%)

118

and alanine (10%)

119

and thus, the resulting devices (e.g., films, nanoparticles) display a hydrophobic

120

character with interesting properties that control the release of the loaded compound

121

20,21

122

biodegradable and can accommodate a great variety of compounds in a non-specific

123

way 22.

124

Therefore, the aim of this work was to prepare, characterize and evaluate a resveratrol

125

nanoparticulate formulation based on zein and to study its oral bioavailability and anti-

126

inflammatory effects in a mouse model of induced endotoxic shock.

.

16

, solid lipid nanoparticles

17

and polymeric nanoparticles

18

, among

19

. Because zein is a natural protein, it is actually a heterogeneous

19,20

. Zein is an amphiphilic protein, possessing

19,20

. Due to this amino acid composition, zein is insoluble in water,

. In addition, as for other nanocarriers of protein origin, these devices are

127 128

Material and Methods

129

Chemicals



130

Zein, resveratrol, lysine, mannitol, sodium ascorbate, poly(ethylene glycol) 400 (PEG

131

400) and Tween 20 were purchased from Sigma-Aldrich (Steinheim, Germany).

132

Resveratrol-3-O-D-glucuronide (Rsv-O-glu) was from @rtMolecule (Poitiers, France).

133

Ethanol, methanol, acetic acid, and acetonitrile HLPC grade were obtained from Merck

134

(Darmastadt, Germany). LPS from Salmonella enterica serovar. Minnesota was

135

purchased from Sigma®, (St. Louis, USA). Deionized reagent water (18.2 MO

136

resistivity) was prepared using a water purification system (Wasserlab, Spain). All

137

reagents and chemicals used were of analytical grade.

138

Preparation of resveratrol-loaded nanoparticles (Rsv-NP-Z)

139

Nanoparticles were prepared by a desolvation method followed by an ultrafiltration

140

purification step and subsequent drying in a spray-drier apparatus. Briefly, 600 mg zein

141

and 100 mg lysine were dissolved in 60 mL of an ethanol:water mixture (65% ethanol

142

by vol.). In parallel, 100 mg resveratrol was dissolved in 10 mL ethanol and 6 mL of this

143

solution was transferred to the zein solution. In addition, 6 mg sodium ascorbate was

144

added to minimize the oxidation of the polyphenol. The mixture was magnetically

145

stirred in the dark for 10 min at room temperature. Nanoparticles were obtained by the

146

continuous addition of 60 mL purified water. The suspension was purified and

147

concentrated by ultrafiltration using a membrane cartridge with a 50 kDa pore size

148

polysulfone (Medica SPA, Italy). Then, 15 mL purified water containing 1.2 g mannitol

149

was added to the resulting suspension of nanoparticles to prevent aggregation and

150

irreversible interactions among the nanoparticles during the drying process. Finally the

151

suspension was dried in a Büchi Mini Spray Dryer B-290 apparatus (Büchi

152

Labortechnik AG, Switzerland) under the following experimental conditions: (i) inlet

153

temperature: 90 °C, (ii) outlet temperature: 45-50 °C, (iii) air pressure: 4-6 bar, (iv)

154

pumping rate: 5 mL/min, (v) aspirator: 100% and (vi) air flow: 400-500 L/h.

155

Control formulations (NP-Z) were prepared as described above but in absence of

156

resveratrol.

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Preparation of resveratrol conventional formulations 6 ACS Paragon Plus Environment

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Two different formulations of resveratrol were also prepared. The first one, a solution of

159

the polyphenol in a mixture of PEG400 and water (1:1 by vol.) was preparing dissolving

160

37.5 mg resveratrol in 5 mL PEG400 under magnetic stirring. Then 5 mL purified water

161

was added and the final mixture was agitated in the dark for 10 min. This formulation

162

was named Rsv-sol.

163

The second one was an extemporary suspension of resveratrol in purified water (Rsv-

164

susp). Briefly, 37.5 mg resveratrol was dispersed in 10 mL purified water under

165

magnetic agitation for 10 min. The size of the resulting suspension was 21.4 ± 9.2 µm.

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The suspension was used after inspection for absence of aggregates.

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Characterization of nanoparticles

168

Size, zeta potential and morphology

169

The mean hydrodynamic diameter and the zeta potential of the nanoparticles were

170

determined by photon correlation spectroscopy (PCS) and electrophoretic laser

171

Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern

172

Instruments Ltd., Worcestershire, UK). The diameter of the nanoparticles was

173

determined after dispersion in ultrapure water (1:10) and was measured at 25 °C with a

174

scattering angle of 90 °C. The zeta potential was measured after dispersion of the dried

175

nanoparticles in 1 mM KCl solution.

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The morphology of the nanoparticles was studied by field-emission scanning electron

177

microscopy (SEM) using a Zeiss DSM940 digital scanning electron microscope

178

(Oberkochen, Germany) coupled with a digital imaging system (Point Electronic GmBh,

179

Germany). The yield of the process was calculated by gravimetry as described

180

previously 22.

181

Resveratrol analysis

182

The amount of resveratrol loaded into the nanoparticles was quantified by HPLC-UV

183

using a previously described analytical method

184

were carried out in an Agilent 1100 Series LC System coupled to a diode-array

185

detector set at 306 nm. The data were analyzed using ChemStation G2171 v. B.01.03

23

with minor modifications. Analyses



186

software (Agilent, USA). The chromatographic system was equipped with a reverse

187

C18 Alltima column (150 mm x 2.1 mm, particle size 5 µm; Altech, USA) and a Gemini

188

AJO-7596 C18 precolumn. The mobile phase, pumped at 0.25 mL/min, was a mixture

189

of water/methanol/acetic acid under gradient conditions. The column was heated to 40

190

°C and the injection volume was 10 µL. Under these conditions, the retention time for

191

resveratrol was 22.8±0.5 min. Calibration curves in 75% ethanol were designed over

192

the range of 1-100 µg/mL (R2≥0.999). Under these experimental conditions, the limit of

193

quantitation was calculated to be 200 ng/mL.

194

For analysis, 10 mg nanoparticles was dispersed in 1 mL water and centrifuged at

195

30,500 g for 20 min. The amount of encapsulated resveratrol was calculated by

196

dissolution of the pellets in 1 mL of 75% ethanol. Each sample was assayed in

197

triplicate, and the results are expressed as the amount of resveratrol (µg) per mg of

198

nanoparticles.

199

The encapsulation efficiency (E.E) was calculated as follows: . . % =

200

× 100 [Eq. 1]

201

where Rsv-t is the total amount of resveratrol in the formulations and, Rsv-p, the

202

amount of resveratrol quantified in the pellet.

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In vitro release study

204

Release experiments were conducted under sink conditions at 37°C using simulated

205

gastric fluid (SGF; pH 1.2) and intestinal fluid (SIF; pH 6.8)

206

20 as a surfactant to increase the aqueous solubility of resveratrol. The studies were

207

performed under agitation in a Slide-A-Lyzer® Dialysis Cassete with a 10,000 MWCO

208

(Thermo Scientific, Rockford, IL, USA). For this purpose, the cassette was filled with 3

209

mg resveratrol nanoparticles previously dispersed in 5 mL water and then introduced

210

into a vessel containing 500 mL SGF (pH 1.2; 37ºC) under magnetic stirring. After 2 h

211

in the SGF, the cassette was introduced into another vessel containing 500 mL

212

thermostatized SIF (pH 6.8; 37ºC, under agitation). At different time points, samples


22

, containing 0.5% Tween

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213

were collected and filtered through filters with a 0.45 µm pore size (Thermo Scientific,

214

Rockford, IL, USA) before quantification by HPLC. Calibration curves for resveratrol in

215

SGF and SIF (0.05-6 µg/mL; R2 ≥ 0.999 in both cases) were generated.

216

To ascertain the resveratrol release mechanism the obtained data were fitted to the

217

Korsmeyer-Peppas and Peppas-Sahlin models. The Korsmeyer–Peppas model is a

218

simple semi-empirical approach which exponentially relates drug release with the

219

elapsed time as expressed in the following equation 24:

220

Mt = K KP ·tn [Eq. 2] M∞

221

where Mt /M∞ is the drug release fraction at time t, KKP is a constant incorporating the

222

structural and geometric characteristics of the matrix and n is the release exponent

223

indicative of the drug release mechanism

224

(Fickian) diffusion mechanism and values between 0.5 and 0.89 indicate anomalous

225

(non-Fickian) diffusion. Values of n between 0.89 and 1 indicate Case II transport,

226

erosion of the matrix.

227

The contributions of Fickian and non-Fickian release were also evaluated by using the

228

Peppas–Sahlin model equation 26:

25

. Values close to 0.5 indicate a Case I

229

Mt = K D ·t 1/2 + K E ·t [Eq. 3] M∞

230

where the first term of the right-hand side is the Fickian contribution (KD is the

231

diffusional constant) and the second term is the Case II erosional contribution (KE is the

232

erosional constant). KD and KE values were used to calculate the contribution

233

percentage of diffusion (D) and erosion (E) as follows 26:

234

235

=

.

[Eq 4]

= . [Eq 5]



236

Only one portion of the release profile (Mt/M∞ ≤ 0.6) was used to fit the experimental

237

data to the previous equation.

238

In vivo pharmacokinetic studies in Wistar rats

239

Pharmacokinetic studies

240

Pharmacokinetic studies were performed in male Wistar rats (200-250 g) obtained from

241

Harlan (Barcelona, Spain). The studies were approved by the Ethical Committee for

242

Animal Experimentation of the University of Navarra (protocol number 028-11) in

243

accordance with European legislation on animal experiments.

244

Prior to the oral administration of the formulations, animals were fasted overnight to

245

avoid interference with absorption, but were allowed free access to water. For the

246

pharmacokinetic studies, rats were randomly divided into 4 groups of 6 animals each.

247

The three experimental groups were: (i) aqueous resveratrol suspension (Rsv-susp),

248

(ii) resveratrol in a PEG400:water mixture (Rsv-sol) and (iii) resveratrol-loaded zein

249

nanoparticles (Rsv-NP-Z). As a control, a group of animals was treated intravenously

250

with the PEG400:water (1:1 by vol.) solution of resveratrol. Each animal received the

251

equivalent amount of resveratrol at a dose of 15 mg/kg body weight either by oral

252

gavage or intravenously via the tail vein.

253

Blood samples were collected at set times after administration (0, 10 min, 30 min, 1 h,

254

2 h, 4 h, 6 h, 8 h, 24 h and 48 h) in specific plasma tubes (Microvette® 500K3E,

255

SARSTEDT, Germany). Samples were immediately centrifuged at 9,400 g for 10 min

256

and plasma aliquots were kept frozen at -80 °C until HPLC analysis of both resveratrol

257

and resveratrol-3-O-D-glucuronide.

258

Determination of resveratrol and resveratrol-3-O-D-glucuronide plasma

259

concentration by HPLC

260

The amount of resveratrol was determined by HPLC-UV using a previously reported

261

analytical method

262

Agilent 1100 Series LC and a diode-array detector set at 306 nm. The data were

263

analyzed in a ChemStation G2171 program (B.01.03). The chromatographic system

27

with minor modifications. Analyses were carried out using an


Page 10 of 45

Page 11 of 45


264

was equipped with a reverse-phase Kromasil C18 column (250 mm x 2.1 mm, particle

265

size 5 µm) and a Gemini AJO-7596 C18 precolumn. The mobile phase, pumped at 0.5

266

mL/min, was a mixture of water, methanol and acetic acid (50:45:5 by vol.) under

267

isocratic conditions. The column was thermostatized at 30°C and the injection volume

268

was 30 µL. Under these conditions, the retention times for resveratrol-3-O-D-

269

glucuronide and resveratrol were 6.2 ± 0.5 min and 12.6 ± 0.5 min, respectively.

270

For analysis, a 100 µL aliquot of plasma was mixed with 50 µL 0.1 N HCl and 500 µL

271

acetonitrile (for protein precipitation) followed by vigorous shaking. The samples were

272

then centrifuged at 4000 rpm for 10 min and the obtained supernatants were

273

evaporated under vacuum in a Speed Vac® system (Holbrook, NY) at 25°C for 30 min.

274

Finally, 100 µL of a mixture of acetonitrile and water (1:1 by vol.) was added and

275

vigorously stirred by vortexing for 10 min. The samples were then filtered through a

276

0.45 µm filter (Thermo Scientific, Rockford, IL, USA) prior to injection.

277

For quantification, calibration curves were prepared over the ranges of 2-70 µg/mL for

278

the metabolite and 50-3,000 ng/mL for resveratrol (R2≥0.99). All of the calibration

279

standards were obtained by adding either resveratrol or resveratrol-3-O-D-glucuronide

280

in acetonitrile (500 µL) to 100 µL plasma from non-treated animals. The polyphenol or

281

its metabolite was then extracted using the same protocol as described above.

282

Under these experimental conditions, the limits of quantification were calculated to be

283

70 ng/mL for resveratrol and 4 µg/mL for the metabolite. Linearity, accuracy and

284

precision values on the same day (intra-day assay) at low, medium and high

285

concentrations of both resveratrol and the metabolite were always within the

286

acceptable limits (relative error and coefficient of variation of less than 15%).

287

Pharmacokinetic data analysis

288

The

289

pharmacokinetic analysis was performed using a non-compartmental model within

290

WinNonlin 5.2 software (Pharsight Corporation, USA). The following parameters were

291

estimated: the maximal serum concentration (Cmax), the time in which Cmax is reached

resveratrol

plasma

concentration

was

plotted


against

time,

and

the


Page 12 of 45

292

(Tmax), the area under the concentration-time curve from time 0 to the last sampling

293

time-point (48 h) (AUC), the mean residence time (MRT), the clearance (Cl), the

294

volume of distribution (V) and the half-life in the terminal phase (t1/2). Furthermore, the

295

relative bioavailability (Fr %) of resveratrol was estimated by the following equation: ! % =

296

"#$ %&'( × "#$ )

100

(Eq. 6)

297

where AUCi.v. and AUCoral are the areas under the curve for the iv and oral

298

administrations, respectively.

299

In vitro/In vivo correlation (INVIC)

300

The eventual correlation between the in vitro and the in vivo results was determined by

301

point-to-point plotting of the amount of resveratrol released from the nanoparticles vs

302

the fraction of resveratrol absorbed (FRA), calculated from the mean plasma

303

concentration-time inputs using the Wagner-Nelson equation 28: *+ =

304

$ ,×"#$- , ×"#$ -.

(Eq. 7)

305

where Ct is the plasma concentration of resveratrol at a time t, k is the elimination rate

306

constant of the polyphenol, AUC0-t is the area under the resveratrol concentration vs

307

time curve from 0 to time t, and AUC0-∞ is the area under the curve from 0 to infinity.

308

Linear regression analysis was applied to the in vitro/in vivo correlation plot, and the

309

coefficient of determination (R2) was calculated.

310

Anti-inflammatory efficacy study

311

Animal model

312

Four-week-old (20-22 g) C57BL/6J female mice were purchased from Harlan

313

(Barcelona, Spain) and housed in standard animal facilities (6 animals per cage with

314

free access to food and drinking water). Housing conditions were maintained with

315

controlled temperature and humidity and with 12 h on/off light cycles. Animals were

316

allowed to acclimate for one week before the experiment.

317

In vivo anti-inflammatory studies were evaluated in an endotoxic shock model set up by

318

intraperitoneal (ip) administration of LPS at a dose of 40 µg per mouse 12 ACS Paragon Plus Environment

29

. Before

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319

administration, the LPS was dissolved in PBS and vortexed for 30 min to complete

320

homogenization.

321

On day 1, mice were randomly distributed into four groups. The first group of animals

322

received an oral dose of 15 mg/kg resveratrol daily as an oral solution (Rsv-sol) for 7

323

days. The second group of animals received the same posology of polyphenol (15

324

mg/kg resveratrol daily for 7 days) but within the zein nanoparticle formulation (Rsv-

325

NP-Z). As controls, a group of animals received LPS treatment (positive-control group),

326

and another group received neither LPS nor resveratrol (negative-control group).

327

Twenty-four hours after the last dose of resveratrol (day 8), the animals were

328

challenged with 40 µg LPS by the ip route. Throughout the study, the rectal

329

temperature of the mice was measured until 24 h after challenge. Clinical anaphylactic

330

reactions were scored 6 h post-challenge by two independent observers. Piloerection

331

was scored as follows: (-) normal mouse, (+) weak reaction and/or scratching of the

332

nose and head, (++) moderate piloerection, and (+++) strong piloerection. Depending

333

on the activity of the animals, their mobility was classified as very low (no reaction after

334

pushing), low (arched back and low movements) or normal.

335

In addition, 90 min after challenge, blood samples were collected into EDTA-K vials

336

(Microvette® 500K3E, SARSTEDT, Germany) from the retro-orbital cavity, centrifuged

337

at 8,000 g for 10 min for serum collection and stored at -20 °C until use.

338

Measurement of plasma TNF-α

339

The concentration of circulating TNF-α in the serum was determined using an enzyme-

340

linked immunosorbent assay kit (Mouse TNF-alpha Quantikine® ELISA Kit, MTA00B,

341

R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

342

Statistical analysis

343

The data are expressed as the mean ± standard deviation (SD) of at least three

344

experiments. The non-parametric Kruskal-Wallis test followed by the Mann-Whitney U-

345

test with Bonferroni correction was used to investigate significant differences. In all



346

cases, p0.96), and the exponent “n” value was 0.75±0.06. All of these results suggest

380

that the release of resveratrol from the nanoparticles was due to a combination of

381

Fickian diffusion and erosion of the nanoparticle matrix. Under these circumstances,

382

the Peppas-Sahlin model was applied, and the erosion (KE) and diffusion (KD)

383

constants were calculated (KD = 0.08±0.02 h-1/2; KE = 0.04±0.01 h-1). Figure 2B

384

displays the contributions of both the diffusion and the erosion mechanisms to the

385

release of resveratrol from zein nanoparticles. The time at which the two mechanisms

386

(diffusion and erosion) similarly contributed to the release of resveratrol was calculated

387

to be 3.5 h.

388

In vivo pharmacokinetics

389

Figure 3A shows the plasma concentration-time profile of a resveratrol solution in

390

PEG400:water (1:1 by vol.) after intravenous (iv) administration of a single dose of 15

391

mg/kg to rats. The data were adjusted to a non-compartmental model. The resveratrol

392

plasma concentration decreased rapidly, in a biphasic way, during the first 8 h post-

393

administration. The peak plasma concentration (Cmax) of resveratrol was 15.2 µg/mL,

394

whereas the AUC and half-life (t1/2) were calculated to be 11.4 µg h/mL and 2.04 h,

395

respectively. The clearance and volume of distribution of resveratrol were 199 mL/h

396

and 569 mL, respectively (Table 2).

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Figure 3B shows the plasma concentration levels of resveratrol when administered

398

orally to rats as a single dose of 15 mg/kg. Interestingly, when resveratrol was

399

formulated as a suspension, no quantifiable levels of the polyphenol were detected in

400

the plasma. In contrast, when resveratrol was administered as a solution (Rsv-sol), the

401

polyphenol plasma levels displayed a maximum concentration (Cmax), or 0.20 µg/mL, 15 ACS Paragon Plus Environment


402

30 min after administration. The plasma levels of resveratrol then decreased rapidly,

403

and quantifiable levels were only detected during the first 4 h post-administration.

404

For resveratrol loaded in zein nanoparticles (Rsv-NP-Z), the amount of the polyphenol

405

in the plasma increased during the first 4 h after administration until reaching a

406

maximum. The resveratrol plasma levels then decreased slowly for the following 20 h.

407

Forty-eight hours post-administration, the amount of resveratrol in the plasma was very

408

close to the quantitation limit of the analytical technique.

409

Table 2 summarizes the main pharmacokinetic parameters estimated via a non-

410

compartmental analysis of the experimental data obtained after administration of the

411

different formulations to rats. The resveratrol AUC values for the zein nanoparticle

412

formulation were significantly higher (p

Zein-Based Nanoparticles Improve the Oral ... - ACS Publications

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