Characterizations on the Stability and Release Properties of Î²-ionone

Subscriber access provided by University of Massachusetts Amherst Libraries

Food and Beverage Chemistry/Biochemistry

Characterizations on the Stability and Release Properties of #-ionone Loaded Thermosensitive Liposomes (TSLs) Ling Chen, Rong Liang, Yihan Wang, Wallace Yokoyama, Maoshen Chen, and Fang Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06130 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49

Journal of Agricultural and Food Chemistry

1

TITLE HEADER

2

Characterizations on the Stability and Release Properties of β-ionone Loaded

3

Thermosensitive Liposomes (TSLs)

4 5

Ling Chena,b, Rong Liangc, Yihan Wange, Wallace Yokoyamad, Maoshen Chena,b, Fang Zhong*a,b

6 7

a

8

Jiangnan University, Wuxi 214122, China

9

b

Key Laboratory of Synthetic and Biological Colloids, Ministry of Education,

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

10

Jiangnan University, Wuxi 214122, P.R. China

11

c

12

of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, P.R. China

13

d

Western Regional Research Center, ARS, USDA, Albany, CA 94710, USA

14

e

Zhejiang Institute for Food and Drug Control, Zhejiang 310000, P.R. China

Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School

15 16 17 18 19 20 21 22 23 24 25 26 27 28

* To whom correspondence should be addressed. Tel: +86（510）85197876, Email:

29

[email protected]. 1 ACS Paragon Plus Environment


30

ABSTRACT:

31

Liposomes with phase transition temperatures, Tm, near pathogenic site

32

temperature are potential chemoprophylactic delivery vehicles. We prepared and

33

characterized the thermal properties of liposomes composed of DPPC and HSPC

34

incorporating β-ionone with Tm at 42℃. Liposomes with β-ionone/lipid ratio (wt/wt)

35

of 1:20 and 1:8 had the necessary stability and released most of the β-ionone. The

36

molecular architecture surround Tm was studied by fluorescent probes, Raman

37

spectroscopy and DSC. β-ionone was found to be preferentially located in the deep

38

regions of lipid bilayer (toward the long chain alkyl of the lipid) at moderate loading.

39

The results showed that β-ionone encapsulated liposomes have a superior release at

40

higher loading amount. Increasing β-ionone leads to disorder in the liquid crystalline

41

state and accelerates the release rate. These studies provide information on the

42

membrane structural properties of β-ionone loaded liposomes that guide rational

43

bioactive molecular delivery systems designing for health product.

44 45

Key words: Thermosensitive liposomes (TSLs), β-ionone, microviscosity, DSC,

46

Raman spectroscopy

47

2 ACS Paragon Plus Environment

Page 2 of 49

Page 3 of 49


48

INTRODUCTION

49

Chemoprevention is effective in treating cancer and inflammation to promote

50

human health. However, a major obstacle is systemic toxicity accompanying effective

51

chemotherapy at target tissues necessitates high delivery concentration of bioactive

52

molecules

53

anticancer component during delivery through the circulatory system while producing

54

a burst release only at the diseased tissue has been a goal of many laboratories.

55

Thermosensitive liposomes (TSLs) that release their encapsulated drug by heat at the

56

tumor site have been demonstrated to be a valid approach to transport and release

57

anticancer drugs to solid tumors (4). This delivery system was first proposed by Yatvin

58

et al. in 1978 for use at sites of mild local hyperthermia

59

to transport a variety of drugs. Agarwal et al. reported a thermosensitive liposomal

60

nanocarrier that held doxorubicin (DXR) up to 24 h and release it at 43℃

61

Melphalan encapsulated TSLs with the phase transition temperature of 42.7℃ were

62

designed by Chelvi for hyperthermia-mediated targeted delivery to murine tumors

63

TSLs reported by Winter et al, were also characterized for delivering the anticancer

64

drug arsenic trioxide (ATO)

65

phospholipid (phosphatidyldiglycerol) based TSLs in a feline sarcoma study has been

66

reported by Zimmermann et al.(9) In addition, one clinical study using combinations of

67

hyperthermia

68

www.ClinicalTrials.gov site, with the Celsion phase I clinical trial for patients with

69

liver tumors been completed, and the phase III trial for treating hepatocellular

(1-3)

. Therefore, research to develop a delivery system that would retain its

and

TSLs

(5)

. Later, TSLs were applied

(6)

.

(7)

.

(8)

. Recently, a preclinical trial using a synthetic

(ThermoDox®)

has

been


listed

on

the

NIH


70

carcinoma and phase II trial for breast cancer patients still in the progress (10).

71

β-ionone, one of the estimated 22,000 isoprenoid products of secondary plant

72

mevalonate metabolism, is widely found in algae, fruits, flowers and vegetables. It is

73

derived from the cleavage of the 9, 10 double bound of β-carotene by a dioxygenase

74

(11)

75

breast cancer and meningioma cells, through suppression of cell division and

76

initiation of apoptosis

77

mammary carcinogenesis via the downregulation of cyclin D1 and Bcl-2 expression

78

and upregulation of Bax expression

79

anticancer properties to gastric adenocarcinoma cells with the IC50 value of 89 µmol/L

80

(13)

81

chemopreventive and antitumor agent, few applications of β-ionone in an antitumor

82

delivery system were developed. It has been reported via numerous research studies

83

that liposome was an effective approach to encapsulate isoprenoids, such as

84

β-carotene, astaxanthin or coenzyme Q10 (17-19). β-ionone, a decomposition product of

85

β-carotene, possessing the similar hydrophobic properties and groups with β-carotene,

86

might has the potential to be incorporated by liposomes. The objective of this research

87

was to investigate the use of TSL as a delivery system for β-ionone to promote it

88

healthy benefits.

. β-ionone has been reported to inhibit the growth of melanoma, gastric cancer,

(12-15)

. For instance, β-ionone could suppress DMBA-induced

(16)

. Additionally, Liu et al. have also studied its

. Although recent research has found that β-ionone may be a potential

89

The precise phase transition temperature, Tm, is the most essential property of

90

TSLs. It is well known that liposomes remain stable and exhibit minimal drug release

91

below their Tm, but release their encapsulated material when the temperature reaches 4 ACS Paragon Plus Environment

Page 4 of 49

Page 5 of 49


92

the Tm (20). TSLs are distinguished from typical liposomes by possessing a narrow Tm.

93

TSLs ideally are selective and release therapeutic levels of drugs only at the targeted

94

temperature at the local tumor site without damaging peripheral noncancerous tissues.

95

Research suggests that Tm in the range of 39-43℃ is optimum to reduce pre-leakage

96

of

97

2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm = 41.3℃) and hydrogenated

98

soy phosphatidylcholine (HSPC, Tm = 52.7℃) have been used to prepare TSLs in this

99

Tm range

drugs

at

body

temperature

(37℃)

(21)

.

1,

(4, 22)

. Previous studies have suggested that the positively charged part of

100

HSPC interacts with the negatively charged part of DPPC by electrostatic interaction

101

resulting in a complex with a single phase transition

102

compositions of PCs, the amount of core material has been reported to affect the Tm as

103

well. The incorporation of temoporfin into liposomes composed of DPPC and

104

dipalmitoyl-phosphatidylglycerol (DPPG) resulted in a distinct decrease of the Tm (24).

105

These studies demonstrate the impact of proportion ratios of wall material and the

106

loading amount of encapsulated drugs on Tm and these properties will be discussed

107

later in detail. In addition, other requirements like the physicochemical stability

108

during storage, ultrafast drug release upon heating and the in vitro stability in serum

109

have been reported to be key roles for TSLs design as well(25, 26), so the storage

110

stability and the release behaviour in biological medium with serum would also

111

characterized in the following study.

(23)

. In addition to the

112

In this study, differential scanning calorimeter (DSC) was used to measure Tm as

113

a function of membrane composition and β-ionone loading. The stability and shelf life 5 ACS Paragon Plus Environment


Page 6 of 49

114

of β-ionone loaded TSLs were characterized by measuring changes in the Z-average

115

particle diameter, phospholipid profiles, TEM images and retention rate during

116

storage. The liposomal release properties were also characterized by in vitro release

117

tests with serum. Release behavior was further analyzed by fluorescence and Raman

118

spectra. The potential application of β-ionone loaded TSLs to deliver effective doses

119

of bioactive molecules, as well as the relationship between β-ionone release behavior

120

and liposomal structural properties were evaluated.

121

MATERIALS AND METHODS

122

Materials

123

HSPC (with the main component of DSPC, 97%) and DPPC were acquired from

124

Shanghai Advanced Vehicle Technology L.T.D. Co (Shanghai, China). β-ionone

125

standard was purchased from ANPEL laboratory Technologies Shanghai Inc

126

(Shanghai, China). The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS, 98%

127

purity),

128

trimethylammonium-DPH (TMA-DPH, 98% purity) were obtained from Sigma

129

Chemical Co. (St. Louis MO, USA). Fetal Bovine Serum (FBS) was purchased from

130

Gibco (Grand Island, NY). Ultra-pure grade water purchased from A.S. Watson Group,

131

Ltd. (Hong Kong, China) was used for HPLC tests and other experiments. Tween 80

132

and other analytical grade reagents were from China Medicine (Group) Shanghai

133

Chemical Reagent Co. (Shanghai, China).

134

Preparation of β-ionone Liposomes

135

1,

6-diphenyl-1,

3,

5-hexatriene

(DPH,

98%

purity),

and

Liposomes of different PC composition were prepared by the thin film hydration 6 ACS Paragon Plus Environment

Page 7 of 49


(27)

136

method developed by Xia et al

. 200 mg of PCs (DPPC, HSPC or the mixture of

137

DPPC and HSPC at different molar ratios), 50 mg of Tween80 and a range of amounts

138

(0-200 mg) of β-ionone were dissolved in ethanol in a round bottom flask. The solvent

139

was evaporated with a rotary evaporator at 50℃ to form a thin film of dry lipid on the

140

wall of the flask. The film was then hydrated by adding 20 mL of phosphate buffer

141

solution (0.05 M phosphate buffer solution, 0.15 M NaCl, PBS, pH 6.8) under

142

vigorous stirring at 50℃ for 30 min to form multilamellar vesicles (MLV). The

143

liposomal suspension was formed by ultrasonic probe processing of the MLV in an ice

144

bath for 4 min at 20% amplifying strength with 1 s of sonication and 1 s rest.

145

Vesicle Size Measurement

146

Z-Average diameter of β-ionone liposomes were evaluated by dynamic light

147

scattering (DLS) with a Nano Particle Analyzer (ZetaPALS, Brookhaven Instruments

148

Ltd., USA), according to methods in our previous report

149

aliquots of liposomal dispersion were suspended in 5 mL of phosphate buffer (0.05 M

150

phosphate buffer solution, 0.15 M NaCl, PBS, pH 6.8) to avoid multiple scattering

151

phenomena due to interparticle interaction. The prepared β-ionone liposomes were

152

then transferred to polystyrene cuvettes, and the Z-Average diameter (𝐷𝑧) and PDI

153

were recorded by DLS using the Nano-Zeta PALS particle size analyzer with a He/Ne

154

laser (λ = 633 nm) and scattering angle of 90°. 𝛥𝐷𝑧 was used to evaluate the

155

physical stability during the storage, calculating by the function as follows:

156

𝛥𝐷𝑧 =

157

Determination of Tm by Differential Scanning Calorimetry (DSC)

(28)

. Briefly, dilute 100 μL

The average diameter after storage-The average diameter initially prepared The average diameter initially prepared


(1)


Page 8 of 49

158

DSC (NETZSCH Instrument Ltd., Germany) thermograms were evaluated to

159

determine the phase transition temperature (Tm) of the liposomes with different

160

HSPC/DPPC ratios and β-ionone/lipid ratios, using an empty aluminum crucible with

161

a lid as a reference

162

transfered to 40 μL aluminum pans, which was subsequently sealed and measured.

163

The samples were scanned from 20 to 60℃ at 5℃/min in duplicate to ensure

164

exemption of the thermal history of the specimens (30).

165

Retention Rate

(29)

. Briefly, 10 μL of the β-ionone liposomal suspension was

166

Total β-ionone in the liposomal suspension was released by completely breaking

167

vesicles with TritonX-100. The concentration of β-ionone was assayed by HPLC

168

(Waters2695, Waters, Milford, MA) at 304 nm compared to a β-ionone standard curve.

169

The retention rate (RR) was calculated by the percentage of total amount of β-ionone

170

after storage relative to the total amount of β-ionone initially prepared.

171

RR(%) = Total amount of β-ionone

172

HPLC Analysis of β-ionone

173

High-performance liquid chromatography (RP-HPLC) was conducted to determine

174

the concentration of β-ionone (Waters, Milford, MA) equipped with a UV-VIS

175

detector, based on the method reported by Waldmann et al (31). In short, sterile syringe

176

filters (0.22 μm) were used to remove particulate contaminants from each sample

177

prior to analysis. Chromatographic separation was performed on a Waters Symmetry

178

C18 (Lichrosphere, 5 mm, 250×4.6 mm, Waters, Milford, MA) column at 30℃. The

179

mobile phase was a mixture of acetonitrile and ultrapure water (70:30). The flow rate

Total amount of β-ionone after storage initially prepared

× 100%


(2)

Page 9 of 49


180

was 1.0 mL/min. The detection wavelength was 304 nm and the sample volume was

181

20 μL.

182

HPLC-MS Analysis of Phospholipid Profile

183

HPLC-ESI-MS/MS technique was applied to analyze the decomposition (32)

Briefly, 1 μL

184

products of the phospholipids according to the method of Zhu et al.

185

lipid samples (with the phospholipid concentration of 1 mg/mL) were injected into a

186

Acquity UPLC Hilic column (2.1 × 100 mm, 1.7 μ m, Waters, Milford, MA) with

187

mobile phase flow rate of 0.3 ml/min. Solvent A and B were hexane and ammonium

188

acetate solution (20 mmol/L), respectively, with the analysis time of 10 min. The

189

thermostat column compartment was operated at 30°C, and the jet stream ESI source

190

was operated in the negative mode. Instrument parameters were set as follows: sheath

191

gas temperature of 100°C; sheath gas flow of 50 L/min; dry gas temperature of 250°C;

192

dry gas flow of 500 L/h; capillary entrance voltage of 3.5 kV; and kimmer voltage of

193

6 V. The MS scan data were collected in the range of m/z 782, 520 and 258. MS data

194

of phospholipids compositions were extracted by MassLynx Software (V4.1).

195

Transmission Scanning Electron Microscope TEM

196

The morphology of empty and β-ionone loaded liposomes were visualized via

197

transmission electron microscopy (TEM, H-7650, HITACHI, Japan) according to our

198

previously reported method.(33) A fresh copper mesh grid was placed onto droplets

199

containing pre-diluted liposomal suspension and excess liquid was removed with filter

200

paper after 4 minutes. Samples were air dried at room temperature, the morphology of

201

the liposomes was recorded by TEM at a voltage of 80 kV. 9 ACS Paragon Plus Environment


202 203

In vitro Release Studies The release rate of β-ionone in vitro was determined according to the method of (34, 35)

204

Yang et al. and Comiskey with minor modification

205

liposome with its β-ionone /lipid ratio of 1:8 was put into a dialysis bag (MW cutoff

206

3000, Sinopharm Co., China), and then transferred to a 250 mL beaker with 200 mL

207

dialysis medium (20% ethanol with 10% serum). This dialysis device was stirred in a

208

thermostatted water bath at 200 rpm under different temperatures of 25, 35, 42 and

209

50℃. At predetermined time intervals (0, 10, 30, 60, 90, 120, 180 min), 200 μL

210

samples of the release medium were withdrawn and replaced with equal volume of

211

fresh release medium. The β-ionone concentration in release medium was analyzed by

212

HPLC, following the methods mentioned. To investigate the release behavior of

213

liposomes loaded with different amount of β-ionone, the release assay were carried

214

out with β-ionone /lipid ratio of 1:20 and 1:8 and the incubation temperature of 42℃.

215

Microviscosity of Liposomal Bilayer Membranes

216

. For the assay, 5 mL of

The liposomal membrane fluidity was measured according to the method of Xia (27, 36)

217

and Tan et al

. Microviscosity is inversely related to membrane fluidity. High

218

microviscosity or lower membrane fluidity values are an indication of higher

219

structural order (37). Microviscosity of the membrane adjacent to a fluorescent probe is

220

positively correlated to the fluorescence polarization of the probe and can be

221

calculated using the Perin-Weber’s equation

222

of the membrane was determined by different fluorescent probes. DPH (oriented in

223

the deep regions of the lipid bilayer), ANS (oriented toward the hydrophilic

(38)

. The microfluidity at different depths


Page 10 of 49

Page 11 of 49


224

headgroup of the lipid) and TMA-DPH (oriented on the surface and glycerol side

225

chain region of the membrane) were fluorescence probes used to investigate the

226

molecular movement in the lipophilic core, superficial region and exterior membrane

227

surface, respectively

228

to a final concentration of 6×10-3 mol/L. The DPH stock solution was prepared by

229

dissolving DPH powder in tetrahydrofuran, followed by adjusting the concentration to

230

2×10-3 mol/L. The DPH stock was kept at 4℃ in the dark. Then, aliquots of DPH

231

stock solution (100 μL) were added to the 10 mL volumetric flask diluted by fresh

232

buffer solution (0.01 M PBS, pH 6.8) and used at room temperature. The final

233

concentration of DPH was 2×10-5 mol/L. TMA-DPH stock solution was prepared by

234

dissolving TMA-DPH powders in tetrahydrofuran/water (1:1, v/v) and the

235

concentration was adjusted to 10-2 mol/L. Aliquots of TMA-DPH stock solutions (100

236

μL) were added into 10 mL volumetric flasks and diluted by fresh PBS. The final

237

TMA-DPH concentration was 10-4 mol/L. When determining the microviscosity of

238

liposomal membranes, a small quantity of probe stock solution was mixed with

239

β-ionone liposomes with their drug/lipid ratios varying from 1:40 to 1:4 to give a

240

lipid/probe molar ratio of 300:1 at room temperature. For investigating the membrane

241

fluidity changing during heating process, the incubation temperature was rise from 35

242

to 50℃. Note that the ANS solution must be dried by nitrogen before analysis to

243

avoid the destruction of the membrane by ethanol

244

which is correlated to microviscosity (η) near the fluorescent probes was calculated

245

using the Perrin-Weber’s equation as follows: (41)

(39, 40)

. Briefly, the ANS probe was dissolved in ethanol solution

(27)

. Fluorescence polarization (P)



(I

-GI

)

I

Page 12 of 49

246

P = (I 0,0+GI0,90 ), G = I 90,0

247

η = 0.46-P

248

where I0,0 and I0,90 are the fluorescence intensities of the emitted light polarized

249

parallel and perpendicular to the excitation light, respectively, and G is the grating

250

correction factor(42). The fluorescence intensities were measured at a range of

251

temperature from 30 to 50℃ with a florescence spectrometer (F-7000, Hitachi Co.,

252

Ltd, Japan). The excitation and emission wavelengths were 350 and 450 nm,

253

respectively, and the slit widths for both excitation and emission were 5 nm.

254

Raman Spectroscopy

0,0

(3)

90,90

0,90

2P

(4)

255

Raman spectroscopy was used to predict the location of β-ionone in the bilayer

256

membrane and characterize the structural properties according to the method of

257

Gardikis et al

258

interval 2 cm-1 via a Raman spectrometer (LabRAM HR Evolution, HORIBA Jobin

259

Yvon S.A.S. France) equipped with a 633 nm frequency stabilized laser source. The

260

collection time was 30 s, and the laser output power was 400 mV. The ordinary

261

Raman spectrum was baseline corrected, and the Raman intensities were measured as

262

peak height. Liposomes with different β-ionone/lipid ratios from 1:40 to 1:4 were held

263

at 25℃ in order to assess the change of the interaction between PCs and β-ionone at

264

increasing amounts, while liposomes with β-ionone/lipid ratios of 1:20 and 1:8 were

265

further investigated from 35 to 50℃. SL represents the changes in both the

266

trans/gauche population ratio and the lateral packing of the chains. ST represents the

267

degree of longitudinal order of liposomes

(43)

. Raman spectra were recorded in the ranges of 600-3000 cm-1 with

(37)

. These parameters were calculated from


Page 13 of 49

268


the equations as follows:

269

SL =

270

ST =

ICH2(Sample) -0.7 1.5

, ICH2 =I2882 /I2847

𝐼1130 /𝐼1086

(5) (6)

1.77

271

where I2882 /I2847 is the height intensity ratios of the peaks at 2882 and 2847 cm-1, and

272

𝐼1130 /𝐼1086 is the height intensity ratios of the peaks at 1130 and 2086 cm-1.

273

Statistical Analysis

274

All the data are expressed as mean±standard deviation (SD). All measurements

275

were performed at least in triplicate. The results were subjected to statistical analysis

276

by one-way ANOVA followed by multiple comparison test with SPSS software (SPSS

277

Inc., (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant

278

when p < 0.05.

279 280

RESULTS AND DISCUSSION

281

Effect of Phospholipid Composition on Tm

282

Tm as a function of membrane composition was determined by DSC. The DSC

283

profiles (Fig. 1) for pure DPPC (trace a), HSPC (trace f) and their mixtures (traces b-e)

284

show phase transitions from 41.30℃ to 53.12℃. HSPC had the highest and DPPC the

285

lowest Tm, agreeing with published data (44, 45). For liposomes composed of both HSPC

286

and DPPC, the greater proportion of HSPC led to higher Tm, which is attributed to the

287

longer acyl chain length and stronger interactions of HSPC (23, 46) This result is highly

288

consistent with that reported by Chen et al. that Tm would rise with increasing HSPC

289

content (with longer length of acyl chains) and decreased after adding DLPC (with

290

shorter acyl chain length) (23). The temperature peak width at half peak height, T1/2, is 13 ACS Paragon Plus Environment


Page 14 of 49

(47)

291

inversely proportional to the co-operativity of the phospholipid bilayers

. The

292

increase of HSPC caused progressive broadening of the temperature peak width as

293

shown in the DSC thermogram, suggesting decreased co-operativity of the transition

294

by increased HSPC concentrations (48). The same results were shown in the studies of

295

Chen’s et al

296

is observed, but as the ratio increases, a shoulder appears, indicating phase

297

segregation. The DSC results suggest that the molar ratio of DPPC: HSPC=8:2 meets

298

the requirements of Tm of 42℃ in target tissue and was selected for further study. This

299

result was consistent with various formulations developed by other research groups.

300

For instance, liposomal formulations composed of DPPC/DSPC/DSPE-PEG2000 in a

301

molar ratio of 80:15:5 reported by Li et al. and of DPPC/DSPC/DPPGOG in a molar

302

ratio of 7:2:1 developed by Lindner et al., with similar DPPC/DSPC ratios, showed

303

the same Tm of 42℃ (49, 50).

304

Effect of Encapsulated β-ionone on Tm

(4)

. Moreover, at lower HSPC/DPPC ratios only a single-phase transition

305

Incorporation of β-ionone in liposomes affects the thermal properties of the

306

bilayers possibly due to some intercalation into the bilayers, adsorption on the surface

307

of liposomes, or interaction with the polar head groups of the PC and was studied by

308

DSC

309

liposomes and liposomes (DPPC: HSPC=8:2) with varying β-ionone/lipid ratios (1:40

310

to 1:1) were characterized by DSC. A symmetrical peak was only occured in the

311

absence of β-ionone with Tm of 43.48℃. Incorporation of β-ionone at even 5 mg

312

(β-ionone/lipid ratio of 1:40) lowered the Tm and broadened the main transition

. Thermograms (Fig. 2) and the Tm and T1/2 (Table 1) of blank

(46, 51-53)


Page 15 of 49


313

temperature peak, with the peak showing asymmetry towards the lower temperature

314

side. Increasing β-ionone content amplified the asymmetry with a shoulder appeared

315

at 1:4 β-ionone/lipid ratio. Further increases of β-ionone to 100-200 mg

316

(β-ionone/lipid ratio of 1:2-1:1) resulted in the Tm shift to 37.38℃ with a larger

317

shoulder spanning 29-42℃, suggesting a phase segregation occurred with the

318

formation of a new membrane species, these liposomes were no longer stable (54).

319

According to the researches of Ichioma Onyesom, the reduction of transition

320

temperature is associated with partial embedding of molecules into the bilayer of lipid

321

or penetration into the core area of liposomes (46). Additionally, Bermudez et al. has

322

reported that molecules interacting with either the polar headgroups, lipophilic

323

hydrocarbon chains or both parts of the lipid bilayer constituents would exert a

324

significant influence on their phase behavior

325

researches as well as our study results inferred that β-ionone has been encapsulated

326

into the liposomal membranes and the embedding of β-ionone would cause the Tm

327

reducing. Further studies about the relationship between loading amounts and

328

liposomal stability were investigated as follows.

329

Storage Stability of β-ionone Loaded Liposomes

(55)

. Information presented in above

330

The stability of shelf life of liposomal vesicles during storage at 4℃ was evaluated

331

by the changes of Z-average diameter (ΔDz) and retention rate (RR) over time. As

332

shown in Fig. 3A, the Z-average diameter of blank liposomes more than tripled to

333

282.9 nm after 80 days storage, with its PDI value changing from 0.155 to 0.298. This

334

tendency to aggregate during storage or manufacture is a thermodynamic property of 15 ACS Paragon Plus Environment


335

liposomes(17). However, this instability was strongly inhibited by β-ionone

336

incorporation. The increases of DZ after 80 days were only 28.48, 8.74, 7.31 and 20.41%

337

at the β-ionone/lipid ratios of 1:40, 1:20, 1:8 and 1:4 (Fig. 3B), with their original

338

particle size of 132.6±2.8, 158.7±3.4, 192.6±1.2 and 203.5±3.1 nm, and PDI values of

339

0.183±0.006, 0.109±0.025, 0.110±0.016 and 0.135±0.012, respectively. These results

340

showed that β-ionone suppressed aggregation on the whole, compared with unloaded

341

ones and in a concentration-dependent manner when the β-ionone/lipid ratio was

342

below 1:8. However, higher β-ionone loading would reduce the suppression properties,

343

which might be attributed to the excess amounts of β-ionone present in the aqueous

344

phase yielding to disruption of the bilayer, thus reduce the dispersion stability of

345

liposomes.

346

The loss of encapsulated β-ionone during storage is shown in Fig. 3C.

347

Liposomes with relatively higher β-ionone/lipid ratios of 1:20 and 1:8 had a retention

348

rate of about 60% after 80 days storage, while samples with high β-ionone/lipid ratio

349

of 1:4 had retention rates of 43%, only. High loss of β-ionone in 1:4 drug/lipid ratio

350

liposomal delivery system might be attributed to the heavier coalescence of liposomes

351

during storage (combining with the results of particle size), which would further

352

destabilize the liposomal bilayers and thus lead to more severe leakage of β-ionone.

353

The initial PC composition of empty and β-ionone loaded liposomes as well as

354

after storage at 38℃ was presented in Table 4. As seen in Table 4, the content of

355

HSPC and DPPC after 4 weeks storage reduced for approximately 6% for all the

356

liposomal samples, but conversely, the lysophospholipids (LPC) content among all 16 ACS Paragon Plus Environment

Page 16 of 49

Page 17 of 49


357

those formulations increased. This result was consistent with the previous reports that

358

the liposomal phospholipids could hydrolyze to LPC during storage, which would

359

destabilize the bilayer structures

360

liposomal formulation was negligible, which would not cause a significant damage to

361

the bilayer structure. In addition, the similar degradation rate of empty liposomes and

362

β-ionone loaded liposomes indicated that the incorporation of β-ionone would not

363

affect the bilayer decomposition.

(56)

. However, the generation of LPC in this

364

The morphology and vesicle shape of empty and β-ionone loaded liposomes were

365

observed by TEM. As presented in Fig. 4A and B, blank liposomes possessed high

366

tendency to aggregate compared with β-ionone loaded ones and the hydrophobic area

367

of β-ionone loaded liposomes were much larger than empty ones (marked with red

368

arrow, the light area surrounding the vesicles), indicating the incorporation of

369

β-ionone into liposomes. The vesicle size around 200 nm showed by TEM were

370

slightly larger than the results by DLS, which might due to the collapse of liposomal

371

vesicles during the progress of taking photos. The variations of vesicle size and shape

372

before and after storage for liposomes with different drug/lipid ratios were shown in

373

Fig. 4C. The size changes of empty and β-ionone loaded samples matched well with

374

the results of DLS presented in Fig. 3A and B, with empty liposomes doubled after

375

storage for about 30 d, while β-ionone loaded liposomes merely changed little.

376

Effect of β-ionone Incorporation on Membrane Fluidity

377

Fluorescence polarization and Raman spectra were utilized to investigate the

378

structural properties of the bilayer membranes when β-ionone was incorporated. The 17 ACS Paragon Plus Environment


379

microviscosity for DPH and TMA-DPH with β-ionone loading is shown in Fig. 5.

380

Low β-ionone/lipid ratios from 1:40 to 1:20 increased the microviscosity of the

381

membrane surrounding both probes indicating β-ionone enhanced membrane rigidity.

382

Further increases in β-ionone to 1:8 did not increase the microviscosity of DPH and

383

TMA-DPH remained constant implying that the hydrophobic interactions had reached

384

saturation. At high loading, with the drug/lipid ratio of 1:4, the hydrophobic core and

385

exterior surface region of the membrane had lower microviscosity or were more

386

highly fluid, compared with 1:20 or 1:8 ones. Over high β-ionone in the bilayer may

387

interfere with acyl chain and/or head group interactions and destabilize the bilayer.

388

The microviscosity measured by the ANS surface probe barely changed at low

389

loading (Fig. 5), suggesting that β-ionone is oriented in the deep regions of the lipid

390

bilayer when incorporated at low levels. The microviscosity for ANS increased with

391

the β-ionone/lipid ratio up to 1:8 followed by a slight decrease at higher concentration.

392

The microviscosity results suggest that β-ionone moves to the surface and may affect

393

the ionic interactions between the polar headgroups at higher β-ionone/lipid ratio

394

(above 1:8). The carbonyl group of the β-ionone molecule has a slight ionic character

395

and may interact with the polar head group of the phospholipid. The location of

396

β-ionone in liposomal bilayers interpreted by our data is shown in Fig. 6.

397

Fluorescence measurements demonstrated that liposomes with β-ionone/lipid

398

ratios of 1:20 or 1:8 are more stable because they had higher microviscosity and lower

399

membrane fluidity (lower ΔDz and higher RR) than other liposomes.

400

Effect of β-ionone Incorporation on the Order Parameters of Lipid Bilayer 18 ACS Paragon Plus Environment

Page 18 of 49

Page 19 of 49


401

Raman spectroscopy has been widely used to determine structural changes at the

402

molecular level of liposomal hydrocarbon chains in membranes by drugs that change

403

molecular conformation and vibrational modes

404

corresponding to inter- and intra- molecular membrane order are shown in Fig. 7.

405

Changes in peak intensities in Raman scattering spectra relate to membrane order (58).

406

The characteristic peaks in Fig. 7A at 2847 and 2882 cm-1 show the symmetrical and

407

asymmetrical vibration of the methylene C-H stretching, respectively

408

calculated from the ratio of peak intensities, I2882/I2847, is associated with the changes

409

in both the lateral interaction between chains and the trans-gauche population ratio (59).

410

The changes of relative peak intensity and SL are shown in Table 3. The data shows

411

that the SL increased with increasing β-ionone/lipid ratio from 1:40 to 1:8 and

412

indicates that the lateral packing and inter-chain order between lipid molecules

413

increased. Nevertheless, SL decreased with further increases in β-ionone to 1:4 ratio.

414

These observations are consistent with the fluorescence microviscosity measurements

415

that β-ionone would incorporate into the membrane at high concentration.

(57)

. Spectra of wavelength range

(43)

. SL,

416

The bands at 1130 cm-1 are associated with the all-trans stretching vibrations of

417

the alkyl C-C bonds, while bands at 1086 cm-1 correspond to the gauche rotations of

418

hydrocarbon chains Fig. 7B. Some researchers found a positive correlation between

419

the longitudinal order and the number of all-trans bonds

420

from the ratio I1130/I1086 may be a sensitive measure of the order in the longitudinal

421

interaction between alkyl chains. The calculated results are in Table 4. The ratios

422

were similar at all β-ionone levels, which might due to the weak intensity of the peaks 19 ACS Paragon Plus Environment

(60)

. Therefore, ST derived


423

that hard to be calculated accurately to ST from the Raman spectra.

424

Release Behavior of β-ionone Loaded Liposomes

425

The release behavior of β-ionone loaded liposomes may be the most important

426

property as an anticancer delivery vehicle and was evaluated using an in vitro release

427

test at incubation temperatures of 25, 35, 42 and 50℃, using the liposomes with the

428

β-ionone/lipid ratio of 1:8. According to the results in Fig. 2 and Table 1, Tm of the

429

liposomes with β-ionone/lipid ratios of 1:20 and 1:8 were all around 42℃, one is of

430

42.32±0.35℃ and another is of 41.24±0.12℃. For the purpose to further clear the

431

differences between these two samples, their release behaviors were also investigated

432

in this study. As shown in Fig. 8A, very little β-ionone was released at 35℃, and

433

almost no release at 25℃. A large release of β-ionone occurs at 42℃, and increases at

434

50℃. Loading amount also affects release as shown in Fig. 8B. Liposomes with

435

β-ionone/lipid ratio of 1:8 were more stable than 1:20 at 42℃. The differences in the

436

release behavior might be attributed to lower Tm at 1:8 ratio, which would be further

437

studied in the following sections.

438

Effect of Heating Temperatures on Membrane Fluidity

439

In order to understand phase transition at the molecular level as a function of

440

temperature, the acyl chain vibration during in vitro release were investigated. The

441

temperature dependence of DPH fluorescence polarization for pure liposome and

442

liposomes with β-ionone/lipid ratios of 1:20 and 1:8 are presented in Fig. 9. The

443

fluorescence polarization of pure liposomes is constant until about 41℃ when there is

444

a sudden decrease at 41-45℃ and then remains constant at temperatures above 45℃. 20 ACS Paragon Plus Environment

Page 20 of 49

Page 21 of 49


445

Liposomes loaded with β-ionone showed similar polarization behavior, but the phase

446

transition temperature was reduced to 39-42℃. On the other hand, the microviscosity

447

of bilayer membranes in the gel state increased when the mole fraction of β-ionone

448

enhanced with the drug/lipid ratio changing from 1:20 to 1:8. The liquid crystalline

449

state decreased with β-ionone loading. The higher microviscosity lowers fluidity of

450

membranes and indicates that the packing density at β-ionone/lipid ratio of 1:8 was

451

lower in the liquid crystalline state, thus β-ionone would release more rapidly. This

452

phenomenon was used to account for the release behavior in previous studies.

453

Effects of Heating Temperatures on the Order Parameters of Lipid Bilayer

454

The order properties of lipid molecules transition from solid to the

455

liquid-crystalline phases were studied with increasing temperature. The phase

456

transition temperature can be measured by the peak height intensity ratio

457

ratio I2882/I2847 in the C-H stretching region and the ratio I1130/I1086 in the C-C

458

stretching region, respectively, as a function of temperature were monitored for

459

liposomes with β-ionone/lipid ratios of 1:20 and 1:8. (Fig. 10). The ratios I2882/I2847

460

and I1130/I1086 remained almost constant below 40℃, followed by an abrupt decline at

461

40-45℃, and remained constant again above 45℃. The phase transition of liposomes

462

with β-ionone/lipid ratio of 1:20 usually took place at 39-40℃ (Fig. 10B and 10D).

463

The incorporation of β-ionone at the drug/lipid ratio of 1:8 resulted in a lower initial

464

transition temperature from 41.43 to 40.50℃ (Fig. 10A) or 41℃ (Fig. 10C). These

465

transition temperatures are very close to the results derived from DSC. The peak ratio

466

at 1:8 β-ionone/lipid ratio was higher than that at 1:10 in the gel state and supports our 21 ACS Paragon Plus Environment

(61, 62)

. The


Page 22 of 49

(63)

467

other data. These ratios correspond to the disorder/order among the chains

. This

468

parameter is further verification that β-ionone increases the disorder in the liquid

469

crystalline state and explains why liposomes at β-ionone/lipid ratio of 1:8 possess

470

superior release properties than those at 1:20.

471

In summary, the release behavior and phase transition mechanisms upon heating

472

of β-ionone loaded thermosensitive liposomes (TSL) were studied in this research.

473

The phase transition temperature, Tm, of β-ionone loaded liposomes composed of

474

DPPC and HSPC was inversely related to the molar ratio of these two PCs and the

475

loading amount of β-ionone. β-ionone incorporation improved the storage stability of

476

liposomes, and those liposomes with β-ionone/lipid ratios of 1:20 and 1:8 were more

477

stable by decreasing membrane fluidity and increasing lateral inner-chain order. The

478

release of β-ionone occurred in the ideal range, 39-43℃. These findings suggest that

479

β-ionone loaded TSLs may be a potential useful delivery system for nutritional

480

supplement or chemopreventive agents.

481

Acknowledgments

482

This research was supported by the National Key R&D Program of China

483

(2016YFD0400801，2016YFD0400802), the National Natural Science Foundation of

484

China (No. 31571891，31401533), The research is also supported by program of

485

“Collaborative Innovation Center of Food Safety Quality Control in Jiangsu

486

Province”, China.

487


Page 23 of 49


488

REFERENCES

489

1. Needham, D.; Anyarambhatla, G.; Kong, G.; Dewhirst, M. W., A new

490

temperature-sensitive liposome for use with mild hyperthermia: characterization and

491

testing in a human tumor xenograft model. Cancer Research 2000, 60, 1197.

492

2. Zhao, L. G.; Zhang, Q. L.; Zheng, J. L.; Li, H. L.; Wei, Z.; Tang, W. G.; Xiang, Y.

493

B., Dietary, circulating beta-carotene and risk of all-cause mortality: a meta-analysis

494

from prospective studies. Scientific Reports 2016, 6, 26983.

495

3. Mehta, R. G., Current paradigms of cancer chemoprevention. Turkish Journal of

496

Biology 2015, 38, 839-847.

497

4. Chen, J.; Cheng, D.; Li, J.; Wang, Y.; Guo, J. X.; Chen, Z. P.; Cai, B. C.; Yang, T.,

498

Influence of lipid composition on the phase transition temperature of liposomes

499

composed of both DPPC and HSPC. Drug Dev. Ind. Pharm. 2013, 39, 197-204.

500

5. Yatvin, M. B.; Weinstein, J. N.; Dennis, W. H.; Blumenthal, R., Design of

501

liposomes for enhanced local release of drugs by hyperthermia. Science 1978, 202,

502

1290.

503

6. Agarwal, A.; Mackey, M. A.; El-Sayed, M. A.; Bellamkonda, R. V., Remote

504

triggered release of doxorubicin in tumors by synergistic application of

505

thermosensitive liposomes and gold nanorods. ACS Nano 2011, 5, 4919-4926.

506

7. Chelvi, T. P.; Jain, S. K.; Ralhan, R., Hyperthermia-mediated targeted delivery of

507

thermosensitive liposome-encapsulated melphalan in murine tumors. Oncology

508

Research 1995, 7, 393-398.

509

8. Winter, N. D.; Murphy, R. K. J.; Schatz, G. C.; O’Halloran, T. V., Development 23 ACS Paragon Plus Environment


510

and Modeling of Arsenic Trioxide Loaded Thermosensitive Liposomes for Anticancer

511

Drug Delivery. J. Liposome Res. 2011, 21, 106.

512

9. Zimmermann, K.; Hossann, M.; Hirschberger, J.; Troedson, K.; Peller, M.;

513

Schneider, M.; Brã¼Hschwein, A.; Meyer-Lindenberg, A.; Wess, G.; Wergin, M., A

514

pilot trial of doxorubicin containing phosphatidyldiglycerol based thermosensitive

515

liposomes in spontaneous feline soft tissue sarcoma. International Journal of

516

Hyperthermia 2016, 33, 1.

517

10. Landon, C. D.; Park, J. Y.; Needham, D.; Dewhirst, M. W., Nanoscale Drug

518

Delivery and Hyperthermia: The Materials Design and Preclinical and Clinical

519

Testing of Low Temperature-Sensitive Liposomes Used in Combination with Mild

520

Hyperthermia in the Treatment of Local Cancer. Open Nanomedicine Journal 2011, 3,

521

38.

522

11. Silva, I.; Rocha, S. M.; Coimbra, M. A., Quantification and potential aroma

523

contribution of beta-ionone in marine salt. Flavour Frag. J. 2010, 25, 93-97.

524

12. Lei, H.; Mo, H.; Hadisusilo, S.; Qureshi, A. A.; Elson, C. E., Isoprenoids

525

Suppress the Growth of Murine B16 Melanomas In Vitro and In Vivo. J. Nutr. 1997,

526

127, 668-674.

527

13. Liu, J. R.; Chen, B. Q.; Yang, B. F.; Dong, H. W.; Sun, C. H.; Wang, Q.; Song, G.;

528

Song, Y. Q., Apoptosis of human gastric adenocarcinoma cells induced by β-ionone.

529

World Journal of Gastroenterology Wjg 2004, 10, 348.

530

14. Duncan, R. E.; Lau, D.; Elsohemy, A.; Archer, M. C., Geraniol and beta-ionone

531

inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in 24 ACS Paragon Plus Environment

Page 24 of 49

Page 25 of 49


532

MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity.

533

Biochem. Pharmacol. 2004, 68, 1739-1747.

534

15. Kim, M. O.; Moon, D. O.; Kang, C. H.; Kwon, T. K.; Choi, Y. H.; Kim, G. Y.,

535

beta-Ionone enhances TRAIL-induced apoptosis in hepatocellular carcinoma cells

536

through Sp1-dependent upregulation of DR5 and downregulation of NF-kappaB

537

activity. Molecular Cancer Therapeutics 2010, 9, 833-843.

538

16. Liu, J. R.; Sun, X. R.; Doug, H. W.; Sun, C. H.; Sun, W. G.; Chen, B. Q.; Song, Y.

539

Q.; Yang, B. F., beta-ionone suppresses mammary carcinogenesis, proliferative

540

activity and induces apoptosis in the mammary gland of the Sprague-Dawley rat. Int.

541

J. Cancer 2008, 122, 2689-2698.

542

17. Tan, C.; Xue, J.; Lou, X.; Abbas, S.; Guan, Y.; Feng, B.; Zhang, X.; Xia, S.,

543

Liposomes as delivery systems for carotenoids: comparative studies of loading ability,

544

storage stability and in vitro release. Food Funct. 2014, 5, 1232.

545

18. Xia, S.; Shiying Xu, A.; Zhang, X., Optimization in the Preparation of Coenzyme

546

Q10 Nanoliposomes. Journal of Agricultural & Food Chemistry 2006, 54, 6358.

547

19. Nakagawa, H.; Shiina, T.; Sekino, M.; Kotani, M.; Ueno, S., Fusion and

548

molecular aspects of liposomal nanocarriers incorporated with isoprenoids. IEEE

549

Transactions on Nanobioscience 2007, 6, 219-222.

550

20. Andresen, T. L.; Jensen, S. S.; Jørgensen, K., Advanced strategies in liposomal

551

cancer therapy: Problems and prospects of active and tumor specific drug release.

552

Progress in Lipid Research 2005, 44, 68-97.

553

21. Kong, G.; Anyarambhatla, G.; Petros, W. P.; Braun, R. D.; Colvin, O. M.; 25 ACS Paragon Plus Environment


554

Needham, D.; Dewhirst, M. W., Efficacy of liposomes and hyperthermia in a human

555

tumor xenograft model: importance of triggered drug release. Cancer Research 2000,

556

60, 6950-6957.

557

22. Chen, J.; Yan, G. J.; Hu, R. R.; Gu, Q. W.; Chen, M. L.; Gu, W.; Chen, Z. P.; Cai,

558

B. C., Improved pharmacokinetics and reduced toxicity of brucine after encapsulation

559

into stealth liposomes: role of phosphatidylcholine. International Journal of

560

Nanomedicine 2012, 7, 3567-77.

561

23. Chen, J.; He, C. Q.; Lin, A. H.; Xu, F.; Wang, F.; Zhao, B.; Liu, X.; Chen, Z. P.;

562

Cai, B. C., Brucine-loaded liposomes composed of HSPC and DPPC at different

563

ratios: in vitro and in vivo evaluation. Drug Dev. Ind. Pharm. 2014, 40, 244-251.

564

24. Kuntsche, J.; Freisleben, I.; Steiniger, F.; Fahr, A., Temoporfin-loaded liposomes:

565

physicochemical characterization. European journal of pharmaceutical sciences :

566

official journal of the European Federation for Pharmaceutical Sciences 2010, 40,

567

305-15.

568

25. V.V, P.; Maharshi., S.; Mathew, S. T.; V.V, P.; Maharshi., S., Liposomes: An

569

Overview. Journal of Drug Delivery & Therapeutics 2012, 67-74.

570

26. Kneidl, B.; Peller, M.; Winter, G.; Lindner, L. H.; Hossann, M., Thermosensitive

571

liposomal drug delivery systems: state of the art review. International Journal of

572

Nanomedicine 2014, 9, 4387-4398.

573

27. Xia, S.; Tan, C.; Zhang, Y.; Abbas, S.; Feng, B.; Zhang, X.; Qin, F., Modulating

574

effect of lipid bilayer–carotenoid interactions on the property of liposome

575

encapsulation. Colloids & Surfaces B Biointerfaces 2015, 128, 172. 26 ACS Paragon Plus Environment

Page 26 of 49

Page 27 of 49


576

28. Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F., Controlled Release of

577

beta-Carotene in beta-Lactoglobulin-Dextran-Conjugated Nanoparticles' in Vitro

578

Digestion and Transport with Caco-2 Monolayers. Journal of Agricultural and Food

579

Chemistry 2014, 62, 8900-8907.

580

29. Habib, L.; Khreich, N.; Jraij, A.; Abbas, S.; Magdalou, J.; Charcosset, C.;

581

Greige-Gerges, H., Preparation and characterization of liposomes incorporating

582

cucurbitacin E, a natural cytotoxic triterpene. International journal of pharmaceutics

583

2013, 448, 313-9.

584

30. Yang, S. Y.; Zheng, Y.; Chen, J. Y.; Zhang, Q. Y.; Zhao, D.; Han, D. E.; Chen, X.

585

J., Comprehensive study of cationic liposomes composed of DC-Chol and cholesterol

586

with different mole ratios for gene transfection. Colloids and surfaces. B,

587

Biointerfaces 2013, 101, 6-13.

588

31. Waldmann, D.; Schreier, P., Stereochemical Studies of Epoxides Formed by

589

Lipoxygenase-Catalyzed

590

4-Hydroxy-.beta.-ionone. Journal of Agricultural & Food Chemistry 1995, 43,

591

626-630.

592

32. Zhu, L.; Wang, P.; Sun, Y. J.; Xu, M. Y.; Wu, Y. J., Disturbed phospholipid

593

homeostasis in endoplasmic reticulum initiates tri-o-cresyl phosphate-induced delayed

594

neurotoxicity. Sci Rep 2016, 6, 37574.

595

33. Rong, L.; Ling, C.; Yokoyama, W.; Williams, P. A.; Fang, Z., Niosomes

596

consisting of tween-60 and cholesterol improve the chemical stability and antioxidant

597

activity of (-)-epigallocatechin gallate under intestinal tract conditions. Journal of

Co-oxidation

of

Retinol,


.beta.-Ionone,

and


Page 28 of 49

598

Agricultural & Food Chemistry 2016, 64.

599

34. Yang, S.; Guo, Y., Preparation of lomustine-iohexol thermosensitive compound

600

liposomes and study on the in vitro release characteristics. Journal of Chemical &

601

Pharmaceutical Research 2014.

602

35. Comiskey, S. J.; Heath, T. D., Serum-induced leakage of negatively-charged

603

liposomes at nanomolar lipid concentrations. Biochemistry 1990, 29, 3626-3631.

604

36. Tan, C.; Zhang, Y.; Abbas, S.; Feng, B.; Zhang, X.; Xia, S.; Chang, D., Insights

605

into chitosan multiple functional properties: the role of chitosan conformation in the

606

behavior of liposomal membrane. Food Funct. 2015, 6, 3702.

607

37. Tan, C.; Xia, S. Q.; Xue, J.; Xie, J. H.; Feng, B. A.; Zhang, X. M., Liposomes as

608

Vehicles for Lutein: Preparation, Stability, Liposomal Membrane Dynamics, and

609

Structure. Journal Of Agricultural And Food Chemistry 2013, 61, 8175-8184.

610

38. Takao,

611

MOLECULAR-INTERACTIONS BETWEEN LIPIDS AND SOME STEROIDS IN

612

A MONOLAYER AND A BILAYER .2. Langmuir 1995, 11, 912-916.

613

39. Marczak, A., Fluorescence anisotropy of membrane fluidity probes in human

614

erythrocytes incubated with anthracyclines and glutaraldehyde. Bioelectrochemistry

615

2009, 74, 236-239.

616

40. JemiolaRzeminska, M.; Kruk, J.; Skowronek, M.; Strzalka, K., Location of

617

ubiquinone homologues in liposome membranes studied by fluorescence anisotropy

618

of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene. Chem. Phys.

619

Lipids 1996, 79, 55-63.

Y.;

Yamauchi,

H.;

Manosroi,

J.;

Manosroi,


A.;

Abe,

M.,

Page 29 of 49


620

41. Shinitzky, M.; Barenholz, Y., Fluidity parameters of lipid regions determined by

621

fluorescence polarization. Biochimica et biophysica acta 1978, 515, 367-94.

622

42. Tan, C.; Zhang, Y.; Abbas, S.; Feng, B.; Zhang, X.; Xia, W.; Xia, S.,

623

Biopolymer-Lipid Bilayer Interaction Modulates the Physical Properties of

624

Liposomes: Mechanism and Structure. Journal of Agricultural & Food Chemistry

625

2015, 63, 7277.

626

43. Gardikis, K.; Hatziantoniou, S.; Viras, K.; Demetzos, C., Effect of a bioactive

627

curcumin derivative on DPPC membrane: A DSC and Raman spectroscopy study.

628

Thermochimica Acta 2006, 447, 1-4.

629

44. Lo, Y. L.; Rahman, Y. E., Protein location in liposomes, a drug carrier: a

630

prediction by differential scanning calorimetry. J. Pharm. Sci. 1995, 84, 805-14.

631

45. Rolland, A.; Brzokewicz, A.; Shroot, B.; Jamoulle, J. C., Effect of penetration

632

enhancers

633

dipalmitoylphosphatidylcholine. A study by differential scanning calorimetry. Int. J.

634

Pharm. 1991, 76, 217-224.

635

46. Onyesom, I.; Lamprou, D. A.; Sygellou, L.; Owusu-Ware, S. K.; Antonijevic, M.;

636

Chowdhry, B. Z.; Douroumis, D., Sirolimus encapsulated liposomes for cancer

637

therapy: physicochemical and mechanical characterization of sirolimus distribution

638

within liposome bilayers. Mol. Pharm. 2013, 10, 4281-4293.

639

47. El Maghraby, G. M.; Williams, A. C.; Barry, B. W., Interactions of surfactants

640

(edge activators) and skin penetration enhancers with liposomes. International journal

641

of pharmaceutics 2004, 276, 143-61.

on

the

phase

transition

of

multilamellar


liposomes

of


Page 30 of 49

642

48. Bolean, M.; Simão, A. M.; Favarin, B. Z.; Millán, J. L.; Ciancaglini, P., The effect

643

of cholesterol on the reconstitution of alkaline phosphatase into liposomes. Biophys.

644

Chem. 2010, 152, 74–79.

645

49. Li, L.; ten Hagen, T. L.; Haeri, A.; Soullié, T.; Scholten, C.; Seynhaeve, A. L.;

646

Eggermont, A. M.; Koning, G. A., A novel two-step mild hyperthermia for advanced

647

liposomal chemotherapy. J. Control. Release 2014, 174, 202-208.

648

50. Lindner, L. H.; Eichhorn, M. E.; Eibl, H.; Teichert, N.; Schmitt-Sody, M.; Issels,

649

R. D.; Dellian, M., Novel temperature-sensitive liposomes with prolonged circulation

650

time. Clinical Cancer Research An Official Journal of the American Association for

651

Cancer Research 2004, 10, 2168-78.

652

51. Cheng, H. Y.; Randall, C. S.; Holl, W. W.; Constantinides, P. P.; Yue, T. L.;

653

Feuerstein, G. Z., Carvedilol-liposome interaction: evidence for strong association

654

with the hydrophobic region of the lipid bilayers. Biochimica Et Biophysica Acta 1996,

655

1284, 20-8.

656

52. Castelli,

657

Indomethacin-Dipalmitoylphosphatidylcholine Interaction. A Calorimetric Study of

658

Drug Release from Poly(Lactide-co-glycolide) Microspheres into Multilamellar

659

Vesicles. Drug Deliv. 1997, 4, 273.

660

53. Montenez, J. P.; Van, B. F.; Piret, J.; Schanck, A.; Brasseur, R.; Tulkens, P. M.;

661

Mingeot-Leclercq, M. P., Interaction of the macrolide azithromycin with

662

phospholipids. II. Biophysical and computer-aided conformational studies. European

663

Journal of Pharmacology 1996, 314, 215.

F.;

Conti,

B.;

Maccarrone,

D.;


Camera,

O.;

Conte,

U.,

Page 31 of 49


664

54. El Maghraby, G. M.; Williams, A. C.; Barry, B. W., Interactions of surfactants

665

(edge activators) and skin penetration enhancers with liposomes. Int. J. Pharm. 2004,

666

276, 143-61.

667

55. Bermudez, M.; Martinez, E.; Mora, M.; Sagrista, M. L.; de Madariaga, M. A.,

668

Molecular and physicochemical aspects of the interactions of the tuberculostatics

669

ofloxacin and rifampicin with liposomal bilayers: a P-31-NMR and DSC study.

670

Colloid Surf. A-Physicochem. Eng. Asp. 1999, 158, 59-66.

671

56. Grit, M.; Crommelin, D. J. A., The effect of aging on the physical stability of

672

liposome dispersions. Chemistry & Physics of Lipids 1992, 62, 113-122.

673

57. Hauser, H.; Phillips, M. C., Conformation of the lecithin polar group in charged

674

vesicles. Nature 1976, 261, 390-4.

675

58. Xynogalas, P.; Kanapitsas, A.; Constantinou-Kokotou, V.; Pissis, P.; Viras, K.,

676

Phase transitions in crystals of racemic long chain 2-amino alcohols. Chem. Phys.

677

Lipids 2005, 135, 83-92.

678

59. Tan, C.; Xue, J.; Eric, K.; Feng, B.; Zhang, X.; Xia, S., Dual effects of chitosan

679

decoration on the liposomal membrane physicochemical properties as affected by

680

chitosan concentration and molecular conformation. Journal of agricultural and food

681

chemistry 2013, 61, 6901-10.

682

60. Mendelsohn,

683

beta-carotene-3-3'diol) as a resonance Raman and visible absorption probe of

684

membrane structure. Biophysical journal 1979, 27, 221-35.

685

61. Gardikis, K.; Hatziantoniou, S.; Viras, K.; Wagner, M.; Demetzos, C., A DSC and

R.;

Van

Holten,

R.

W.,

Zeaxanthin


(

3R,3'R

-beta,


Page 32 of 49

686

Raman spectroscopy study on the effect of PAMAM dendrimer on DPPC model lipid

687

membranes. International journal of pharmaceutics 2006, 318, 118-23.

688

62. Craig, N. C.; Bryant, G. J.; Levin, I. W., Effects of halothane on

689

dipalmitoylphosphatidylcholine

690

Biochemistry 1987, 26, 2449-58.

691

63. O'Leary, T. J.; Ross, P. D.; Levin, I. W., Effects of anesthetic and nonanesthetic

692

steroids on dipalmitoylphosphatidylcholine liposomes: a calorimetric and Raman

693

spectroscopic investigation. Biochemistry 1984, 23, 4636-41.

liposomes:

a

Raman

694


spectroscopic

study.

Page 33 of 49


Figure Captions Fig. 1 DSC thermograms of liposomes with different DPPC/HSPC ratios (a, pure DPPC; b, DPPC: HSPC=9:1; c, DPPC: HSPC=8:2; d, DPPC: HSPC=6:4; e, DPPC: HSPC=5:5; f, pure HSPC). Fig. 2 DSC thermograms of liposomes (DPPC/HSPC=8:2) at varying β-ionone/lipid ratios (a, 0; b, 1:40; c, 1:20; d, 1:8; e, 1:4; f, 1:2; g, 1:1). Fig. 3 Changes in z-average diameter (ΔDz/%) of (A) pure liposomes and (B) β-ionone encapsulated liposomes with different drug/lipid ratios during storage at 4℃ in the dark for 80 d; Changes in the (C) retention rate (RR) of β-ionone liposomes with different drug/lipid ratios during storage at 4℃ in the dark for 80 d. Fig. 4 TEM morphology of (A) empty and (B) β-ionone loaded liposomes with the drug/lipid ratio of 1:8 (magnification 10000× and 30000×); the TEM images of (C-1) empty liposomes (magnification 80000×), (C-2) liposomes with drug/lipid ratio of 1:20 (magnification 100000×) and (C-3) liposomes with drug/lipid ratio of 1:8 (magnification 60000×) before (left) and after (right) storage for 4 weeks. Fig. 5 Microviscosity (η) in liposomes for ANS, TMA-DPH and DPH as a function of β-ionone/lipid ratios from 0 to 1:4. Fig. 6 Schematic representation of the main patterns about the localization of β-ionone molecules in liposomal vesicles. Fig. 7 Raman spectra in the range from (A) 2800 to 3000 cm−1 and (B) from 1000 to 1200 cm−1 of pure liposomes and liposomes loading with different β-ionone/lipid



ratios from 0 to 1:4. Fig. 8 The release curve of β-ionone loaded liposomes (A) at different incubation temperatures from 25 to 50℃; (B) with different β-ionone/lipid ratios of 1:20 and 1:8 at 42℃. Fig. 9 Relationship between fluorescence polarization of DPH and temperature for (A) pure liposomes and (B) liposomes with different β-ionone/lipid ratios of 1:20 and 1:8. Fig. 10 I2882/I2847 vs. temperature graph for liposomes at β-ionone/lipid ratios at (A) 1:8 and (B) 1:20; I1130/I1086 vs. temperature graph for liposomes at β-ionone/lipid ratios at (A) 1:8 and (B) 1:20.


Page 34 of 49

Page 35 of 49


Table Captions Table.1 Tm and △T1/2 of liposomes with different β-ionone/lipid ratios. PC composition (mol/mol)

80%DPPC+20%HSPC

β-ionone/lipid Tm(℃)

△T1/2(℃)

0

43.48±0.20a

1.80±0.12a

1:40

42.33±0.13b

2.40±0.09a,b

1:20

42.32±0.35c

2.76±0.21b

1:8

41.24±0.12d

3.52±0.18c

1:4

40.07±0.86e

5.09±0.07d

1:2

38.57±0.08e

5.60±0.92d

1:1

37.20±0.08f

7.28±0.30e

ratios (wt/wt)

Data are presented mean ±SD, n = 3. Different letters in the same row indicate significant difference (P < 0.05, Duncan analysis)



Page 36 of 49

Table.2 Phospholipid composition of liposome dispersions with different β-ionone/lipid ratios during storage. β-ionone/lipid ratios (wt/wt)

0

1:20

1:8

Peak Area

Storage time at 38℃ (d)

PC

LPC

0

1208

110

14

1194

122

28

1180

134

0

1232

117

14

1196

139

28

1148

168

0

1286

84

14

1247

123

28

1232

137


Page 37 of 49


Table.3 Order parameters of pure liposomes and liposomes with different β-ionone/lipid ratios and variations percent as deduced from Raman spectra in the range between 1000 to 1200 cm−1. β-ionone/lipid ICH2(I2882/ I2847)

SL

(SL-SL.0)/SL.0

0

1.0675

0.245

-

1:40

1.0852

0.2568

4.82%

1:20

1.0941

0.2627

7.24%

1:8

1.1396

0.2931

19.63%

1:4

1.0836

0.2557

4.38%

ratios (wt/wt)



Page 38 of 49

Table.4 Order parameters of pure liposomes and liposomes with different β-ionone/lipid ratios and variations percent as deduced from Raman spectra in the range between 2800 to 3000 cm−1. β-ionone/lipid IC-C(I1130/ I1086)

ST

(ST-ST.0)/ST.0

0

0.9374

0.5296

-

1:40

0.918

0.5186

-2.07%

1:20

0.9594

0.542

2.35%

1:8

0.9751

0.5509

4.02%

1:4

0.9681

0.5469

3.28%

ratios (wt/wt)


Page 39 of 49


Figure graphics

Fig. 1



Fig. 2


Page 40 of 49

Page 41 of 49


Fig. 3



Fig. 4 42 ACS Paragon Plus Environment

Page 42 of 49

Page 43 of 49


Fig. 5



Fig. 6


Page 44 of 49

Page 45 of 49


Fig. 7



Fig. 8


Page 46 of 49

Page 47 of 49


Fig. 9



Fig. 10


Page 48 of 49

Page 49 of 49


Graphic Abstract

Thermosensitive liposomes was fabricated to rapidly release the bioactive molecule β-ionone at certain temperature of 42℃


Characterizations on the Stability and Release Properties of Î²-ionone

Recommend Documents