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Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol

Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol
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  Characterization of   v esicles prepared with  v arious non-ionicsurfactants mixed with cholesterol Aranya Manosroi a,b, *, Pa v eena Wongtrakul c , Jiradej Manosroi a,b ,Hideki Sakai d,e , Fumio Sugawara d , Makoto Yuasa d,e , Masahiko Abe d,e a Pharmaceutical Cosmetic Raw Materials and Natural Products Research and De v elopment Center (PCRNC), Chiang Mai, Thailand  b Faculty of Pharmacy, Institute for Science and Technology Research and De v elopment, Chiang Mai Uni  v ersity, Chiang Mai 50200,Thailand  c Faculty of Pharmacy, Chiang Mai Uni  v ersity, Chiang Mai, Thailand  d Faculty of Science and Technology, Tokyo Uni  v ersity of Science, Noda, Chiba, Japan e Institute of Colloid and Interface Science, Tokyo Uni  v ersity of Science, Tokyo, Japan Recei v ed 6 February 2003; accepted 14 March 2003 Abstract The  v esicles (niosomes) prepared with hydrated mixture of   v arious non-ionic surfactants and cholesterol werestudied. The bilayer formation was characterized by X-cross formation under light polarization microscope and theability of the  v esicles to entrap water-soluble substance. Membrane rigidity was measured by means of mobility of fluorescence probe as a function of temperatures. The entrapment efficiencies of the  v esicles and micro v iscosities of the v esicular membrane depended on alkyl chain length of non-ionic surfactants and amount of cholesterol used to prepare v esicles. The stearyl chain (C 18 ) non-ionic surfactant  v esicles showed higher entrapment efficiency than the lauryl chain(C 12 ) non-ionic surfactant  v esicles. Cholesterol was used to complete the hydrophobic moiety of single alkyl chain non-ionic surfactants for  v esicle formation. Niosome prepared with Tween 61 bearing a long alkyl chain and a largehydrophilic moiety in the combination with cholesterol at 1:1 molar ratio was found to ha v e the highest entrapmentefficiency of water soluble substances. #  2003 Else v ier Science B.V. All rights reserved. Keywords:  Non-ionic surfactants, Niosomes; Micro v iscosity; Calcein; DPH 1. Introduction Liposomes prepared with a  v ariety of phospho-lipids were introduced in 1965 and ha v e beenextensi v ely studied as drug carriers and drugdeli v ery. The  v esicles are unilamellar or multi-lamellar spheroid structure composed of lipidmolecules assembled into bilayers. They can carryhydrophilic drugs by encapsulation in water phaseor hydrophobic drugs by intercalation them intohydrophobic domains. Liposomes ha v e been re-ported to increase drug stability, enhance thera- * Corresponding author. Tel.:  / 66-53-94-4338/89-4806; fax:  / 66-53-89-4169. E-mail address:  pmti005@chiangmai.ac.th (A. Manosroi).Colloids and Surfaces B: Biointerfaces 30 (2003) 129    / 138www.else v ier.com/locate/colsurfb0927-7765/03/$ - see front matter # 2003 Else v ier Science B.V. All rights reserved.doi:10.1016/S0927-7765(03)00080-8  peutic effects, prolong circulation time and pro-mote uptake of the entrapped drugs into target sitewhile drug toxicity is diminished [1    / 8]. They mayser v e as a solubilization matrix as local depot forsustained release or permeation enhancers of dermally acti v e compounds or as a rate-limitingmembrane barrier for the modulation of systemicabsorption of drugs  v ia the skin [9,10]. Howe v er,there are problems in the general applications of liposomes. In an aqueous system, liposomes ha v eproblems regarding degradation by hydrolysis of phospholipid molecules. Problems with the physi-cal and chemical stabilities of aqueous suspensionsof liposomes ha v e been addressed by many re-searchers who introduced a dry free-flowing gran-ular product that could be immediately hydratedbefore use. Although this is an impro v ement o v ercon v entional liposomes, a  v acuum and nitrogenatmosphere is still recommended during prepara-tion and storage to pre v ent oxidation of phospho-lipids. Therefore, the alternati v e substances areextensi v ely studied to prepare bilayer  v esiclesinstead of phospholipids in order to a v oid suchproblems.One alternati v e of phospholipids is the hydratedmixture of cholesterol and non-ionic surfactantssuch as alkyl ethers, alkyl esters or alkyl amidesnon-ionic surfactants [11    / 14]. This type of   v esicleformed from the abo v e mixture has been known asniosomes or non-ionic surfactant  v esicles. Thestructure and properties of niosomes are similarto those of liposomes. Both hydrophobic andhydrophilic substances can be embedded in nioso-mal  v esicles. The chemical stability as well as therelati v ely low cost of the materials used to prepareniosomes make this  v esicle more attracti v e thanliposomes for industrial productions both inpharmaceutical and cosmetic applications.Furthermore, there are large numbers of non-ionicsurfactants a v ailable for the design of   v esicles ondemand. This  v esicle has been reported to decreaseside effects, gi v e sustain release and enhancepenetration of the trapped substances throughskin. Se v eral mechanisms has been used to explainthe ability of niosomes to modulate drug transferthrough skin, e.g. (1) adsorption and fusion of niosomes on the surface of skin leading to highthermodynamic acti v ity gradient of drug at theinterface, which is the dri v ing force for permeationof lipophilic drug, (2) reduction of the barrierproperties of stratum corneum resulting from theproperty of   v esicles as a penetration enhancer [15].Many drugs such as estradiol [16,17], tretinoin[18], dithranol [10] and enoxacin [19] ha v e beensuccessfully encapsulated in niosomes for topicalapplication. Niosomes as deli v ery de v ices ha v ealso been studied with anticancer, anti-tubercular,anti-leishmanial, anti-inflammatory, hormonaldrugs and oral  v accine [20    / 28]. The encapsulationof drugs in niosomes can decrease drug toxicity,increase drug absorption and retard remo v al of drug from the circulation due to slow drug release.Howe v er, characteristics of   v esicles prepared withnon-ionic surfactants such as dispersion stability,particle size and microfluidity ha v e not yet beenmade clear. Most of the technical researches in thisarea pay attention to the formulations of drugs inthis  v esicle as a deli v ery system.In this study, the ability of se v eral non-ionicsurfactants mixed with cholesterol to form bilayer v esicles was studied. The  v esicle suspensions werecharacterized with optical microscope for mor-phology, light polarization microscope (LPM) forlamellar structure formation and thermal analysisfor the gel    / liquid transition temperature. Entrap-ment efficacy of fluorescence substance (calcein)was determined to confirm  v esicle formation andmicro v iscosity of membrane was measured tostudy packing structure of the  v esicular mem-brane. 2. Materials and methods  2.1. Materials Brij 30 (polyoxyethylene 4 laurylether), Brij 72(polyoxyethylene 2 stearylether), Span 60 (sorbitanmonostearate), Tween 61 (polyoxyethylene sorbi-tan monostearate), cholesterol were obtained fromSigma Chemical Co. Diglyceryl monolaurate,glyceryl distearate, glyceryl monostearate, Span80 (sorbitan monooleate), and tetraglyceryl mono-laurate were presents from Nikkol Company,Japan. Molecular formulae of these non-ionicsurfactants are shown in Table 1. 1,6 diphenyl- A. Manosroi et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 129    /  138 130  Table 1Vesicle formation ability, HLB  v alues, phase transition temperatures of   v arious non-ionic surfactants*Vesicle formation was determined by maltese cross formation under light polarization.  a HLB  v alues were gi v en by suppliers.**Phase transition temperatures of the unhydrated samples. A. Manosroi et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 129    /  138  131  1,3,5-hexatriene (DPH) and calcein were pur-chased from Wako Pure Chemical Industrial,Ltd, Japan. All other chemicals and sol v entswere of analytical grade.  2.2. Vesicle preparation The  v esicles were prepared by con v entionalchloroform film method [29]. The non-ionic sur-factants and cholesterol were dissol v ed in chloro-form, which was then e v aporated at 60  8 C under v acuum by rotary-e v aporator. The resulting sur-factant film was dried o v er night in desiccatorsunder  v acuum at room temperature (25 9 / 2  8 C).The obtained film was hydrated with PBS (phos-phate buffered saline) under mechanical agitationfor about 1 h at 60 9 / 1  8 C. The  v esicle formingability of all surfactants was in v estigated in theabsence and presence of different molar ratio of cholesterol. The bilayer  v esicle formation wasconfirmed by maltese cross formation under aLPM (type IMT2-NIC2, Olympus Optical Co.,Ltd, Japan) and the ability of the  v esicles to entrapwater-soluble substance. The shape of   v esicles wasobser v ed by using a differential interference op-tical microscope (type IMT2-NIC2, OlympusOptical Co., Ltd, Japan).  2.3. Characterization of   v esicles Vesicles prepared with different non-ionic sur-factants were studied in term of the ability of the v esicles to entrap water-soluble fluorescence sub-stance (calcein) and micro v iscosity of   v esicularmembrane.  2.3.1. Determination of entrapment efficiency The entrapment efficiency of a water-solublefluorescent marker (calcein) in niosomal  v esicleswas determined by spectrofluorophotometer (Shi-madzu, RF-5300PC, Japan). Calcein concentra-tion used to prepare niosomes was 0.1 mM. Theniosomal dispersions were diluted to 1:50000 withPBS, when the total fluorescence intensity wasmeasured ( I  total ). Calcein in the bulk aqueousphase was quenched by complexation with cobaltions using cobalt chloride. Then, the fluorescentintensity was measured ( I  in ). Subsequently, theniosomal membrane was ruptured by Triton X-100, while  I  tx  was consequently obtained. Theexcitation and emission wa v elengths were 490and 520 nm, respecti v ely. The entrapment efficacyof calcein was calculated according to the follow-ing equation: Entrapment efficiency (%)  I  in  I  tx  r  100 I  total  I  tx  r where r, the  v olume correction factor, was 1.04.  2.3.2. Determination of micro v iscosity of bilayermembrane DPH (1,6 diphenyl-1,3,5-hexatriene) was usedas a fluorescent probe. The solution of 1 mM DPHin tetrahydrofuran was added to niosomal disper-sion. The mixture was then incubated for 1 h at37 9 / 1  8 C. The molar ratio of surfactant/choles-terol to DPH was 300:1. The micro v iscosity of niosomal membrane was determined by fluores-cence polarization ( P  ), which can be calculatedaccording to the following equation. P   ( I  p  GI  v ) = ( I  p  GI  v ) where  I  p  and  I  v  were the fluorescence intensity of the emitted light polarized parallel and  v ertical tothe exciting light, respecti v ely, and  G   is the gratingcorrection factor. The fluorescence intensities  I  p and  I  v  were measured at  v arious temperatures withspectrofluorophotometer. The excitation andemission wa v elengths were 350 and 450 nm,respecti v ely. 3. Results and discussion 3.1. Niosome formation Table 1 shows HLB  v alues and phase transitiontemperatures of the non-ionic surfactants used inthis study. The non-ionic surfactant  v esicles wereprepared by a con v entional chloroform filmmethod, which has been generally used to prepareliposomes. Since cholesterol has been known tostabilize phospholipid-based  v esicles, effects of cholesterol addition to non-ionic surfactant sys-tems on niosome formation was also in v estigated. A. Manosroi et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 129    /  138 132  The total concentration of surfactant and choles-terol was adjusted to 20 mM. Similar to liposomalstructure, the hydrophobic parts of the surfactantsare shielded from water molecules and the hydro-philic head groups contact with water to obtainclosed bilayer structure. Niosome formation cap-ability of the non-ionic surfactants was alsosummarized in Table 1. Surfactants with a singlealkyl tail normally form micelles in diluted aqu-eous solutions. But, some non-ionic surfactantswith single alkyl tail in this study (Span 60, Brij 72,glyceryl monostearate) can form  v esicular struc-ture since they ha v e relati v ely large hydrophobicmoieties with low water solubility. Surfactantspreferably forming micelles need additi v es suchas cholesterol to achie v e suitable molecular geo-metry and hydrophobicity for bilayer  v esicle for-mation.Fig. 1(a) shows a differential interference opticalmicroscopic image of spherical  v esicles preparedwith 1:1 molar ratio of Span 60 and cholesterol.Large  v esicles with about 5  m m in diameter wereobser v ed. Fig. 1(b) is a light polarization micro-scopic image of the Span 60  v esicles. The X-crossimage obser v ed under polarization confirmed thelamellar structure of bilayer membrane.Without cholesterol,  v esicle formation was ob-ser v ed for only surfactants with stearyl chain (C 18 )such as Brij 72, glyceryl monostearate and Span 60at the concentration of 20 mM. Addition of cholesterol into these  v esicular suspensions leadsto an increase of physical stability (i.e. aggregatesize and dispersibility in aqueous solutions). Onthe other hand, the lauryl (C 12 ) chain surfactantssuch as Brij 30, diglyceryl monolaurate and tetra-glyceryl monolaurate, cannot form  v esicles with-out cholesterol. This must be attributed to thehigher HLB (hydrophilicity) and the smaller CPP(critical packing parameter) of the surfactantmolecules. Moreo v er, these surfactants can form v esicles only in the presence of suitable amounts of cholesterol. 3.2. Trapping efficiency of niosomes Fig. 2 shows the effects of Span 60/cholesterol in v arious molar ratios on entrapment efficiency of calcein. Intercalation of cholesterol in the bilayermembrane was reported to stabilize membrane butdecrease particle size of the  v esicles leading to the Fig. 1. Optical micrographs of niosomes prepared with 1:1molar ratio of   v arious non-ionic surfactants to cholesterol. (a)Span 60  v esicles, (b) Maltese formation of Span 60  v esicles, (c)tetraglyceryl monolaurate  v esicles. A. Manosroi et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 129    /  138  133
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