For Peer Review Only - University of Malaya · For Peer Review Only ... 23 CVC when mixing with the palmitoleic acid. ... (RES) and accumulated mostly in the liver and spleen within

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Effect of PEGylated lipid and Lecinol S-10 on physico-

chemical properties and encapsulation efficiency of

palmitoleic acid vesicles

Journal: Journal of Liposome Research

Manuscript ID: LLPR-2013-0046

Manuscript Type: Original Paper

Date Submitted by the Author: 11-Jul-2013

Complete List of Authors: Teo, Yin Yin; University Malaya,

Misran, Misni; University Malaya, Chemistry Low, Kah Hin; University Malaya, Chemistry

Keywords: palmitoleic acid, PEGylated lipid, Lecinol S-10

URL: http:/mc.manuscriptcentral.com/llpr Email: [email protected]

Journal of Liposome Research

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Effect of PEGylated lipid and Lecinol S-10 on physico-chemical properties 1

and encapsulation efficiency of palmitoleic acid vesicles 2

3

Yin Yin Teo*, Misni Misran and Kah Hin Low 4

5

Department of Chemistry, Faculty of Science, University Malaya 6

50603 Kuala Lumpur, Malaysia 7

8

Abstract: 9

10

The type of phospholipid plays an important role in the formation of fatty acid vesicle. 11

PEGylated lipid such as 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-12

[methoxy(polyethylene -glycol)-2000] (DPPE-PEG2000) as well as 1,2-dipalmitoyl-sn-13

glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene -glycol)-5000] (DPPE-PEG2000) 14

and Lecinol S-10 are mixed with palmitoleic acid to form vesicle. PEGylated lipid is a type 15

of phospholipid that covalently bonded to the polyethylene glycols (PEG) as a polar 16

headgroup whereas Lecinol S-10 is a mixture of phospholipid. The effect of polymerization 17

degree of PEGylated lipid on the formation of palmitoleic acid vesicle is studied. In addition, 18

the effect of Lecinol S-10 to the vesicle system is also investigated. Critical vesiculation 19

concentration (CVC) for the formation of palmitoleate-palmitoleic acid vesicle with 20

incorporation of PEGylated lipid is higher than the vesicle formed from pure palmitoleic acid. 21

In contrast to PEGylated lipid, Lecinol S-10 demonstrated a result that reduces the value of 22

CVC when mixing with the palmitoleic acid. Stability of vesicle suspension has been 23

examined by using particle size and zeta potential at 30 °C. DPPE-PEG2000 and Lecinol S-24

10 have effectively promoted stabilization for palmitoleate-palmitoleic acid vesicles. In 25

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contrast, DPPE-PEG5000 is unable to stabilize the palmitoleate-palmitoleic acid vesicle 26

suspension. It has been observed that magnitude of zeta potential is relatively lower in the 27

suspension of vesicle with the presence of Lecinol S-10 and/or PEGylated lipid. Although 28

some of the mixed vesicles formed were not very stable, they have displayed the potential in 29

encapsulating the active ingredients calcein. The results show that the encapsulation 30

efficiencies of calcein are of encouraging value. 31

32

Keywords: PEGylated lipid, Lecinol S-10, palmitoleic acid, vesicle, stability 33

34

Corresponding author: 35

[email protected] 36

Tel: +603 7967 7022 Ext 2546 37

Fax: +6 03 79674193 38

39

1.0 Introduction 40

Fatty acid vesicles were first successfully prepared by Gebicki and Hicks in 1973 as an 41

additional invention to the formation of vesicle from phospholipid. Although fatty acid 42

possess only a single hydrocarbon chain compare to phospholipid with two hydrocarbon 43

chains, this factor is not preventing the potential of fatty acid as a precursor for the formation 44

of vesicle. However, the vesicle formed from fatty acids is restricted to the matrix 45

environment such as pH, ionic composition of the solution and concentration of the fatty 46

acid(Haines, 1983) 47

. This is due to the formation of vesicle from fatty acid is related to the hydrogen bond 48

interaction between the carboxylate ion and carboxylic acid head group. Vesicles formed 49

from unsaturated fatty acid such as oleic acid (cis-9-octadecenoic acid) (Hargreaves and 50

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Deamer, 1978, Gebicki and Hicks, 1973), linoleic acid (cis,cis-9,12-octadecadienoic 51

acid)(Gebicki and Hicks, 1976) and cis-4,7,10,13,16,19-docosahexaenoic acid have been 52

reported elsewhere(Rogerson et al., 2006, Namani et al., 2007). Furthermore, vesicles may 53

also be prepared from saturated fatty acids such as decanoic acid(Namani and Walde, 2005). 54

Nevertheless, vesicles facing a drawback like other colloidal particles that is they are 55

tend to form aggregate. Besides, they are also rapidly cleared from the circulation by the 56

recticulo endothelial system (RES) and accumulated mostly in the liver and spleen within a 57

few minutes or a few hours(Lasic, 1995). Many attempts have been made in order to 58

overcome the above mentioned disadvantages. One of the attempts is to incorporate 59

PEGylated lipid in the formation of vesicles. PEGylated lipid is a type of phospholipid that 60

covalently bonded to the polyethylene glycols (PEG) as a polar headgroup. The efficiency in 61

prolonging the circulation time of PEGylated vesicles has been explained by steric repulsive 62

barrier around the vesicles(Torchilin et al., 1994). The successful formation of stable vesicle 63

suspension has promoted them to become vehicles for drug delivery due to their 64

biocompatibility and the possibility in loaded with both hydrophilic and hydrophobic 65

compounds. Thus far, information on the factors affecting the capability and the amount of 66

drug being loaded in the vesicle is still limited. Besides, the physicochemical properties of 67

vesicles have not much been explored. Therefore, in the present study, investigation on the 68

effect of two different types PEGylated lipid on palmitoleate-palmitoleic acid vesicles was 69

carried out. Their physicochemical properties were studied by means of loading efficiency, 70

particle size and zeta potential. In order to reduce the cost of production, Lecinol S-10 as a 71

mixture of phospholipid was used and their effect on the formation of vesicle was also 72

studied. 73

74

2.0 Experimental 75

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Palmitoleic acid (9-cis-hexadecenoic acid, ≥ 98.5 %), boric acid minimum 99.5 %, sodium 76

tetraborate minimum 99.5 % were purchased from Fluka (Buchs, Switzerland). 1,2-77

dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] 78

(DPPE-PEG2000) and 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-79

[methoxy(polyethylene glycol)-5000] (DPPE-PEG5000) were from Avanti Polar Lipids Inc. 80

(Alabama, USA). Hydrochloric acid, sodium hydroxide 98 % and chloroform of analytical 81

grade were purchased from HmBG Chemicals. Calcein ≥ 93 % were from Fluka. Lecinol S-82

10 was a gift from NIKKO company. All chemicals were used as received. Deionized water 83

with resistivity 18.2 MΩ cm was obtained from Barnstead NANOpure

® Diamond

TM ultrapure 84

water system. Deionized water was doubly distilled and deaerated with nitrogen gas prior to 85

use. 86

87

Preparation of stock solution 88

A stock solution of 12.5 mM palmitoleic acid and 27.5 mM NaOH was prepared by mixing 89

NaOH solution to palmitoleic acid and stirred until a transparent solution was obtained. On 90

the other hand, preparation of vesicle solution containing 1,2-dipalmitoyl-sn-glycerol-3-91

phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dipalmitoyl-sn-glycerol-92

3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] and Lecinol S-10 were done 93

by firstly mixed fatty acid with DPPE-PEG2000 or DPPE-PEG5000 in the mole ratio of 50 to 94

1 in a small amount of chloroform. The mixture solution was sonicated in order to dissolve 95

PEGylated lipid followed by remove of the chloroform under reduced pressure through rotary 96

evaporator. Gel like mixture were obtained and rehydrated with warm deionized water (50 °C) 97

and NaOH solution. Stock solution containing Lecinol S-10 were prepared as accordingly to 98

the above mentioned method with the amount of palmitoleic acid to Lecinol S-10 in the ratio 99

of 10 to 3 (w/w). 100

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101

Titration of the stock solution with HCl 102

A stock solution of 1.500 mL was pipetted into a 14.5 ml sample vial followed by addition of 103

(1.500 – x) mL deionized water and x mL HCl (0.125 M). The solution was mixed for 1 104

minute by vortex mixer Uzusio VTX 3000L then the pH of the solution was measured by a 105

Mettler Toledo pH meter. Calibration was performed at the titration temperature with buffer 106

of pH 4.0, 7.0 and 9.2. An average of 3 measurements was carried out. 107

108

Transmission electron microscopy 109

The images of vesicle at 40 mM were obtained by using Hitachi H-7100 transmission 110

electron microscope with negative-staining method. The sample was prepared by immersed 111

the formvar-coated copper grid into a drop of the vesicle solution and allowed to stand for 10 112

minutes. The excess vesicle solution was blotted with filter paper and followed by staining 113

process with 3% phosphotungstic acid. The grid was allowed to stand for another 10 minutes 114

and air dried. The specimens were viewed and photographed with a transmission electron 115

microscope operating at accelerating voltage 100 kV. 116

117

Critical vesiculation concentration (CVC) determinations 118

A series of solutions with different concentration of palmitoleic acid at pH 8.5 in 50 mM 119

borate buffer were prepared. The solutions were filter through Minisart®

NY nylon filter 120

(Germany) with diameter 25 mm and 0.2 µm pore size prior to the measurement of 121

equilibrium surface tension. The measurements were performed at 20.0 °C to 40.0 °C using a 122

tensiometer balance from KRUSS with K12 tensiometer processor by Du Nouÿ ring method. 123

In order to maintained the temperature of solutions, the solutions were thermal equilibrated in 124

a water bath with the desired temperature and a thermostated water circulation bath was 125

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connected to the vessel that surrounding the solutions while the measurement was going on. 126

Surface tension of water was (71.50 ± 0.05) mNm−1

. Measurements were repeated five times 127

and an average was calculated. 128

129

Particle size and zeta potential measurement 130

The vesicle solutions containing 2 mM fatty acid were first filtered through 0.20 µm nylon 131

membrane filter prior to the size measurement. All of the sample solutions were kept at room 132

temperature (28 °C) for the study of stability. Measurement of the mean diameter and zeta 133

potential were performed throughout a period of 30 days. The z-average diameter (the mean 134

hydrodynamic diameter based upon the intensity of the scattered light) of the vesicle was 135

measured by the dynamic light scattering method at 30 °C. Moreover, the electrophoretic 136

mobility of the vesicle was measured at 30 °C and the zeta potential of the vesicle solutions 137

ware calculated by applying Henry equation. Malvern NanoZS zetasizer from Malvern 138

Instruments Ltd. United Kingdom fitted with a 633 nm ‘red’ laser and the scattered light 139

detected at the angle of 173° was used for the measurement of particle size and zeta potential 140

of the vesicle solutions. 141

142

Encapsulation of calcein 143

Calcein at concentration of 3 mM were dissolved in 50 mM borate buffer pH 8.5 then added 144

into 25 mM fatty acid. The mixture solution was kept stirring until all of the fatty acid were 145

dissolved. The resulting mixture was then adjusted to pH 8.5 by 0.5 M NaOH and 0.5 M HCl 146

solution. The encapsulation method for fatty acid containing PEGylated lipid and/or Lecinol 147

S-10 was almost similar to the encapsulation of calcein in pure fatty acid vesicle. The only 148

difference being that the fatty acid with PEGylated lipid and/or Lecinol S-10 was firstly 149

dissolved in chloroform followed by removal of chloroform using rotary evaporator. This 150

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resulted in the formation of a viscous mixture. Then, borate buffer solution and deionized 151

water at ~ 50 °C containing calcein was added into the viscous mixture and stirred. The 152

resulting mixture solution was then adjusted to pH 8.5 by using NaOH (0.5 M) and HCl (0.5 153

M). Separation of the free calcein from those loaded in vesicles was achieved by gel 154

permeation chromatography method. Sepharose 4B as a solid phase was pretreated with the 155

appropriate fatty acid solution just above the CVC and packed in a glass column of 30 cm × 1 156

cm. 200 microlitre of the mixture solution was introduced into the column and every 2 mL of 157

eluate was collected followed by dilution with ethanol to a total volume of 5 mL. The 158

absorbance for both calcein loaded in vesicles and free calcein in the bulk medium were 159

measured by Varian Cary 50 UV-Vis spectrophotometer at 496 nm which is the maximum 160

absorbance for calcein(Namani et al., 2007). The loading efficiency was calculated from the 161

percentage value of absorbance at 496 nm for calcein loaded in vesicle over the absorbance 162

for total amount of calcein in the sample. 163

164

3.0 Results and discussions 165

Titration curve 166

The formation of palmitoleate-palmitoleic acid vesicle was first studied by acid-base titration 167

curve (Figure 1) while the result from here will be utilized in deciding the suitable pH for 168

CVC determination. A series of fully ionized palmitoleic acid solutions was achieved by 169

adding excess amount of sodium hydroxide to the palmitoleic acid followed by titration with 170

hydrochloric acid. At pH greater than pH 9.5, all of the solutions were observed as clear, 171

regardless the composition of solutions. This is an indication that all of the fatty acid 172

molecules were in the form of fully ionized and soluble in the solution. At an increased 173

concentration of hydrochloric acid with pH around 9, the solutions appeared to be slightly 174

turbid that arise from scattering of larger particle in the aqueous suspension. At this region, 175

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only small change of pH was observed with respect to the amount of hydrochloric acid. This 176

is due to buffering effect of fatty acid with the present of both ionized and non-ionized fatty 177

acid molecules in solution. It has been reported that vesicles were observed at the pH regions 178

that is close to pKa of fatty acids. At the pH range approximate to pKa, about half amount of 179

the corresponding acids are ionized and promote the formation of pseudo-double-chain 180

amphiphile through hydrogen bonding. This pseudo-double-chain amphiphile is smaller in 181

head group size compared to the apparent fatty acid monomer and brings to a more 182

cylindrical molecular shape that favoring the formation of bilayer and hence vesicles. Further 183

addition of hydrochloric acid into the solution results in the formation of milky solution and 184

oil droplet. This is due to the formation of emulsion and followed by phase separation 185

whereby all of carboxylate groups were protonated to carboxylic acid in the presence of 186

excess hydrochloric acid. 187

The trend of titration curves did not show a significantly change with the addition of 188

DPPE-PEG2000 or DPPE-PEG5000. On the other hand, addition of Lecinol S-10 to the 189

mixture solution reduces the amount of hydrochloric acid required in obtaining the buffering 190

region. In other words, Lecinol-S10 has the potential to act as an acid. As reported 191

elswhere(Bae et al., 2009), Lecinol S-10 contains 32 % of phosphatidylcholine, 31 % 192

phosphatidylethanolamine, 17 % phosphatidylinositosol and 9 % phosphatic acid. Therefore, 193

the quaternary ammonium ion may be possibly forming an ionic interaction with palmitoleate 194

ion or the negatively charged ion from the DPPE-PEG2000 or DPPE-PEG5000. As a result, 195

less hydrochloric acid is required on achievement of the buffering region. Altough the 196

different amount of hydrochloric acid required in achieving the buffering region, pH 8.5 was 197

selected in this studied as vesicles are found at this pH. 198

199

CVC determinations 200

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Critical vesiculation concentration (CVC) is a parameter that is being applied to measure the 201

ability of a substance to form vesicle with respect to the concentration of the substance. An 202

isotherm of surface tension (σ) as a function of logarithm concentration of fatty acid was 203

plotted to obtain the value of the critical vesiculation concentration (CVC). The CVC value is 204

calculated from the intersection point of the two straight lines that fitted the data points in the 205

range of concentration with a linear decrease of surface tension and the concentration region 206

where the surface tension are almost constant. Value of CVC at temperature range from (20.0 207

± 0.5) °C to (40.0 ± 0.5) °C for palmitoleate-palmitoleic acid solution with incorporation of 208

DPPE-PEG2000, DPPE-PEG5000 and/or Lecinol S-10 are shown in figure 2. The effect of 209

temperature on CVC was observed in the formation of vesicle from palmitoleic acid either 210

with or without incorporation of PEGylated lipid. We found that increase of temperature 211

results in higher CVC. This can be explained by the mobility of the molecule at higher 212

temperature is faster and this may hinder the aggregation of the molecule. Therefore, the 213

amount of molecules required to form vesicle is increase in order to enhance the probability 214

of aggregation. On the other hand, CVC for the formation of palmitoleate-palmitoleic acid 215

vesicle with incorporation of PEGylated lipid is higher than the vesicle formed from pure 216

palmitoleic acid. This is due to the present of negatively charged at phosphate head group 217

from the PEGylated lipid induces electrostatic repulsion among the palmitoleate-palmitoleic 218

acid and hence hindered the ability of the molecules aggregate into vesicles. Similarly, the 219

bulkier of the PEGylated lipid, the higher of the CVC as the tendency of the molecules to 220

aggregate is hindered. In contrast to PEGylated lipid, Lecinol S-10 demonstrated a result that 221

reduces the value of CVC when mixing with the palmitoleic acid. This phenomena implicates 222

that Lecinol S-10 promotes the formation of vesicle by reduce the electrostatic repulsion 223

effect among the head group through the positively charge ammonium group of the 224

phosphotidylcholine and phosphatidylethanolamine. As a result, although both PEGylated 225

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lipid and Lecinol S-10 compose of phospholipid, however, they display an oppositely role in 226

the formation of palmitoleate-palmitoleic acid vesicle. 227

228

Pariticle size and zeta potential 229

230

In this study, the stability of vesicle was accessed through their ability against 231

aggregation as shown in figure 3. Surprisingly, palmitoleate-palmitoleic acid vesicles manage 232

to maintain their vesicle size up to 28 days without incorporation of PEGylated lipid or 233

Lecinol S-10. This is obviously contrasted with the result obtained for vesicle formed from 234

oleate-oleic acid vesicle that had been carried out, however the result is not shown here. 235

Incoporation of DPPE-PEG2000, DPPE-PEG5000 and Lecinol S-10 have successfully 236

reduced the mean vesicle size. There is a significant reduction of vesicle size for vesicle 237

formed from mixture of palmitoleic acid and Lecinol S-10. The presence of Lecinol S-10 in 238

the formation of fatty acid vesicles causes suppression in the size of vesicles. This suggests a 239

coorperative participation of Lecinol S-10 in the formation of fatty acid bilayer. The 240

reduction in particle size of vesicle is directly related to the curvature principle of the bilayer. 241

Lower bilayer curvature will form larger size vesicles whereas higher bilayer curvature will 242

result in smaller size vesicles. The spontaneous curvature of a bilayer is determined by the 243

uneven distribution of composition in both the inner leaflet and outer leaflet of the bilayer. If 244

both layers are symmetric either physically or chemically, the resulting spontaneous 245

curvature is equal to zero. Therefore, bilayer with only one component cannot result in a non-246

zero spontaneous curvature. However, spontaneous curvature may exist in the environment 247

with asymmetry distribution of composition in a bilayer(Safran et al., 1990). In our study, 248

Lecinol S-10 with the major component composing of 39 % PC and 38 % PE that attached to 249

two long saturated hydrocarbon fatty acid chains may distribute asymmetrically in the bilayer 250

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of fatty acid vesicle. This is due to the properties of PE with an inverted cone shape may pack 251

efficiently in the inner monolayer. Therefore, it has a higher preferential to occupy the inner 252

leaflet of the bilayer. As a result, the curvature difference between the two monolayers is 253

increased compared to the system composing pure fatty acid thus favoring the formation of 254

smaller size vesicle(Risselada and Marrink, 2009) 255

The plausible reason for the formation of smaller size vesicles is due to the presence 256

of long PEG chain that is more dynamic in an aqueous solution. As a result, the effective 257

head group area is larger and results in smaller packing parameter compared to the packing 258

parameter for vesicle formed from pure fatty acid molecules. However, the reduced value of 259

packing parameter is expected to be within the value for formation of vesicle that is greater 260

than 0.5 but smaller than 1. Therefore, formation of vesicle with high bilayer curvature is 261

expected for packing parameter that reduced to the value approximately 0.5. Besides, the 262

formation of smaller size vesicle with incorporation of PEGylated lipid is intentionally to 263

decrease the steric repulsion force between the head group. This is due to the presence of 264

bulky polyethoxylate group at the surface of the bilayer has induced an extensive hydration 265

around the polar head group that cause steric repulsion among the polyethoxylate group. In 266

order to reduce the degree of repulsion force, an area expansion was obtained by bending the 267

bilayer into a higher degree of curvature(Sriwongsitanont and Ueno, 2004). Therefore, in 268

view of the above mentioned explanation, DPPE-PEG5000 with bulkier PEG tends to form 269

smaller size vesicles compared to DPPE-PEG2000. Similar results were observed for ternary 270

mixture of fatty acid, DPPE-PEG2000 and Lecinol S-10. In contrary, ternary mixture of C18 271

fatty acid, DPPE-PEG5000 and Lesinol S-10 demonstrated larger size of vesicle compared to 272

those in their binary mixed system. This might be owing to the structural incompatibility 273

between palmitoleic acid with the bulky DPPE-PEG5000 and Lecinol S-10. 274

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It has been observed that DPPE-PEG2000 has effectively promoted stabilization to 275

certain extend for palmitoleate-palmitoleic acid vesicles. However, incorporation of DPPE-276

PEG5000 into palmitoleate-palmitoleic acid vesicle only manages to stabilize the vesicle 277

system for less than 8 days. The vesicle size increased after day 8 which may be due to the 278

process of aggregation and this is further confirmed from the decrease of magnitude of zeta 279

potential. On the other hand, DPPE-PEG2000 was added into the binary system of 280

palmitoleate-palmitoleic acid and Lecinol S-10. The results were similar to those in binary 281

mixture of palmitoleate-palmitoleic acid and Lecinol S-10 but with formation of smaller size 282

vesicle. Similarly, addition of DPPE-PEG5000 into the binary mixture of palmitoleate-283

palmitoleic acid and Lecinol S-10 also results in a smaller size vesicle. However, limited 284

stability is observed for this ternary mixed fatty acid vesicle. This may be due to formation of 285

flocs in the solution as can be observed through polarizing microscope in figure 4. In addition, 286

polydispersity index for vesicle prepared from ternary mixture were found increase from ~0.3 287

to ~0.6 throughout the storage time. 288

A relatively lower magnitude of zeta potential is observed for fatty acid vesicles in the 289

presence of Lecinol S-10 compare to pure fatty acid vesicles as shown in figure 5. Similar 290

result is also found in the mixture of fatty acid vesicle made up of PEGylated lipid and 291

mixture of PEGylated lipid-Lecinol S-10. Although both Lecinol S-10 and PEGylated lipid 292

do reduce the magnitude of zeta potentials, the reduction of zeta potential to a lesser negative 293

value is much more pronounce for vesicle containing PEGylated lipid. There are two 294

possibilities that may explain this phenomenon. One of the reasons could probably due to a 295

long and bulky polyethoxylate group that is coating around the vesicles surface. This in turns 296

also cause slower vesicle mobility and hence lower their zeta potential(Sakai et al., 2002, 297

Heurtault et al., 2003, Centis and Vermette, 2008). Another possible explanation is that the 298

presence of bulky polyethoxylate group with different degree of polymerization, choline and 299

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inositol group from Lecinol S-10 have effectively shielded the negatively charge on the 300

surface of vesicle. Therefore, extension of these bulky groups from the surface of vesicle has 301

shifted the shear plane far apart from the surface of vesicle and results in a lower magnitude 302

in zeta potential (Woodle et al., 1992). On the other hand, magnitudes of zeta potential for 303

vesicle prepared from ternary mixture were found higher than those from binary mixture. The 304

plausible reason is the interaction between choline group, inositol group with polyethoxylate 305

group could reduce the distance of shear plane from the vesicle surface. As shown from our 306

findings, we can deduce that Lecinol S-10 and PEGylated lipid have shown cooperative 307

participation in the bilayer formation. The overall trend in zeta potential were found 308

consistent for both of the fatty acid vesicle suspensions either in the pure system or binary 309

mixture with Lecinol S-10, DPPE-PEG2000 or their ternary mixture. However, a gradual 310

decrease in the magnitude of zeta potential is shown in the vesicle system containing 311

palmitoleate-palmitoleic acid-DPPE-PEG-5000 as well as in the ternary mixture of 312

palmitoleate-palmitoleic acid-DPPE-PEG5000-Lecinol S-10. We can deduce from the result 313

of zeta potential that addition of DPPE-PEG2000 or Lecinol S-10 into the formation of fatty 314

acid vesicle help in stabilization of the vesicle system. On the other hand, addition of DPPE-315

PEG5000 with higher degree of polymerization at polyethoxylate chain into the vesicle 316

system may render destabilization of the vesicle. The results obtained from zeta potential 317

measurements are also in agreement with those found in the particle size measurements. 318

Transmission electron microscopy technique has been the best available option to 319

observe the presence of vesicles in the solution. This is due to most of the vesicles that have 320

been produced by our method were well below 500 nm which could not be viewed using 321

normal light or polarizing microscope. TEM micrograph of palmitoleate-palmitoleic acid 322

vesicles at 40 mM are shown in figure 6. Most of the vesicles were present on a bright 323

background as spherical rings although some oval in shape or oil droplets. Deeper colour is 324

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observed at region inside the ring compared to the background. Observation of oil stain in the 325

images of vesicle is possibly related to the elasticity of the membrane bilayer in vesicles. As a 326

particle is exposed to an environment of very low pressure, the shearing effect may occur. If 327

the bilayer were high in elasticity, it remains unperturbed by the vacuum condition. 328

Nonetheless, collapse of the bilayer could also be observed as oil droplet stains arising from 329

collapse of vesicles with low bilayer elasticity. 330

The results showed that encapsulation efficiency of palmitoleate-palmitoleic acid 331

vesicles is higher compared to those with incorporation of Lecinol S-10 and/or PEGylated 332

lipids. This is possibly due to small trapping volume in PEGylated vesicle with their smaller 333

hydrodynamic size. As reported elsewhere, the size of vesicle is significantly correlated with 334

its loading efficiency(Nii et al., 2003). Another possible reason is that palmitoleic acid 335

leading to a close packing of the bilayer that results in fewer leakage of calcein, while 336

inclusion of Lecinol S-10 in the formation of vesicle may possibly affect the packing 337

geometry in bilayer and cause a reduction in the loading efficiency compared to the pure fatty 338

acid vesicle. Although different composition of vesicle displays variation in calcein loading 339

efficiency, an average of 10 % calcein was loaded in the prepared vesicles. The loading 340

efficiencies of calcein in vesicles may vary from 0.1 % to 39.5 %(Memoli et al., 1994, 341

Manosroi et al., 2003, Bahia et al., 2010, Liu, 2010). 342

343

4.0 Conclusions 344

345

The present work reported the effect of Lecinol S-10 and PEGylated lipid on 346

palmitoleic acid vesicle. Although both are from the group of phospholipid, their effect on the 347

physico-chemical properties of palmitoleate-palmitoleic acid vesicle is varied. Additionally, 348

the bulkiness of the PEGylated lipid is also found to influence the stability of the vesicle 349

(c) (a)

OS

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formed. CVC for the formation of palmitoleate-palmitoleic acid vesicle with incorporation of 350

PEGylated lipid is higher than the vesicle formed from pure palmitoleic acid. The bulkier of 351

the PEGylated lipid, the higher of the CVC as the tendency of the molecules to aggregate is 352

hindered. Lecinol S-10 promotes the formation of vesicle by reduce the electrostatic 353

repulsion effect among the head group result therefore it reduces the value of CVC. The 354

presence of DPPE-PEG2000 or Lecinol S-10 in the palmitoleate-palmitoleic acid has 355

effectively promoted stabilization to palmitoleate-palmitoleic acid vesicles compared to 356

DPPE-PEG5000. 357

358

Acknowledgements 359

This research work was supported by the Department of Higher Education Malaysia under 360

the Fundamental Research Grant Scheme (FRGS) with project number FP001-2013A. 361

362

Declaration of interest 363

There are no known conflicts of interest in connection with this work. 364

365

References 366

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Haines, T. H. (1983). Anionic lipid headgroups as a proton-conducting pathway along the 384

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873x674mm (96 x 96 DPI)

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286x201mm (150 x 150 DPI)

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873x674mm (96 x 96 DPI)

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Optical polarizing micrograph of 2 mM palmitoleate-palmitoleic acid-DPPE-PEG5000-Lecinol S-10 vesicle at pH 8.5 after 30 days of storage

202x151mm (96 x 96 DPI)

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Mean zeta potential of palmitoleate-palmitoleic acid vesicle with their mixture for a period of 30 days at 30 °C. pure fatty acid, ⊞ FA+Lecinol S-10, FA + DPPE-PEG2000, FA + DPPE-PEG2000 + Lecinol S-10,

FA + DPPE-PEG5000, FA+ DPPE-PEG5000 + Lecinol S-10. Error bars indicate the 95% confidence

interval.

873x674mm (96 x 96 DPI)

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TEM micrographs of (a) palmitoleate-palmitoleic acid vesicle, (b) palmitoleate-palmitoleic acid-DPPE-PEG2000 vesicle (c) palmitoleate-palmitoleic acid-DPPE-PEG5000

99x108mm (96 x 96 DPI)

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Table 1. Loading efficiency of 3 mM calcein in 25 mM palmitoleate-palmitoleic acid vesicle

and their mixtures at pH 8.5.

Composition of

palmitoleate-palmitoleic

acid vesicle

Loading efficiency, %

Calcein

Palmitoleic acid 36 ± 1

Lecinol S-10 12 ± 1

DPPE-PEG2000 12 ± 1

Lecinol S-10 + DPPE-

PEG2000

7 ± 1

DPPE-PEG5000 4 ± 1

Lecinol S-10 + DPPE-

PEG5000

2 ± 1

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List of figures 1

Figure 1. Equilibrium titration curve for (12.50 ± 0.05) mM palmitoleic acid with their 2

mixture solutions at 28 °C. pure fatty acid, ⊞ FA+Lecinol S-10, FA + DPPE-PEG2000, 3

FA + DPPE-PEG2000 + Lecinol S-10, FA + DPPE-PEG5000, FA+ DPPE-PEG5000 4

+ Lecinol S-10. 5

6

Figure 2. CVC of palmitoleate-palmitoleic acid and their mixtures at temperature 20 ºC to 40 7

ºC. pure fatty acid, ⊞ FA+Lecinol S-10, FA + DPPE-PEG2000, FA + DPPE-PEG2000 8

+ Lecinol S-10, FA + DPPE-PEG5000, FA+ DPPE-PEG5000 + Lecinol S-10. 9

10

Figure 3. Mean particle size of palmitoleate-palmitoleic acid vesicle with their mixture for a 11

period of 30 days at 30 °C. pure fatty acid, ⊞ FA+Lecinol S-10, FA + DPPE-PEG2000, 12

FA + DPPE-PEG2000 + Lecinol S-10, FA + DPPE-PEG5000, FA+ DPPE-PEG5000 + 13

Lecinol S-10. Error bars indicate the 95% confidence interval. 14

15

Figure 4. Optical polarizing micrograph of 2 mM palmitoleate-palmitoleic acid-DPPE-16

PEG5000-Lecinol S-10 vesicle at pH 8.5 after 30 days of storage 17

18

Figure 5. Mean zeta potential of palmitoleate-palmitoleic acid vesicle with their mixture for 19

a period of 30 days at 30 °C. pure fatty acid, ⊞ FA+Lecinol S-10, FA + DPPE-PEG2000, 20

FA + DPPE-PEG2000 + Lecinol S-10, FA + DPPE-PEG5000, FA+ DPPE-PEG5000 21

+ Lecinol S-10. Error bars indicate the 95% confidence interval. 22

23

Figure 6. TEM micrographs of (a) palmitoleate-palmitoleic acid vesicle, (b) palmitoleate-24

palmitoleic acid-DPPE-PEG2000 vesicle (c) palmitoleate-palmitoleic acid-DPPE-PEG5000 25

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vesicle and (d) palmitoleate-palmitoleic acid-Lecinol S-10-DPPE-PEG2000 vesicle. Arrows 26

indicate the vesicle, OS = oil droplet stain. 27

28

29

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For Peer Review Only - University of Malaya · For Peer Review Only ... 23 CVC when mixing with the palmitoleic acid. ... (RES) and accumulated mostly in the liver and spleen within