Effects of different stabilizers on colloidal properties and encapsulation efficiency of vitamin D3 loaded nano-niosomes

Journal Pre-proof Effects of different stabilizers on colloidal properties and encapsulation efficiency of vitamin D3 loaded nano-niosomes Vahideh Talebi, Babak Ghanbarzadeh, Hamed Hamishehkar, Akram Pezeshki, Alireza Ostadrahimi PII:

S1773-2247(19)30426-5

DOI:

https://doi.org/10.1016/j.jddst.2019.101284

Reference:

JDDST 101284

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 26 March 2019 Revised Date:

31 August 2019

Accepted Date: 11 September 2019

Please cite this article as: V. Talebi, B. Ghanbarzadeh, H. Hamishehkar, A. Pezeshki, A. Ostadrahimi, Effects of different stabilizers on colloidal properties and encapsulation efficiency of vitamin D3 loaded nano-niosomes, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/ j.jddst.2019.101284. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

2

Effects of different stabilizers on colloidal properties and encapsulation efficiency of vitamin D3 loaded nano-niosomes

3 4 5

Vahideh Talebi1, Babak Ghanbarzadeh*1,2, Hamed Hamishehkar3,

6

Akram Pezeshki1, Alireza Ostadrahimi 4

1

7 8

1-

9 10

Tabriz, P.O. Box 51666-16471, Tabriz, Iran 2-

11 12 13 14 15

Department of Food Science and Technology, Faculty of Agriculture, University of

Department of Food Engineering, Faculty of Engineering, Near East University P. O. Box 99138, Nicosia, Cyprus, Mersin 10, Turkey

3-

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 4- Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

* Corresponding author: [email protected], [email protected] Tel: 00989123039836, Fax: 00984133356005

16 17 18 19 20 21 22 23 24 1

25

Abstract

26

The vitamin D3 loaded nano-niosomes were prepared by thin layer hydration and sonication

27

methods. Influence of stabilizers (vitamin E acetate, polyethylene glycol and, cholesterol) on

28

the different physicochemical properties of resulted niosomes including particle size

29

distribution, zeta potential, encapsulation efficiency (EE%), antioxidant activities, turbidity,

30

sedimentation, and pH were evaluated. The images of scanning electron microscopy (SEM)

31

confirmed the results of the particle size data which were obtained using dynamic light

32

scattering (DLS). Fourier transform infrared spectroscopy (FT-IR) and differential scanning

33

calorimetry (DSC) analysis demonstrated the incorporation of vitamin D3 into niosomes.

34

Incorporation of vitamin E-acetate and cholesterol to the niosome formulation (N2) caused a

35

significant increase (P<0.5) in niosome size. Addition of vitamin E-acetate caused a

36

significant effect on increasing of encapsulation efficiency.The highest efficiency was

37

observed in the samples containing cholesterol and polyethylene glycol. The niosome

38

formulation containing 0.02 mmole vitamin E acetate with the size of 99 nm was selected as

39

the optimal formulation. This formulation had the highest antioxidant capacity (56%), the

40

lowest pH changes and high encapsulation efficiency (92.9%).

41

Keywords: nano-niosome, vitamin E-acetate, vitamin D3, encapsulation, stabilizer

42

2

43

1. Introduction

44

Nutraceuticals are food ingredients that, in addition to having basic nutritional properties,

45

have valuable biological activities such as enhancing the immune system and inhibitory

46

effects against important disorders such as cancer, cardiovascular diseases, diabetes, macular

47

degeneration, and other age-related diseases. Vitamins are among the most important groups

48

of nutraceutical compounds and are naturally present in foods. Low doses of vitamins are

49

essential for growth, maintaining the normal state of cells and body function. By growing

50

tendency of people to low-fat or non-fat foods due to the changing the lifestyle, and also the

51

destruction of nutraceuticals during the processing and food preservation, concerns about

52

diseases caused by essential hydrophobic nutrients deficiencies have been increased.

53

Therefore, the enrichment of some foods and beverages with these essential nutrients can be

54

an effective way to solve this problem.

55

Vitamin D is a lipid-soluble vitamin with two main chemical forms, vitamin (ergocalciferol)

56

D2 and vitamin D3 (cholecalciferol) [1]. Vitamin D3 (cholecalciferol) is synthesized in the

57

human epidermis after light exposure [2]. Recent studies showed a high prevalence of

58

vitamin

59

diabetes [4], metabolic bone diseases, osteoporosis, hypertension and others [5]. Due to poor

60

food sources containing this vitamin, food fortification with vitamin

61

[6].

62

The role of vitamin E as an important factor and its antioxidant and non-antioxidant

63

biological activities in the normal metabolism of all cells and reduction of cardiovascular

64

disease, diabetes, and cancer has been proven. Because of higher chemical stability of

65

vitamin E acetate (the esterified form of α-tocopherol) compared to the non-esterified form

66

(α-tocopherol), this form is often used in foods and beverages [7].

67

Encapsulation of nutraceutical compounds by lipid or polymer-based carriers is an effective

deficiency, especially in women [3] which causes cardiovascular diseases and

3

is the priority deal

68

way to protect their properties and also increase their solubility and bioavailability and

69

decrease their possible off flavor [2]. Niosome, non-ionic surfactants vesicle (NSV) is one of

70

the nanocarriers that is formed from non-ionic surfactants. It is structurally similar to

71

liposomes and consists of an aqueous core enclosed by a bilayer membrane of non-ionic

72

surfactants [8]. In their vesicular structure, the hydrophobic groups are placed in the interior

73

of the membrane while the hydrophilic ones are exposed to the aqueous core. The niosome

74

and vesicular liposome structures do not form spontaneously, and they need some energy

75

input (e.g., hand-shaking, agitation, ultrasound, heating) and this has led to the development

76

of different preparation methods, during the last two decades. Niosomes, as substitutes of

77

liposomes, [9] are chemically more stable and have lower costs than liposomes. In addition, a

78

more wide range of surfactants is available for the formulation of these vesicular carriers.

79

These vesicles can encapsulate a large amount of different functional components (lipophilic,

80

hydrophilic, and amphiphilic) in their small volumes [10]. Span and Tween, non-ionic

81

surfactants, have some interesting properties such as biodegradability, biocompatibility, and

82

low toxicity, so they are used in some foods and pharmaceuticals [11].

83

In recent years, several types of research have worked on the production of nutriceutical

84

loaded noisomes such as capsaicin [11], resveratrol [12], gallic acid, ascorbic acid, curcumin

85

and quercetin [13], resveratrol/curcumin and alpha-tocopherol/curcumin [14], vitamin D3 and

86

ferrous sulfate [15], α-Tocopherol [16]. However, the comparison of effects of different

87

stabilizers on the noisome systems has not extensively studied.

88

The main objective of the present work was to preparing vitamin D3 loaded nano-niosome by

89

thin layer hydration and sonication method and then study of effects of some food grade

90

stabilizers such as vitamin E acetate, polyethylene glycol and cholesterol on physicochemical

91

properties of developed nano-niosomes.

92 4

93

Materials and Method

94

1.1.

95

Vitamin D was provided by DSM. (Swiss). Vitamin E acetate was obtained from Zahravi.

96

(Iran) and Cholesterol was purchased from Sigma (Germany). Polyethylene glycol 400 was

97

purchased from Sacharlau (Spain). Other chemicals including Span 60, Tween 80, 2,2-

98

diphenyl-1-picrylhydrazyl (DPPH) powder and 2-propanol were analytical grade and were

99

provided from Merck (Germany).

Materials

100

1.2.

Preparation of nano-niosomes

101

Nano-niosomes were prepared according to thin layer hydration- sonication method described

102

by Pando, et al. [17] with some modification. Span 60, a non-ionic surfactant was used as the

103

main component in the formulations. Span 60 and Tween 80 were dissolved in isopropyl

104

alcohol (5ml) at 50 ºC for 20 min. Vitamin

105

isopropyl alcohol (5 ml). Two solutions were mixed and thin layer formation was performed

106

in the rotary evaporator LABOROTA 4002- digital model (Heidolph, Germany) at 60 ºC, 90

107

rpm, 100 mmHg for 60 min. Hydration was done by 10 ml distilled water in rotary at 65 ºC

108

for 30 min. Sonication was performed for 6 min by 1 min intervals at 120 Hz (UP200H

109

model, Hielscher, Germany). Finally, the prepared nano niosomes were filtered through 0.2

110

µm Whatman filter.

111

Due to the increased encapsulation efficiency in the range of HLB = 8.6 [18], two types of

112

surfactants were used to obtain the desired HLB. The percentages of the used amounts of

113

surfactants were obtained from Equation 1 [19]: HLB = =

114

(

and Vitamin E- acetate were dissolved in

)+( +

)

× 100

(1)

=

The formulation used for the preparation of nano-niosomes is shown in 5

115

Table 1.

116 117

1.3.

Mean size, polydispersity index, and zeta potential of niosomes

118

The average diameter, polydispersity index (PDI) and zeta potential of samples was measured

119

using a dynamic laser scattering (DLS) device, the Nanotrac Wave Microtrac (Germany)

120

model. For this purpose, each sample was diluted 20 times with distilled water. All specimens

121

were measured in three replicates. This system determined the particle size by measuring

122

their propulsion and dynamic light diffraction (DLS), and then the size is determined by

123

applying proven theories.

124

The average particle diameter was reported in terms of the volumetric average. The mean

125

particle diameter or DeBroukere mean was calculated from Equation 2 [20]:

!" !

∑ 4,3 = ∑

(2)

126

1.4.

127

Morphology of nano-niosomes was investigated by scanning electron microscopy (SEM).

128

The sample was diluted 20 times with water and observed under KYKY-EM3200 at an

129

accelerating voltage of 26 kV [21].

130

Morphological analysis (scanning electron microscopy)

1.5. Differential scanning calorimetry (DSC)

131

Differential scanning calorimetry of blank sample and nanoniosome containing vitamin D3

132

(N1) and vitamin D3 was performed on a DSC device (PerkinElmer, Jade DSC model, US

133

manufacturing). The device was calibrated with indium, and an empty aluminum container

134

was used as a reference. Samples with a mass of approximately 4 milligrams were scanned at

135

20 °C/min at a temperature range of 30 to 300 °C, and the melting point of the compounds

136

was determined [22].

137

1.6.

Fourier transform infrared spectroscopy (FTIR) 6

138

Infrared spectroscopy was performed using an FTIR device TENSOR 27, in the range of 400-

139

4000 cm-1. The samples (Span 60, Tween 80, Vitamin D3, Vitamin E-acetate, blank nano-

140

niosome and nano-niosome containing vitamin D3) were separately mixed with pure

141

potassium bromide (KBr) in a ratio of 1 to 100 and this mixture was used in

142

spectrophotometry [23].

143

1.7.

144

The antioxidant capacity (AOC) was determined using the 2,2-diphenyl-1-picrylhydrazyl

145

(DPPH) method. DPPH is a free radical widely used to determine the ability of antioxidant

146

molecules to react as free radical scavengers [11]. First, nano-niosomes were broken down

147

using chloroform. For this purpose, 2 ml of nano-niosome solution was mixed with 2 ml of

148

chloroform and stirred for 20 minutes. Then the white upper liquid (water) was discarded. A

149

chloroform solution containing DPPH (0.01mM) was prepared and mixed with samples in the

150

ratio of 1:1, then the adsorption changes were read after 2 hours of incubation in darkness at

151

517 nm with UV-vis Ultrospec 2000 (UK) spectrophotometer. The antioxidant capacity with

152

the DPPH inhibitory content was determined according to Equation 3 [24]:

Antioxidant capacity

Inhibition% =

+, − +. × 100 +,

(3)

153

where A0 is absorbance of control sample at 517 nm and A1 is sample absorbance at 517 nm.

154

1.8.

155

To obtain a qualitative amount of oxidation and hydrolysis of niosomes over a month, the pH

156

of the nano-niosome solutions was measured. The pH was measured by direct injection of a

157

pH meter (Metrohm, Switzerland) into a new mass solution, and setting the sample

158

temperature.

159

1.9.

160

To obtain a qualitative dimension of variation in the size and release rate of nano-niosomes

pH

Turbidity

7

161

during a month, the turbidity was measured. First, 1 ml of the sample was diluted with 2 ml

162

of distilled water and stored at room temperature for 30 minutes. Then, the turbidity of the

163

sample was measured in a spectrophotometer of the Ultrospec 2000 produced by the UK in

164

quartz tubes at a wavelength of 600 nm [25].

165 166

1.10.

Sedimentation

167

In order to determine the stability of nano-niosome solutions, their sediment was measured. 5

168

ml of each formulation was stored for 60 days and 30 days at 2 °C and room temperature and

169

their sedimentation height was measured. Sediment volume was obtained using Equation 4

170

[26]: S% =

V1 2V × 100 ,

(4)

171

where Vu is sediment volume at a specified time and V0 is Total volum of nano-niosome

172

solution.

173

1.11.

174

To determine encapsulation efficiency (EE%), 3 ml of nano-niosome solution was mixed

175

with 3 ml of isopropyl 50% solution (to dissolve only soluble vitamins). Amicon filter was

176

used by centrifugation at 4000 rpm for 2 minutes [27] to isolate free and encapsulated

177

vitamins. From each of the upper and lower sections of the filter, 1 ml samples were

178

removed, and 500 µl of chloroform was added to each. The test tube containing nano-

179

niosome was shaken for 20 minutes to degrade the nano-niosome and free the vitamin.

180

Sample containing free vitamin was also shaken for several minutes. The chloroform phase of

181

the solutions was separated, and the samples were dried under nitrogen gas. EE was

182

calculated using Equation 5:

Loading parameters

8

Encapsulation efficiency% =

Capsulated vitamin (mg) × 100 Free + encapsulated vitamin (mg)

(5)

183

The loading capacity (LC) calculated as the ratio of the active ingredient to the whole lipid

184

(stabilizing agent + surfactant + vitamin E-acetate).

185

1.11.1. High-Performance liquid chromatography (HPLC)

186

HPLC (Waters 1525 Binary, USA) was used to determine the free and encapsulated vitamin

187

in nano-niosomes. The optimal condition of the column and the HPLC device to determine

188

the encapsulation efficiency was as follows: C18 silica gel column (5 µm, 250mm × 4.6mm),

189

room temperature, flow rate 1 ml/min, wavelength 270 nm, injection valium 20 µl, and

190

mobile phase chloroform + 0.5% amyl alcohol (95 ml), ethyl acetate (5 ml).

191

In order to plot the calibration curve, 100 mg of a standard solution of 1 million units was

192

transferred to a 50-caliber balloon and chloroform was added to reach the specified valium

193

(2000 IU / ml); 20 µl of the standard solution was injected to the device, and the sub-curved

194

surface was calculated.

195

The concentration of vitamin D3 was determined using Equation 6: CD =

AD × CF AF

(6)

196

where At is the area below the vitamin D3 curve in the sample solution, As is the area under

197

the curve of vitamin D3 in the standard solution, Cs is standard concentration and Ct is sample

198

concentration

199

1.12.

200

Physical and chemical tests were performed on a completely randomized design with three

201

replications. Data were analyzed by one-way ANOVA and Duncan's comparison test at 5%

202

level. The statistical software of SPSS 23 was used for data analysis.

203

2.

Results and discussion

204

2.1.

Determination of niosome mean size, PDI and zeta potential

Statistical analyses

9

205

The particle size in a colloidal carrier system plays an important role in determining its

206

stability, bioavailability, solubility, turbidity and rheological properties. Decreasing particle

207

size can lead to lower gravitational separation and turbidity, and higher solubility and

208

bioavailability. The results of particle size, PDI and the zeta potential measurements of blank

209

niosome and vitamin D3 loaded nano-niosomes with or without stabilizing agents (vitamin E

210

acetate, cholesterol and polyethylene glycol 400) are presented in Figure 1.

211

In the first day, the particle sizes of different niosome samples were 74-197 nm. The particle

212

size of vitamin D3-loaded noisome (N2) did not show a significant difference with the control

213

sample (without vitamin, N0) (74 vs 76 nm). Incorporation of vitamin E-acetate to the

214

niosome formulation (N2) caused a significant increase (P<0.5) in niosome size (93 nm) and

215

increasing of vitamin E concentration enhanced particle size nonsignificantly (93 to 99 nm).

216

According to Figure 1 (a), the particle sizes of niosomes containing cholesterol (G" ) and

217

polyethylene glycol (GH ) were larger compared to other formulations and G" sample had the

218

largest size (197±5 nm).

219

Presence of cholesterol increased particle size probably due to the spacing in surfactant

220

molecule in the hydrophobic layer. Similar results were reported for α-tocopherol-loaded

221

niosomes [16] and ascorbic acid and α-tocopherol niosomes [28].

222

The vesicle size depends on the concentration and nature of surfactant (HLB value),

223

stabilizer, bioactive component (hydrophobicity) and method of preparation.

224

After 40 days, the change in the size of G" and GH was significant and their size increased

225

(P<0.05); however, the size of G. and GI samples were reduced which could be due to the

226

separation of the particles interconnected during storage time. Beugin, et al. [29] in a study of

227

nano-niosomes containing cholesterol and polyethylene glycol observed the reduction of

228

particle size after one week. The size of their nano-niosomes was between 63-141 nm

229

depending on the formulation. 10

230

The polydispersity index of samples is shown in Figure 1 (b). The PDI value of samples was

231

in the range of 0.32-0.51, implying a relatively broad size distribution. N0 showed the

232

minimum PDI and N2 had the widest size distributions. At the first day, the PDI of nano-

233

niosome was significantly increased by incorporation of vitamin D3 and vitamin E. However;

234

Adding cholesterol and polyethylene glycol (G and G" ) caused a significant decrease on

235

polydispersity. During the 40 days of storage, the increase of PDI could be observed in the

236

samples containing stabilizer agents except for N2.

237

One of the factors affecting the physical stability and surface features of vesicles is the

238

surface charge which is usually evaluated by zeta potential [30]. The ionic atmosphere around

239

the charged particle causes an electrostatic repulsion between the particles, and when two

240

particles approach each other with the same load; it creates a repulsive force that prevents the

241

flocculation of particles. Therefore, by measuring zeta potential, the physical stability of the

242

colloidal systems could be estimated. On the other hand, the surface charge of vesicles is

243

important in adhering to active encapsulated compounds and cellular membrane.

244

According to Figure 1 (c), the zeta potential of samples except for the control sample (N0)

245

and the sample containing vitamin D3 (N1) were negative. This positive or negative zeta

246

potential is probably due to the different nature of the stabilizer agents. The highest zeta

247

potential (+29 mV) was related to the sample containing vitamin D3 (N1) and the lowest zeta

248

potential (-15mV) was observed in the sample contains vitamin D3 and vitamin E-acetate

249

(N3). All samples showed a decrease in zeta potential for 40 days, which was high in sample

250

GH and less in control samples (P<0.05). Among the various formulations, after 40 days, the

251

zeta potential of the sample containing vitamin D3 (N1) was higher than the rest (+19 mV).

252

Ionic strength and pH are two factors that can have the most profound impact on the zeta

253

potential values [31]. The zeta potential is decreased by leakage of active substances from the

254

inside of the niosomes and decreasing of medium’pH. 11

255

In general, in all of the formulations, the zeta potential was not high which is similar to the

256

previous reports [16, 32]. Zeta potential of particles in the colloidal system depends on type

257

and concentration of surfactants, active material, and stabilizer. Also, environmental and

258

process condition such as temperature, ionic strength of solvent and mechanical treatment can

259

be effective. Low zeta potential indicating the low electrostatic force of the vesicles which

260

can be attributed to the use of non-ionic and neutral surfactants [33]. Also, the observed zeta

261

potential can be due to the vitamin D3, stabilizers, pH of the hydration water, application of

262

heat and subsequent sonication. In general, the important parameter in long term stability of

263

the nano-niosomes could be related to repulsive steric between the vesicles. Also, vitamin D3

264

could induce relatively good positive charge density and selected stabilizers decreased this

265

stabilizing effect.

266

2.2.

267

Scanning electron microscopy (SEM) is used to provide more accurate information with

268

respect to size, size distribution, particle aggregation and shape of nano-niosomes.

269

Figure 1 (d) shows SEM images of nano-niosomes containing vitamin D3 (N1) which was

270

determined by size distribution analysis as the best niosome. These images showed spherical

271

and elliptical nanometer-sized particles in the vesicular carriers, which was almost

272

ascertained by the results of the particle size measurements. In some parts, vesicles had been

273

stuck together and reduction in the size of particles over time can be attributed to the

274

separation of these masses.

275

2.3.

276

An important application of DSC in delivery system researches is the study of the changes in

277

thermal behavior and phase transition (melting, crystallization and glass transition) of carriers

278

by an encapsulant, stabilizer, and other ingredients. These changes, by incorporation of

279

bioactive compounds, may relate to interactions between surfactants and encapsulant that

Morphological analysis by SEM

Differential scanning calorimetry (DSC)

12

280

affect bioavailability. Thermodynamic parameters, such as enthalpy and melting temperature,

281

are very important because they are not only indicating the complexation and kinetics of the

282

active material release but also potentially show the stability of the nano-niosomes during

283

storage.

284

DSC analysis was performed to evaluate the entry of vitamin D3 to the nano-niosome

285

structure and its encapsulation. Figure 2 (a) shows the DSC curve of vitamin D3, the control

286

sample (N0), and nano-niosome containing vitamin D3 (N1). Also, the melting temperature

287

and enthalpy of them are presented in

288

Table 2.

289

Analyses of all three samples were carried out at a temperature of 30-300 °C. As seen in

290

Figure 2 (a), there was only one melting peak in the vitamin D3 loaded nano-niosome (N1)

291

graph, and there was no peak of vitamin melting peak; This is in accordance with a previous

292

study [34]. The absence of vitamin D3 melting peak and observing only one melting peak in

293

the curve of nano-niosomes containing vitamin D3 can indicate that the two compounds are

294

co-crystallized and their compatibility is good.

295

Also, by incorporation of vitamin D3 enthalpy and the melting temperature of nano-niosome

296

increased slightly (5 °C) compared to the control sample. According to this evidence, it can

297

be said that vitamin D3 can create hydrogen (by the free hydroxyl group in the structure of

298

vitamin D3) and hydrophobic interactions with surfactant compounds and which in turn

299

changes the thermal properties of niosomes and gives higher stability. Also, the increase in

300

melting temperature of nano-niosome containing vitamin D3 indicates an increase in

301

crystallite size and crystalline order than the control sample (blank).

302

The colorimetric studies of Sezgin-Bayindir, et al. [26], on the niosomes containing

303

Candesartan, showed that Candesartan melting peak was removed from the DSC curves of

304

loaded niosomes, indicate that the active agent was encapsulated into the niosome and has the 13

305

amorphous state. Adversely, Varshosaz, et al. [35] reported that insulin loading had no

306

significant effect on the melting point of niosomes.

307

2.4.

308

In Figure 2 (b), the IR spectra of the nano-niosome components are presented. As shown in

309

the figure, vitamin D3 has a peak in 3300-3600 cm-1, which is related to stretching vibrations

310

of OH groups. Also, vitamin D3, blank nano-niosome and nano-niosome containing vitamin

311

D3 have a peak in 2900-3000 cm-1, which is related to stretching vibrations of CH groups.

312

As Figure 2 (b) shows all peaks regarding niosomes and vitamin D3 are present with a slight

313

displacement and intensity. In nano-niosome containing vitamin D3, the intensity of the

314

hydroxyl group peak has been declined in comparison to blank niosome, indicating hydrogen

315

bond formation. The intensity of the ester, ether and Alkanes group peaks have increased.

316

The increase in the intensity of these peaks is indicative of the formation of the interaction

317

among the components. Mehta, et al. [36] examined the IR spectra of blank and drug

318

(rifampicin, isoniazid, pyrazinamide) encapsulated niosomes. Their results did not show

319

significant differences between the control sample and the drugs containing samples.

320

2.5.

321

DPPH is a free radical widely used to measure the ability of antioxidant molecules to react as

322

a free radical scavenger or hydrogen donor [13]. The aim of this study was to evaluate the

323

antioxidant properties of nano-niosomes. Therefore, the DPPH test was performed in equal

324

volume from different formulations of nano-niosomes. The inhibitory concentration of 1 ml

325

of the samples is shown in Error! Reference source not found. (a).

326

The potential role of vitamin

327

work [37]; So all samples, although in a little amount, have anti-oxidant properties.

328

According to Error! Reference source not found. (a), the highest inhibitory effect in 1 ml of

329

nano-niosome solution belonged to nano-niosomes containing vitamin E-acetate (N3: 56%

Fourier transform infrared spectroscopy (FTIR)

Antioxidant properties

as a membrane antioxidant has been reported in previous

14

330

and N2: 42%) and lowest inhibitory belonged to blank sample (N0: 15%) due to the lack of

331

vitamin D3. The higher antioxidant properties in vitamin E-acetate samples compared to those

332

without it could be due to the break-up of the ester bond in vitamin E-acetate and the retrieval

333

of antioxidant properties at very low levels. Yang, et al. [7] studied vitamin E-acetate-

334

enriched nanoemulsions. Their results showed that approximately 35% of vitamin E-acetate

335

was converted to a free form (vitamin E) in the simulated intestine. Also, Di Mambro, et al.

336

[38] showed that Vitamin E acetate had almost no antioxidant activity (7% in 200 µg/ml) in

337

vitro, whereas for vitamin E at the same concentration the inhibitory concentration was 72%.

338

After 30 days the inhibitory effect of N0, N1 and N5 samples did not change significantly.

339

However, in samples containing vitamin E-acetate (N2 and N3) and a sample containing

340

cholesterol (N4), after 30 days the antioxidant properties decreased. Probably, vitamin E-

341

acetate and cholesterol was oxidized after a long period of time, which reduced the

342

antioxidant properties of these samples.

343

2.6.

344

Due to the exposure of binary bands to light and oxygen in nano-niosomes, lipid oxidation

345

may be easily accomplished by free radicals. In addition, with the presence of water,

346

hydrolysis may also occur and with releasing of free fatty acids, the pH is reduced [39]. Since

347

pH is one of the instability factors in these nano niosomes, it is possible to estimate the

348

chemical and physical instability in a qualitative way by measuring pH.

349

Error! Reference source not found. (b) shows the pH variations of different nano-niosome

350

formulations during one-month storage. The pH of the hydration water for all samples was

351

about 7.5, which was changed after the preparation of nano-niosomes.

352

The pH was decreased by incorporation of vitamin D3 and stabilizer agents. After a month,

353

the pH of nano-niosomes containing stabilizers was much lower. The pH of N4 and N5 was

354

almost identical on the first day, but over time, the nano-niosome containing polyethylene

pH

15

355

glycol decreased further. Probably the stabilizing effect of cholesterol is more, and prevents

356

the active substance leakage; So it causes less decrease in pH.

357

Considering that pH reduction is one of the instability factors in these samples, decreasing pH

358

can effect on physical and chemical stability. Soto-Jover, et al. [40] showed that the low pH

359

significantly reduced the physical and chemical stability of the nano-niosomes containing

360

canthaxanthin prepared by Tween 80 and Span 60.

361

2.7.

362

Turbidity refers to the light scattering properties by the particles in suspension. It depends on

363

nature, number, and size of colloidal particles in the liquid phase, on the difference in

364

refractive index between the particles and the medium, and on the particle size distribution.

365

To obtain a qualitative measure of particle size variations during 30 days, the turbidity of a nano-

366

niosome solution was measured [25]. The results of the optical properties of nano-niosomes

367

are illustrated in Error! Reference source not found. (c). Blank and N1 samples showed the

368

least turbidity and sample N4 had the highest turbidity.

369

The difference in turbidity among the samples of the first day can be due to the nature of the

370

stabilizers and correlated well with the size of nano-niosome particles. Cui, et al. [41]

371

reported that turbidity increases with increasing particle size.

372

Particle migration (i.e. creaming or sedimentation), and particle size variation or aggregation

373

(i.e. coalescence and flocculation) are the two major destabilization phenomena which affect

374

the homogeneity of dispersions [42]. As expected, the turbidity of all samples decreased over

375

a month. The decrease in turbidity could be due to the degradation of the active substance,

376

particle size variation, and flotation of particles to the surface of the solution [43].

377

Turbidity

2.8. Sedimentation

378

The effect of temperature on the amount of sediment during the storage period was

379

investigated. (70 days for samples in the refrigerator and 30 days for samples outside of the 16

380

refrigerator). Due to the higher instability of samples outside of the refrigerator, their

381

sediment percentage was measured on the 30th day. According to Error! Reference source

382

not found. (d), the results showed that the storage temperature was effective in the

383

sedimentation of the nano-niosomes. Sun, et al. [44] reported the effect of increasing the

384

temperature on aggregation in nanostructured lipid carriers. Also, Ravaghi, et al. [45] showed

385

that the high temperature significantly reduced the physical and chemical stability of the

386

nano-niosomes containing canthaxanthin.

387

Instability of samples at high temperatures may be due to the breakdown of hydrogen bonds

388

of surfactants. As seen in Error! Reference source not found. (d), sedimentation was observed

389

in all formulations (room and refrigerator temperature) except control sample in refrigerator

390

temperature. With increasing temperature and increasing time, the amount of sediment

391

increased. The samples in the refrigerator were flocculated on the 50-55th day and returned to

392

their original state with a little shaking, but after the 65-70th day, the phenomenon of

393

coalescence happened. Also in the cholesterol-containing sample, a cake after the 60th day

394

was formed, and flocculation phenomenon was not observed. In the samples outside the

395

refrigerator, after 12-20 days, the flocculation phenomenon occurred, and after the 25th day,

396

the coalescence and sedimentation occurred. The main factor for aggregation in the vesicular

397

systems can be attributed to the van der Waals attractions. Also, this aggregation in niosomes

398

may be due to the reduction of electrostatic repulsion between niosomes, the ion charge

399

density of the dispersion medium, high input energy (sonication, etc.), widespread

400

distribution of particles, reduction of water-binding sites in the membrane, etc., which causes

401

thermodynamic instability [26].

402

In some formulations, despite the higher zeta potential, more sediment was observed than the

403

other samples. Chemical instability may be affected by physical instability over time. The

404

results of Bozó, et al. [30] showed that the amount of surface charge was unaffected by 17

405

sedimentation.

406

2.9.

407

One of the most important parameters in the evaluation of niosomal formulations is

408

entrapment efficiency. Figure 4 (a) shows the encapsulation efficiency (EE%) of the samples

409

which varies from 75.3% to 94.3%. Addition of vitamin E-acetate caused a significant effect

410

on increasing of encapsulation efficiency (75.3% vs 82.4%). Also, increasing the amount of

411

vitamin E-acetate caused the increase of EE (2%). The highest efficiency was observed in the

412

samples containing cholesterol and polyethylene glycol. Cholesterol can decrease the

413

permeability of the bilayer with a better chain order and stability. The encapsulation

414

efficiency of vitamin D3 containing cholesterol was 94.2%. This is in accordance with a

415

previous study [15]. In a sample containing PEG, the EE% could be increased by entrapping

416

some molecules in the PEG chains [45]. These results show that the EE was strongly

417

dependent on the type and amount of the stabilizer agent (p < 0.05). Overall, a stabilizer can

418

increase EE% by changing hydrophobicity (HLB), rigidity, chain order and spacing between

419

tails of the lipid membrane.

420

The results of Wagner [46] showed higher EE of vitamin

421

sulfate (25.1%) in nano-niosomes. Palozza, et al. [47], in the study of nano-niosomes

422

containing beta-carotene, showed that the addition of cholesterol increased the encapsulation

423

efficiency.

424

Figure 4 (b) shows the loading capacity of the nano-niosomes. In general, the LC of all

425

samples was low which could be due to the initial degradation of vitamin

426

sonication and hydration). The highest LC is related to the sample containing cholesterol

427

(7%) and a sample containing polyethylene glycol (6.2%). The lowest LC is assigned to

428

sample N1` (2.4%). Considering that there was no significant difference in the LC of samples

429

containing vitamin E-acetate (N2 and N3).

Loading parameters

18

(95.9%) compared to iron

(during

430

3.

Conclusion

431

Niosomes provide a non-toxic and inexpensive vehicle for encapsulation of different

432

functional components (lipophilic, hydrophilic, and amphiphilic). In this study, the vitamin

433

D3 loaded nano-niosomes were produced by using a thin layer hydration method. The

434

resulted niosomes had nanometric size (<200 nm) and high encapsulation efficiency (>75%).

435

The different stabilizers were effective in enhancing of encapsulation efficiency and vitamin

436

E-acetate was the best stabilizer for this system due to the highest increasing effects on

437

encapsulation efficiency and antioxidant capacity.

438

4.

439

The authors gratefully acknowledge the support of the University of Tabriz and the support of

440

the Drug Applied Research Center of the Tabriz University of Medical Science.

441

5.

442

We wish to confirm that there are no known conflicts of interest associated with this

443

publication and there has been no significant financial support for this work that could have

444

influenced its outcome.

Acknowledgments

Conflict of Interest

445 446

6.

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photodynamic therapy, Int. J. Pharm. 494 (2015) 258-263.

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540

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551

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552

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553

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556

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558

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560

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561

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562

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563

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564

[45] M. Wagner, Application Of Supercritical Carbon Dioxide In Lipid Vesicle Design And

565

Extraction Of Oil From Potato Chips, (2015).

566

[46] P. Palozza, R. Muzzalupo, S. Trombino, A. Valdannini, N. Picci, Solubilization and

567

stabilization of β-carotene in niosomes: delivery to cultured cells, Chem. Phys. Lipids. 139

568

(2006) 32-42.

569 570

24

571

Figure Captions

572

Figure 1. Effect of formulation on a) size; b) PDI; and c) zeta potential of nano-niosomes by

573

calculating standard deviation in three replicates (different letters indicate a significant

574

difference at the 5% probability level of Duncan test); d) Scanning electron microscopy of

575

nano-niosomes containing vitamin D3 (N1).

576

Figure 2. a) DSC curve of the control sample, the sample containing vitamin D3 and vitamin

577

D3;

578

containing Vitamin D3 (N1).

579

Figure 3. a) Percentage of inhibition of one ml of nano-niosome specimens on day one and

580

on day 30 after production; by calculating the mean and standard deviation in three replicates

581

(different letters indicate a significant difference at the 5% probability level according to

582

Duncan test); b) Changes in pH of formulations over a month; c) Turbidity of nano-niosomes

583

in the first and 30th days (similar letters indicate no significant difference at the 5% Duncan

584

test); d) The percentage of deposition of formulations at room temperature and inside the

585

refrigerator after 70 days.

586

Figure 4. Encapsulation efficiency and loading capacity of the formulation by calculating the

587

mean and standard deviation with three repetitions (different letters representing a significant

588

difference at the 5% probability level of Duncan's test).

b) FTIR spectrum for vitamin D3, blank nano-niosome (N0) and nano-niosome

589

25

b

a a

300

0.6

b

a a

0.5 d

200

0.4

150 gh gh

100

a

bc

f

gh h

d

ef e 0.2

g

50

0.1

0

0 N1

N2

N3

1st day

N4

N0

N5

40th day

d

cd

cd e

10 0

-20

f

f

f

-10

f

e bc

-30 N0

N1

N2

1st day

30 20

N1

N2

N3

d

b

N4

N5

Formulation 1st day

N3

N4

Formulation

c

a

b

cd

cd

0.3

Formulation

Zeta potential ( mV)

a

d

N0

590 591 592

b bc

c

PDI

Particle size (nm)

250

40th day

Figure 1

26

40th day

N5

593 594 595

Figure 2

596 597

27

a

60

a 6.8

50 40

6.6

c cd

fe

6.2

g

fg

30

6.4

cde def pH

Inhibition%

b

20

b

7

h

6 5.8

h h

5.6 5.4

10

5.2 0

5 N0

N1

N2

N3

N4

N5

0

5

10

N0 N3

30th day

b

d

0.7

e

0.6

e f

0.5 0.4 0.3 0.2 0.1

g h

N1 N4

N2 N5

d

25 20 15 10 5

g h

0

0 N0

N1

N2

N3

N4

N0

N5

1st day

N1

N2

N3

N4

N5

Formulation

Formulation

T=4 - 70th day

30th day

598 599

30

30

sedinantation%

Absorbance

35

b c

0.9 0.8

25

a

c

1

20

Day

Formulation 1st day

15

Figure 3

600 601 602 603 604 28

T=25 - 30th day

605 606 607 608 609 610 611 612

100 90 80 70 60 50 40 30 20 10 0

b a

e

d

c

b

Loading capacity %

Encapsulation efficiency%

a

N1

N2

N3

N4

a b

d

N1

N5

Formulation

613 614

10 9 8 7 6 5 4 3 2 1 0

c

c

N2

N3 Formulation

Figure 4

615 616 617 618 619 620 621 622 623 29

N4

N5

624

Table 1. The formulation used for the preparation of nano-niosomes Formulation JKL 60⁄NOPP 80 (mole ratio) Vitamin D3 (mmole) Vitamin E- acetate (mmole) Cholesterol (mmole) Polyethylene Glycol 400 (mmole)

N0

N1

N2

N3

N4

N5

3:2

3:2

3:2

3:2

3:2

3:2

-

0.02 -

0.02 0.01 -

0.02 0.02 -

0.02 0.06 -

0.02 0.06

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648

30

649

Table 2. Melting and enthalpy temperature of the control sample, sample G. and vitamin Sample

Melting peak (oC)

Onset temperature (oC)

Endset temperature (oC)

Enthalpy (J/g)

N1

53

33.6

62.8

-36.4

Control (N0)

48

30.16

56.3

-10.7

Vitamin D3

91

82.98

98

-35.47

650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689

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