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Colloids and Surfaces B: Biointerfaces 95 (2012) 144– 153

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

j our na l ho me p age: www.elsev ier .com/ locate /co lsur fb

reparation of vesicle drug carrier from palm oil- and palm kernel oil-basedlycosides

urul Fadhilah Kamalul Aripina, Jae Won Parkb, Hyun Jin Parka,∗

School of Life Sciences and Biotechnology, Korea University, Seoul, South KoreaSeafood Research and Education Center, Oregon State University, United States

r t i c l e i n f o

rticle history:eceived 17 November 2011eceived in revised form 21 February 2012ccepted 21 February 2012vailable online 3 March 2012

eywords:iosomelycosideesicle

a b s t r a c t

A new mixture of alkyl glycosides derived from palm oil (PO) or palm kernel oil (PKO) was synthesised.This mixture contains glycosylated disaccharide of either maltose or lactose with aliphatic chain thatvaries according to the PO or PKO fatty acids composition. The synthesis method produced no poly-merised sugar unlike the production of the commercial glycosides (APG). The mixture only containsvarious glycosides differing by the alkyl chain and stereoisomers. Three anomeric mixtures can be pro-duced depending on the reaction time and catalyst: �-dominant mixture, �-dominant mixture and equalmixture. The PO and PKO derived glycosides were able to form a stable vesicle with a small amount ofdicetyl phosphate (DCP) and showed high vitamin E encapsulation efficiency. Low packing density ofthe membrane bilayer enabled more vitamin E to participate in the membrane formation. The anomeric

tereochemical effectalm oilalm kernel oil

mixtures of the maltosides provide no difference in membrane packing behaviour as it was governed bythe hydrophilic region. Significant difference in membrane packing density was observed for lactosidesanomeric mixtures because the packing behaviour was influenced by the hydrophobic region. Inclusionof cholesterol led to decrease in vitamin E encapsulation as well as reducing the stability of the vesicle sys-tem. The vesicular formulations of the glycosides were stable for 3 months when stored at refrigerationtemperature.

. Introduction

Niosomes have been studied extensively in recent years as drugarriers other than the liposomes. Non-ionic surfactants such asugar esters, glycosides and polyoxyethylene glycol can form lamel-ar phase at high water concentration, some with the help of lipiddditives. The closed lamellar phase is the typical formation of theesicles. Non-ionic surfactants provide a few advantages over thehospholipids because they are more economical and are chem-

cally more stable as they are not easily hydrolysed or oxidiseduring storage. Moreover, most commercial non-ionic surfactantsre relatively non-toxic: they are categorised as GRAS (generallyegarded as safe) and are listed in many pharmacopoeias such asuropean Pharmacopoeia 6.0 [1]. These advantages have led to theide application of non-ionic surfactants in food, cosmetic and

harmaceutical industries.

Niosomal formulations have been studied for drug delivery ofral and intravenous administrations as well as cutaneous and

∗ Corresponding author at: #217, College of Lifescience and Biotechnology (Westuilding), Korea University, 5-Ka, Anam-dong, Sungbuk-ku, Seoul 136-701,outh Korea. Tel.: +82 02 3290 4149; fax: +82 02 953 5892.

E-mail address: hjpark@korea.ac.kr (H.J. Park).

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2012.02.032

© 2012 Elsevier B.V. All rights reserved.

transcutaneous applications. However, the focus of research hasbeen on dermal and transdermal applications as niosomes haveproven potential for cosmetics. Studies showed that niosomes fromcommercial non-ionic surfactants e.g. ethylene oxide, sugar estersand glycosides can enhance drug delivery through the stratumcorneum, the impermeable barrier in skin [2,3].

Amongst the non-ionic surfactants, sugar esters are most com-monly used in the development of vesicular drug delivery systemsbecause they are inexpensive, commonly available and since notoxicity has been reported [4–9]. However, the recent develop-ment of drug delivery systems from glycosides has also capturedthe attention of the scientific community to produce novel glyco-sides and to study their properties [10–18]. Glycosides comprise ofsugar head group and aliphatic chain connected via ether linkage ismore stable compared to the ester linkage. In nature, glycosides orglycolipids are found as component of the cell membrane of livingorganisms such as plants, animals and microorganisms like bacteriaas well as fungi [19]. A carbohydrate head group allows cell recogni-tion and high specific interactions with the antibody receptor [20].Commercial glycosides known as alkyl polyglucosides (APG) com-

prised of a mixture of glucosides with a degree of polymerisationapproximately 1.5 sugar units. APG is considered a natural surfac-tant because it is synthesised from natural renewable resourcessuch as starch and vegetable oil. Thus, it is readily biodegradable

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nd according to the acute toxicity tests, APG is not toxic or harm-ul [21,22]. It also has high skin compatibility. Therefore, APG haseen used in the formulations of cosmetic and personal skin careroducts for many years.

Development of glycoside-based drug delivery systems was firsteported by Kiwada et al. [23]. Results of tissue distribution andharmacokinetics experiments have demonstrated that glycosidesre more effective drug carrier for liver, lung and kidney com-ared to the phosphotidylcholine. Vesicles of galactoside have highffinity towards the liver specifically in the binding of hepatocyteshrough specific galactose interactions. Manconi et al. conductedtudies on cutaneous delivery system of tretinoin using APG vesi-les and reported increased efficacy as compared to liposome [24].wo types of APG were studied, Oramix NS10 (HLB = 11) showedhe highest skin accumulation whereas more hydrophilic OramixG110 (HLB = 16) displayed high fluxes for better in vitro diffusionhrough the skin layers.

A new mixture of alkyl glycosides derived from palm oil (PO) andalm kernel oil (PKO) was synthesised. This mixture contains gly-osides that vary only on the alkyl chain moiety whereas the sugaread group remains the same with initial starting sugar as no poly-erisation of the sugar occurred. Disaccharides such as maltose and

actose were chosen based on economical reason and the availabil-ty of the material. Having a disaccharide as the hydrophilic moietyrovides better HLB for the glycosides to accommodate long alkylhains from plant resources.

PO and PKO have different fatty acid compositions. PO containslmost 50% monounsaturated and polyunsaturated derivativeshilst PKO is composed mostly of saturated fatty acids [25]. These

ils are the major resources of fatty acids. A previous study charac-erised the liquid crystal phases of PO and PKO derived glycosides26]. The targeted application for these glycosides is for vesicularrug delivery system as they form lamellar phases at low water con-entration. Since these glycosides are made from food materials andontain alkyl glycosides that are similar to APG, they are assumedo be non-toxic and biocompatible. Thus, preparation of vesicularystems from these glycosides for cutaneous drug delivery systemas investigated.

The aim of this research was to prepare vesicular formulationsrom PO and PKO based glycosides in order to study their per-ormance as drug carriers. The investigation covers the physicalharacteristic of the vesicles such as morphology, size, zeta poten-ial and membrane fluidity, as well as encapsulation efficiency andtorage stability. The main focus was to study the effect of thelycosides’ chemical structures (e.g. sugar head group, aliphaticixtures and stereochemical effect) on the vesicle properties.

he glycosylation method used to synthesise these glycosides canroduce different anomeric mixtures depending on the reactionime and catalyst. Formulation parameters were also investi-ated to achieve optimised conditions for the glycoside carrierystem.

. Materials and methods

.1. Materials

�-d-Lactose, d(+)-maltose monohydrate and boron trifluorideBF3) required for glycosides syntheses were purchased from Sigmaldrich (USA). PO was from processed palm oil (palm olein) andKO was obtained from Golden Jomalina Food Industries Sdn.hd. (Malaysia). Dicetyl phosphate (DCP), cholesterol and dl-

-tocopherol used in the vesicle preparations were purchased

rom Sigma Aldrich. The fluorescence probe, 1,6 diphenyl-1,3,5-exatriene (DPH) was also purchased from Sigma Aldrich. Solvents

or synthesis and vesicle preparation were AR grade and HPLC grade

: Biointerfaces 95 (2012) 144– 153 145

methanol was used in HPLC analyses which were all used withoutfurther purification.

2.2. Synthesis method

The glycosides were synthesised according to a previouslydescribed glycosylation procedure by Hashim et al. with minormodifications [27]. The synthesis method consisted of three steps;peracetylation, glycosylation and deacetylation. PO and PKO werereduced to alcohol using a general reduction method [28]. A sus-pension of lithium aluminium hydride (LiAlH4; 33 mmol) in 40 mldiethyl ether was stirred in a two-neck round bottom flask in aclosed condition. PO or PKO (22 mmol) in 30 ml diethyl ether wasadded slowly to the suspension through a dropping funnel to avoidvigorous reflux. Ethyl acetate followed by water was then addeddrop wise to deactivate excess LiAlH4 after the final addition ofthe oil. Heat produced from the reaction was cooled down by anice-water bath. The PO or PKO alcohol mixture was washed with100 ml cold aqueous sulphuric acid (10%) and the organic solventwas evaporated.

In order to have a selective glycosylation at the anomeric carbonas well as to activate it, hydroxyl groups of the sugar were per-acetylated [29,30]. Sodium acetate (121 mmol) and 100 ml aceticanhydride were stirred and heated to reflux. Sugar (58 mmol) wasadded in a small fraction to the hot suspension. Sugar and sodiumacetate will generate heat due to the exothermic reaction thereforecontinuous heating was not necessary. After all sugar was added,the solution was further heated at 120 ◦C for 1 h. The hot solutionwas poured into ice-water and stirred until sticky white solid wasformed. A white powder was obtained after several washes withdistilled water. Pure peracetylated sugar was recrystallised fromethanol.

Peracetylated sugar (15 mmol) and the alcohol (17 mmol) weredissolved in 60 ml dichloromethane and stirred in a closed appara-tus at room temperature. Boron trifluoride (18.2 mmol) was slowlyinjected into the solution and the glycosylation reaction took sev-eral hours to complete depending on the desired mixture. The�-dominant mixture took 48 h and �-dominant was 6 h, whilst theequal mixture took 24 h to complete. The reaction was stoppedby neutralising the solution with saturated sodium bicarbonatesolution and the organic layer was washed two times with water.Dichloromethane was evaporated and acetonitrile was added laterto the product. Hexane extractions were done a few times to com-pletely remove the excess alcohol. Acetonitrile was evaporatedfrom the peracetylated glycosides.

Finally, deacetylation step was performed by adding a cat-alytic amount of sodium methoxide to induce a basic mediuminto a methanolic solution of peracetylated glycosides (50 ml). Thesolution was stirred for 3 h and the progress of the reaction wasmonitored by TLC (thin layer chromatography). After the reactionwas completed, methanol was evaporated and the product waspurified from unreacted sugar with n-butanol and water extrac-tions. A small amount of diluted sulphuric acid was added toneutralise the excess sodium methoxide. N-butanol was evapo-rated and the product was dried in a vacuum oven at 50 ◦C for48 h.

2.3. Nuclear magnetic resonance (NMR)

PO and PKO based glycosides were characterised by 1H NMR.NMR spectra were recorded on Varian NMR Systems spectrom-

eter at 500 MHz. Methanol-d4 (CD3OD) was used in the samplepreparation. The maltosides were measured at room temperaturewhilst the lactosides required higher temperature of 55 ◦C due toinsolubility problem at lower temperature.

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PO maltoside (MPO): ı = 5.33–5.36 (m, 1.2H, CH ),.15–5.16 (d, 1H, H-1′′), 4.77 (d, �H-1, J = 3.8 Hz), 4.26 (d, �H-, J = 7.6 Hz), 3.20–3.92 ((m, bulk sugar signals), 2.76–2.79 (m, 0.1H,CH CH2 CH ), 2.01–2.07 (m, 2.5H, CH2 CH ), 1.53–1.64 (m,H, CH2 ), 1.29–1.32 (m, 23H, CH2 ), 0.90 (t, 3H, CH3).

PKO maltoside (MPKO): ı = 5.35 (m, 0.3H, CH ), 5.15 (m, 1H,-1′′), 4.77 (d, �H-1, J = 3.6 Hz), 4.27 (d, �H-1, J = 8.0 Hz), 3.20–3.90

m, bulk sugar signals), 2.02–2.07 (m, 0.6H, CH2 ), 1.53–1.64 (m,H, CH2 ), 1.29 (m, 20H, CH2 ), 0.90 (t, 3H, CH3).

PO lactoside (LPO): ı = 5.33–5.36 (m, 1H, CH ), 4.77 (d, �H-1, = 4.0 Hz), 4.37 (d, 1H, H-1′′), 4.28 (d, �H-1, J = 7.6 Hz), 3.25–3.89 (m,ulk sugar signals), 2.76–2.79 (m, 0.1H, CH CH2 CH ) 2.02–2.05m, 2H, CH2 ), 1.61–1.65 (m, 2H, CH2 ), 1.29–1.33 (m, 23H,CH2 ), 0.90 (t, 3H, CH3).

PKO lactoside (LPKO): ı = 5.35 (m, 0.2H, CH ), 4.77 (d, �H-, J = 3.6 Hz), 4.35–4.38 (m, 1H, H-1′′), 4.28 (d, �H-1, J = 8.0 Hz),.23–3.92 (m, bulk sugar signals), 2.02–2.04 (m, 0.5H, CH2 ),.60–1.65 (m, 2H, CH2 ), 1.29–1.32 (m, 20H, CH2 ), 0.90 (t, 3H,H3).

.4. Vesicle preparation

Glycosides vesicles were prepared by the thin film methodeported by Bangham et al. [31]. All formulations were preparedt 2 �mol/ml total lipid concentration except for higher total lipidoncentration formulations. The formulation consisted of glyco-ide, DCP and cholesterol. Ratio of glycosides to DCP was always:0.2 whilst cholesterol and vitamin E were added depending onhe investigated amount. First, the glycosides, DCP, cholesterol anditamin E were dissolved in a mixture of chloroform and methanol4:1, v/v). The solvent was then evaporated to form a thin film andried under vacuum condition for another hour to ensure com-lete removal of organic solvents. Later, the thin film was hydratedsing 25 ml deionised water in a shaking water bath at 55 ◦C (which

s above the transition temperature of the glycosides) for an hournd hydration period was extended to overnight at room tempera-ure. Finally, the vesicles were sonicated to obtain small unilamellaresicles (SUV) using a Sonics Vibra-Cell CV33 ultrasonic probe (Son-cs & Materials, USA) for 10 min (pulse on, 10 s; pulse off, 10 s) at50 W. All formulations were protected against light at all timesuring the preparation process.

.5. Transmission electron microscopy (TEM)

The morphology of the glycosides vesicles was observed using Tecnai G2 F30 microscope (Philips-FEI, Holland). The dispersedesicles were dropped onto a carbon film-covered copper grid. Thexcess dispersion was blotted off with filter paper and air-driedvernight. TEM studies were conducted at KBSI (Seoul).

.6. Size and zeta potential measurements

Vesicle average size, polydispersity index (PDI) and zeta poten-ial were determined by quasielastic laser light scattering using aetasizer ZEN 3600 Nano Series apparatus (ZEN, UK). The measure-ents were made with dilute solutions at 25 ◦C.

.7. Fluorescence anisotropy measurements

DPH loaded vesicles were prepared by adding a small amount ofPH solution in tetrahydrofuran into the mixture of glycosides andCP in chloroform and methanol (4:1, v/v). The molar ratio of gly-

osides to DPH was 200:1. The organic solvents were evaporatednd thin film was hydrated with water at 55 ◦C for 30 min. The for-ulation was protected from the light during preparation to avoid

egradation of DPH. Fluorescence anisotropy measurements were

: Biointerfaces 95 (2012) 144– 153

carried out using a Cary Eclipse spectrofluorometer (Varian, USA)with a manual polariser at room temperature (25 ◦C). The excita-tion and emission wavelengths were 360 and 429 nm respectively.Membrane fluidity of the vesicles were determined from the mea-surement of fluorescence anisotropy (r), which was calculated bythe following equation:

r = IVV − IVH

IVV + 2IVH

where IVV and IVH were the fluorescence intensity of the emit-ted light polarised parallel and perpendicular to the exciting light,respectively.

2.8. Determination of encapsulation efficiency

The vesicle formulations were purified by separating the freevitamin E using gel permeation chromatography (GPC) withSephadex G25. The vesicles were disrupted by adding isopropanol(sample to isopropanol, 1:1, v/v) into the formulation to extractthe vitamin E and samples were analysed using HPLC. Vitamin Ewas determined at 295 nm via reverse phase C18 column (MightysilRP-18 GP, 4.6 mm × 150 mm (5 �m), Kanto Chemical) operating atroom temperature on a Waters 2690 Separation Module (WatersAssociates, USA) and a Waters 996 photodiode Array Detector(Waters Associates). The mobile phase was 100% methanol at aflow rate of 1.0 ml/min. Different concentrations of vitamin E inmethanol were used to construct the calibration curve. Encapsula-tion efficiency was defined as:

encapsulation efficiency (%) = encapsulated vitamin E (mol)initial vitamin E (mol)

× 100

2.9. Storage stability studies

Glycosides vesicle formulations with and without cholesterolwere stored in the dark at 4 ± 1 ◦C and 25 ± 3 ◦C for 3 months.Released vitamin E was separated from the encapsulated vitaminE by GPC. The remaining vitamin E was measured with HPLC. HPLCsamples were prepared the same way as mentioned above.

2.10. Statistical analysis

Data analysis was performed using the software packageMicrosoft Excel, version 2007. Results are expressed as themean of three experiments ± S.D. Statistical data were ana-lysed using Student’s t-test with p ≥ 0.05 as a minimal level ofsignificance.

3. Results and discussion

3.1. Synthesis and characterisation of PO and PKO glycosides

PO and PKO based glycosides were synthesised using glycosyla-tion method adapted from the method used to synthesis branchedchain glycosides [27]. The synthesis route consists of three majorsteps: preparation of the starting materials (reduction of PO andPKO and peracetylation of sugar), glycosylation and deacetylation(Fig. 1). In this case, the purification step involving the separationof anomers using the chromatography technique was omitted. Byomitting the purification step, the costs of production could bereduced because chromatography consumes a lot of organic sol-vents.

Glycosylation of PO and PKO glycosides were done at a certainreaction time using boron trifluoride (BF3) as the catalyst. The reac-tion produced a mixture of � and � anomers which configurationdiffers at C1 carbon. Three types of mixtures could be obtained from

N.F.K. Aripin et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 144– 153 147

PO and PKO based glycosides.

tmmswt�ecab[

i�raniba

Fig. 1. Synthesis steps of

his synthesis method: �-dominant and �-dominant mixtures andixture containing equal amount of anomers (denoted as equalixtures). The ratio of � and � anomers was controlled by factors

uch as reaction time and type of catalyst because the anomersere obtained in different pathways. The � anomer formed from

he thermodynamic pathway required a strong catalyst, whilst the anomer is more kinetically favoured, can be produced by a mod-rately active catalyst. Tin tetrachloride (SnCl4) can also be used toatalyse the reaction as it produced more � anomers. However, thispproach is not propitious since it tends to react with the doubleond thus reducing the unsaturation degree of the PO glycosides26].

For a �-dominant mixture, a reaction time of 6–7 h gave a sat-sfying yield of 35–75%. An equal mixture took 24 h whereas an-dominant mixture needed a longer reaction time of 48 h. These

eaction times were applicable for both maltosides and lactosidespart from a slight difference on the �/� ratio of the � domi-

ant mixture. The �/� ratio was determined by comparing the

ntegration of nuclear magnetic resonance (NMR) spectra peak ofoth anomers. An � anomer showed a double peak at 4.77 ppmnd � anomer at 4.26–4.27 ppm (Fig. 2). Estimations were done

Fig. 2. NMR spectra showing integrations of � and � anomers peaks for a � dominantmixture.

1 faces B: Biointerfaces 95 (2012) 144– 153

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ith proper baseline and phase correction to reduce the systematicrror.

Quality of the product was checked with the NMR spec-ra. To ensure problems from starting material contaminationid not arise, the ratio of sugar to lipid chain was calculated.xcess alcohol was used during synthesis and was eliminated bycetonitrile–hexane extraction. Free alcohol elimination after theeacetylation of the sugar was impossible due to the polarity ofoth compounds is quite similar. Nevertheless, alcohol peaks wereot easily detected in the spectra. This was because most of theignals overlapped with the alkyl chain of the glycosides. Thus, thentegration of sugar peaks was compared to the CH2 peak.

Other information obtained from the NMR spectra were thensaturation degree and average alkyl chain length of the glyco-ides. Analyses from the spectra showed that no degradation ofhe double bond occurred during the synthesis. The unsaturationegree of PO and PKO derived glycosides was according to the initialmount found in the oil. The average alkyl chain for PO glycosidess estimated from the NMR spectra was 17 carbons and PKO glyco-ides consisted of 14 carbons. The results were in agreement withhe previous work [26].

.2. Vesicle formation, morphology and size

PO and PKO based glycosides formed vesicles by swelling thery surfactant mixture deposited in the round bottom flask afterhe evaporation of the organic solvents. This was done with theddition of water at an elevated temperature along with moderatehaking to ensure proper hydration. Hydration at higher tempera-ure was necessary because the glycosides have a high gel to liquidransition temperature in order to form a more fluid membrane. Theesicular formulations were then sonicated to obtain unilamellarnd smaller-sized vesicles.

Table 1 shows the characteristics of the vesicles for all glyco-ides. Mean size of the PO glycosides was relatively alike, whilsthe LPKO formed smaller vesicles than its maltoside counterpart.n this case, vesicles formed from lactosides or maltosides did notiffer much in terms of size. The mean sizes of octyl glucoside andctyl galactoside micelles were similar despite the use of differentugar head group [32]. Furthermore, even though PO had longerverage aliphatic chain, no significant difference in size was evi-ent (p > 0.05). This was probably due to the heterogeneity of theliphatic chains in the oil, since chain length can influence vesicleize of the sugar esters, as vesicles are larger with longer alkyl chain33]. The polydispersity index (PDI) indicated that all formulationsere reasonably multi-dispersed niosomes. The zeta potential of

he formulations showed more or less similar surface charges pro-ided that both sugars had a total of two sugar units.

All anomeric mixtures of PO and PKO lactosides formed similar-ized vesicles (Table 1). On the other hand, PO and PKO maltosideshowed a slight difference in size amongst the mixtures. Whilst-dominant and equal mixtures were similar, the �-dominantixture formed larger vesicles. In contrast, in the case of LPKOaltosides, � dominant mixture had larger vesicles than vesicles

ormed from equal and �-dominant mixtures’.Inclusion of cholesterol in a vesicle formulation is required to

orm a stable vesicle in terms of reduced permeability of waternto the vesicle. Preparation of PO maltoside without cholesterolemonstrated that the glycoside could not form the vesicles, but

nstead formed micelles. Based on the critical packing parameterCPP) theory, a surfactant tends to assemble as vesicle when the

PP value is 0.5–1 (a surfactant with CPP value more or less than theiven range will formed inverted and noninverted micelles respec-ively) [34]. The reported CPP value for tetradecyl maltosides is.877 [35].

Fig. 3. Effect of cholesterol on the vesicle size.

The effect of cholesterol addition on the vesicle sizes of the gly-cosides is shown in Fig. 3. MPO displayed similar sizes at mol ratioof cholesterol of 0.2 and 0.4. But as more cholesterol was added,sizes decreased markedly. As for MPKO vesicles, the size decreasedwith addition of cholesterol at mol ratio of 0.4 and 0.8. The samewas not found with the lactosides. Whilst LPO maintained similarvesicle size, LPKO had an increase in size as the cholesterol amountincreased. It is known that cholesterol can strengthen the packingassembly in the hydrophobic region by filling in the crevices causedby incompact surfactant packing. Therefore, in less packed mem-brane such as the maltosides, cholesterol provided close packingthus reducing the size. As for lactosides particularly LPKO whichhas a compact membrane assembly, addition of cholesterol dis-turbed the packing formed by the glycosides thus increasing thevesicle radius to establish a stable vesicle. A similar profile has beenobserved with Span 40 vesicles [36].

TEM observations (Fig. 4) showed that vesicles of all glyco-side formulations were nearly spherical. LPO displayed microscopicaggregations apart from the vesicles. LPO was the least stable for-mulations when it flocculated into large masses after 6 days. Sincethe process of preparing the TEM samples involved drying of themedium, the flocculation might happen faster than when in solu-tion. Drying also might have increased the vesicle sized marginallyfor all samples.

3.3. Membrane fluidity

Thermotropic liquid crystal studies have shown that packingdensity of the lipophilic region is mainly attributed to the stabil-ity of the glycosides’ liquid crystal phase [37]. A close or compactpacking requires stronger energy to disrupt the assembly, thusincreasing the thermal stability of the phase. In order to relatethis to the packing density of the vesicle membrane, membranefluidity was studied by the fluorescence anisotropy measurement.This method has been widely applied to monitor structural changesin synthetic membranes as well as biomembranes [38]. In theapproach, a fluorescence probe is embedded in the hydrophobicregion of the vesicle, allowing it to sense the fluidity gradient inthe bilayer membrane [39]. In this study, DPH was selected, as itis one of the most efficient probes for studying the fluidity in thehydrophobic region [40].

For disaccharide glycosides, �-anomers showed higher stabilitycompared to the anomers [41]. This is because an aliphatic � link-age makes the glycoside structure more linear, thus resembling a

rod shape which enables close packing assembly. �-Dominant mix-tures of LPKO showed a higher value of anisotropy compared to the�-dominant mixture (Fig. 5) implying that the former had morea rigid membrane due to a closer packing assembly within the

N.F.K. Aripin et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 144– 153 149

Table 1Mean size, polydispersity index (PDI) and zeta potential of the glycosides.

Mean size (nm) PDIa Zeta potentiala (mV)

� dominant Equal � dominant

MPOb 428 ± 49 356 ± 106 585 ± 97 0.44 ± 0.09 −49 ± 4MPKOc 454 ± 41 394 ± 37 455 ± 60 0.42 ± 0.03 −41 ± 1LPOd 497 ± 251 287 ± 101 356 ± 25 0.38 ± 0.05 −38 ± 3LPKOe 379 ± 80 340 ± 2 312 ± 39 0.41 ± 0.05 −44 ± 8

a PDI and zeta potential values were of equal formulations.b PO maltoside.c PKO maltoside.d PO lactoside.e PKO lactoside.

Fig. 4. TEM micrograph of the vesicle formulation

Fig. 5. Fluorescence anisotropy, r of various glycoside formulations differing sugarhead group, alkyl chain mixture and dominating anomers.

s (a) MPO, (b) MPKO, (c) LPO and (d) LPKO.

membrane. However, this was not the case with anomeric mix-tures of MPO, MPKO and LPO where no significant changesin the membrane fluidity between � and � dominant mix-tures (MPOa: 0.16 ± 0.01, MPOb: 0.16 ± 0.01; MPKOa: 0.14 ± 0.01,MPKOb: 0.12 ± 0.01; and LPOa: 0.29 ± 0.01, LPOb: 0.27 ± 0.01)(p > 0.05).

In order to explain this phenomenon, it is necessary to examinethe stereochemical effect of longer alkyl chain length. Stereochem-ical effect is reduced with longer alkyl chains’ maltosides. In onestudy, the difference in thermal stability decreased from 40 to 33 Kwhen comparing pure anomers of 12 and 14 carbon chain malto-sides [42]. Plus both anomeric mixtures formed similar lyotropicphases in water [26]. This can be further explained from the struc-tural perspective. A �1 → 4 linkage between two glucose units inthe sugar head group led to the bending of the molecular structure

and so form a less compact assembly for maltosides. Moreover, thepacking assemblies in the membrane bilayers has weaker intralayerhydrogen bonding and relies more on interlayer hydrogen bond-ing [42]. Therefore, having a � or � configuration for the aliphatic

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hain does not provide any significant changes in membraneexibility because the packing behaviour of the membrane isoverned mostly by the sugar moiety. Ribier and Handjani-Vilaoncluded that the fluidity is not only affected by the hydrophobicegion but by the hydrophilic region as well [43].

As for anomeric mixtures of LPO, the anisotropy profile can-ot be explained in the same way as the maltosides. Contrary toaltosides, lactosides display an increased difference in thermal

tability as alkyl chains lengthen [41]. Structure wise, lactose isore linear in shape due to the �1 → 4 linkage between the two

ugars. Therefore, a compact packing assembly can form. In addi-ion, the axial hydroxyl group at the C4 of galactose unit favoursntralayer hydrogen bonding. A previous study reported that theransition temperature for lactosides of the same alkyl chain isigher than for maltosides [30]. Anisotropy measurements indi-ate that the membrane of lactosides anomeric mixtures is lessuid than that of the maltosides. However, this does not explainhy LPO anomeric mixtures did not show differences between- and �-dominant mixtures. One way to look at it is the pack-

ng arrangement in the hydrophobic region. LPO is a PO-derivedlycoside, which contained high unsaturated alkyl chains. Vill ando-workers reported a decreased in the clearing temperature of annsaturated derivative of 18-carbon chain maltosides compared toaturated ones [41]. In one study, a difference in thermal stabilityas more apparent for palmitoyl lactosides than oleoyl lactosides

y more than 90 K [26]. This was due to the presence of a doubleond alkyl chain in the hydrophobic region created a disturbance

n the packing arrangement. It prevented close packing due to lowan der Waals interactions. Therefore in this case, although the �-nomer of the lactosides had a linear shape and was able to form alose packing, the presence of unsaturated component produced aacking behaviour similar to the bent-shaped �-anomer.

Looking at equal mixtures of the maltosides, the anisotropyalue for MPO was 0.19 ± 0.01 whilst that for MPKO was.13 ± 0.01. The same trend was observed for the lactosides, wherePKO (0.13 ± 0.01) had a higher membrane fluidity than LPO0.203 ± 0.005). For the equal mixtures, the packing behaviour wasikely influenced mainly by the hydrophobic region. Regardless ofhe sugar head group, glycosides derived from PO displayed higheruorescence anisotropy than the glycosides derived from PKO.espite having high amount of unsaturated compounds, PO basedlycosides managed to form a rigid membrane. This was due tohe chain length effect because PO consists of long aliphatic chainsith an average of 17 carbons. Another interesting observation is

hat the all three lactosides showed a distinct degree of membraneuidity greater than the maltosides. It seems that the impact ofynergistic effect was greater in lactosides.

.4. Encapsulation efficiency

In order to study the performance of these glycosides as drugarriers, vitamin E (�-tocopherol) was used as a model in encap-ulation of a hydrophobic compound. Encapsulation efficiency isn important parameter to determine the performance of a drugesicle. Factors such as surfactant type, formulation componentnd preparation method greatly influence the drug loading. In thisaper, the impacts of glycosides structure and formulation werevaluated.

All maltosides mixtures showed high encapsulation efficienciesFig. 6a). In spite of the dominating anomers and alkyl chain com-ositions, no significant differences were observed (p > 0.05). As for

actosides, the �-dominant formulations displayed higher encap-ulation efficiencies than the �-dominant formulations for both POnd PKO glycosides. These results indicate the hydrophobic vitamin

was encapsulated more readily within the hydrophobic region of

: Biointerfaces 95 (2012) 144– 153

membranes with a lower density and so could participate in theformation of vesicles membranes along with the glycosides.

The encapsulation efficiency results agreed with the membranefluidity results. Mixtures with high membrane fluidity have lowerpacking density therefore provide more space for vitamin E toparticipate in the membrane formation. A closely packed mem-brane, such as the �-dominant mixture of LPKO, will tend toexclude most of the vitamin E, leading to lowered encapsulationefficiency. In case of LPO, the �- and �-dominant mixtures didnot concur with the fluorescence anisotropy results. Although bothanomeric mixtures had similar membrane fluidities the encapsu-lation efficiencies were totally different. The �-dominant mixtureencapsulated only 38% of vitamin E, whereas encapsulation was100% for the �-dominant mixture. Despite high packing densityof the � dominant mixture, somehow the vitamin E was able toincorporate in the membrane as much as with the other mixtures.

Apart from the impact of the surfactant structure, vesicu-lar formulation preparation factors were also considered. Factorssuch as cholesterol amount, drug quantity and total lipidconcentration were observed in order to optimise encapsulationefficiency. Cholesterol and dicetyl phosphate were applied in theglycosides formulations to increase the stability of the vesicle bystabilising the membrane bilayers and to prevent the aggregationwithin the vesicle dispersions. Cholesterol has been reported toincrease the encapsulation efficiency of various hydrophilic drugsin vesicular formulations, as it reduces the permeability of the drugfrom the vesicle core [44–46]. However, for hydrophobic com-pounds, it was the opposite. Lipid additives like cholesterol anddicetyl phosphate occupied the hydrophobic region, thus compet-ing with vitamin E for packing space in the membrane explainingthe decreasing pattern as the cholesterol ratio increased for theglycosides (Fig. 6b). On the other hand, LPO showed a rather incon-sistent encapsulation efficiency trend with increasing addition ofcholesterol. A formulation devoid of cholesterol was selected forfurther investigations, as it provided the highest encapsulation effi-ciency.

MPO and MPKO showed increased encapsulation efficiency ofvitamin E up to a mol ratio of 1.0 (Fig. 6c). Decreased encapsulatedvitamin E was observed as the vitamin E ratio exceeded the gly-cosides ratio. In case of LPO, the encapsulation efficiency startedto drop with a 1:1 mol ratio of vitamin E to glycosides. Althoughthe efficiency decreased, the amount of encapsulated vitamin Ewas similar from ratio 0.75 to 1.25 showing that an optimal condi-tion has been achieved. The LPKO showed a similar encapsulationefficiency of 93–98% at a different ratio. This could mean thatLPKO is able to encapsulate more than a mol ratio 1.25. At a molratio of 0.75, all the glycosides formulations showed almost thesame amount of encapsulated vitamin E (MPO: 33 ± 2 �mol, MPKO:36.9 ± 0.7 �mol, LPO: 36 ± 3 �mol, LPKO: 37 ± 1 �mol).

The total lipid concentration was increased from previous con-centration of 2 �mol/ml to 6 �mol/ml to study the impact of totallipid concentration (Fig. 6d). Formulations with concentrationsbeyond this range were not favourable since the vesicles were eas-ily aggregated and precipitated. Even formulations of 4 �mol/mland 6 �mol/ml were also unstable for more than a week. All gly-cosides formulations produced high encapsulation efficiency at2 �mol/ml. MPO and MPKO displayed a decrease as the concen-tration increased. LPO showed decreased encapsulation efficiencyat 4 �mol/ml and a slightly increased efficiency at 6 �mol/ml. LPKOshowed similar results regardless of the concentration, with avitamin E encapsulation efficiency of 100%. Total lipid has beenreported to increase the encapsulation efficiency of flurbiprofen

in Span 60 [44]. However Yoshioka et al. observed no significantchanges in the amount of carboxyfluorescein encapsulated in Span80 [45]. But, in case of salicydic acid and p-hydroxyl benzoic acid inboth Span 60 and 80, an increased–decreased profile was reported

N.F.K. Aripin et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 144– 153 151

Fig. 6. Encapsulation efficiency of various glycosides’ vesicular formulations (a) different chemical structure, (b) cholesterol amount, (c) vitamin E amount and (d) total lipidconcentration.

Fw

ig. 7. Storage stability of the vesicles encapsulating the vitamin E for various glycosidesithout cholesterol at 25 ± 3 ◦C, (c) formulation prepared with cholesterol at 4 ± 1 ◦C and

, (a) formulation prepared without cholesterol at 4 ± 1 ◦C, (b) formulation prepared (d) formulation prepared with cholesterol at 25 ± 3 ◦C.

1 faces B

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awai

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52 N.F.K. Aripin et al. / Colloids and Sur

n another study [47] suggesting that the total lipid concentrationas a different effect depending on the drug’s chemical structure.

.5. Storage stability

The stability of the glycoside vesicles during storage wasbserved at a refrigerated temperature (4 ± 1 ◦C) and room tem-erature (25 ± 3 ◦C). Released vitamin E was separated using GPCnd the remaining vitamin E encapsulated within the vesicle wereuantified (Fig. 7). Two types of formulations were prepared for thetudy: vesicular formulations with and without cholesterol.

For formulations without cholesterol, all glycosides except LPOhowed a similar stability profile when stored at low temperatureFig. 7a). More than 90% of the vitamin E was retained inside the

PO, MPKO and LPKO vesicles. On contrary for LPO, large massesere observed after 6 days, indicating the instability of the system

hus the formulation was no longer observed. At room tempera-ure (25 ± 3 ◦C), all vesicles formulations became less stable. MPOnd LPKO vesicles released more 40% vitamin E after 3 monthstorage. However MPKO still remained with 83% encapsulated vita-in E. Room temperature storage also increase the instability of

iposomes [48].Inclusion of cholesterol in vesicular formulations will provide

ore compact packing that reduces the permeability of the drug.owever, based on the findings shown in Fig. 7c, vesicles of MPO,PKO and LPKO remained stable up to 22 days before more vita-in E was released. At the end of the study period, only about 50%

itamin E remained encapsulated. Storage at room temperatureccelerated the release of vitamin E, with decreased encapsulateditamin E after 9 days for MPO, MPKO and LPKO formulationsFig. 7d). Stability of LPO vesicles remained the same. From thebservation, inclusion of cholesterol somehow affected the struc-ural integrity of the vesicles.

Alamelu and Panduranga Rao reported that liposomes contain-ng unsaturated fatty acids can easily disintegrate compared to theiposomes constructed of saturated acids [49]. It was suggested thatnsaturated compounds led to degradation due to the atmosphericxidations occurred on the double bond. Since PO is comprised oflmost 50% unsaturated compounds, it could explain the lowestemaining vitamin E of MPO vesicles for all conditions. However, its uncertain whether the instability shown by these glycosides wasue to the same cause. Storage stability of various liposomes andiosomes systems also showed similar profile despite the fact thatpan 60 was used in this study [50].

. Conclusions

Presently, equal mixtures of all glycosides possessed the bestesicle properties. The glycosides had high encapsulation efficien-ies for vitamin E, provided by high membrane fluidity. The vesicleizes were comparable to commercial APG vesicles and the vesi-les were stable for at least 3 months when stored at refrigerationemperature.

Packing behaviour of the maltosides was governed by theydrophilic moieties. Therefore there was no significant difference

n vesicles criteria between the anomeric mixtures. However, in thease of lactosides, packing behaviour of the hydrophobic region wasreatly affected when a perturbation such as unsaturated compo-ent was present. PKO glycosides were a better carrier for vitamin

compared to the PO glycosides.Addition of cholesterol to vesicular formulations reduced the

mount of encapsulated vitamin E as it competed for packing spaceithin the membrane. In case of maltosides, cholesterol acted

s filler for a less compact packing, reducing vesicle sizes withncreasing amounts of cholesterol. However, the opposite effect

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: Biointerfaces 95 (2012) 144– 153

was evident for lactosides. A mol ratio of 1:1 (glycoside:vitaminE) was the optimal amount of vitamin E encapsulation for mostglycosides. High total lipid concentration is not recommended forvesicles preparations as it will decrease encapsulation efficiencyand reduce the stability of the system.

Acknowledgements

This work is supported by Korea University, Republic of Korea.We are very grateful to Prof. Choi Yong Seok (Korea University) forproviding the facilities for the synthesis process. We are also deeplygrateful to Chemistry Department, Faculty of Science, University ofMalaya, Malaysia for the access to the spectrofluorometer.

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