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A new crown ether as vesicular carrier for 5-fluoruracil: Synthesis, characterization and drug delivery evaluation

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Niosomes have shown promise as cheap and chemically stable drug delivery systems. In this paper a novel crown ether amphiphile, 1,16-hexadecanoyl-bis-(2-aminomethyl)-18-crown-6 (Bola A-16), has been synthesized with the aim of developing a long time
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  Colloids and Surfaces B: Biointerfaces 58 (2007) 197–202 A new crown ether as vesicular carrier for 5-fluoruracil:Synthesis, characterization and drug delivery evaluation Rita Muzzalupo ∗ , Fiore Pasquale Nicoletta, Sonia Trombino,Roberta Cassano, Francesca Iemma, Nevio Picci  Dipartimento di Scienze Farmaceutiche, Universit`a della Calabria, Via P. Bucci, Ed. Polifunzionale, Arcavacata di Rende, 87030 Rende, CS, Italy Received 19 February 2007; received in revised form 9 March 2007; accepted 9 March 2007Available online 14 March 2007 Abstract Niosomes have shown promise as cheap and chemically stable drug delivery systems. In this paper a novel crown ether amphiphile, 1,16-hexadecanoyl-bis-(2-aminomethyl)-18-crown-6 (Bola A-16), has been synthesized with the aim of developing a long time stable controlledrelease system. Niosomes have been prepared with different molar ratios of amphiphile and cholesterol and their morphological properties havebeen determined by quasi-elastic light scattering and transmission electron microscopy. The composition of niosomes affects the entrapmentefficiency and the release rate of 5-fluorouracil, a well-known antineoplastic molecule. In addition, other two known azacrown ether amphiphiles(4,7,10,13-pentaoxa-16-aza-cyclooctadecane)-hexadecanedioc acid diamide (Bola D-16) and  ,  -(4,7,10,13-pentaoxa-16-aza-cyclooctadecane)-hexadecane (Bola C-16), have been synthesized and the obtained vesicles have been characterized for comparison. Furthermore, the release profileof 5-fluorouracil  in vitro , from these niosomes, has been studied over a period of 6h in order to simulate a hematic adsorption.© 2007 Elsevier B.V. All rights reserved. Keywords:  Niosomes; Crown ether; Drug delivery; 5-Fluorouracil;  In vitro  delivery 1. Introduction Colloidal drug carrier systems such as micellar solutions,vesicles, liquid crystal dispersions and nanoparticles all seemto be promising drug delivery systems (DDSs) and have ledto increasing attention over the past 20 years. In particular,vesicular systems (liposomes and niosomes) play an increas-ing important role since they can be used as membrane models,in chemical reactivity studies, or in drug delivery and tar-geting [1,2]. Niosomes are formed by the self-assembly of  non-ionic amphiphiles in aqueous media resulting in closedbilayer structures. An increasing number of non-ionic surfac-tants has been found capable of entrapping hydrophilic andhydrophobic solutes [3]. The greater stability, lower cost of sur- factants and less storage problems [4–6] have prompted for the exploitation of these vesicles as an alternative to phospholipidones. ∗ Corresponding author. Tel.: +39 0984493173; fax: +39 0984493298.  E-mail address:  rita.muzzalupo@unical.it (R. Muzzalupo). Liposomesandniosomeshavebeenverysuccessfullyappliedfor cosmetic purposes [7,8] and experimentally evaluated ascarriers of many antiblastic drugs, such as metotrexate, dox-orubicin and cisplatin [9–11], glucocorticoid [12], hemoglobin [13], indomethacin [14] and the antipsoriatic dithranol [15]. The encapsulation of pharmaceutical materials in niosomes candecrease drug toxicity, increase drug absorption, stability oractivity and retard its removal from the circulation in the caseof slow release [16–18].Crown ethers belong to the class of macrocycles and pos-sess an outstanding ability to complex ions and small organicmolecules, and to behave as ionophores capable of transportingionsacrosslipophilicbarriers.Typicalapplicationsareprovidedbydevicesfortheextractionandseparationofheavymetalsfromaqueous solutions and for the stabilization of metal cations inorganic media and in crown ether-based sensors. The ability of crown ethers to bind metal cations depends on the cavity size,on the nature of heteroatoms (oxygen, nitrogen or sulphur), onthe kind of substituents in the macrocycle, and on the particularused solvent [19]. There is also growing interest in the use of  crown ethers for their antitumor activity [20]. 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2007.03.010  198  R. Muzzalupo et al. / Colloids and Surfaces B: Biointerfaces 58 (2007) 197–202 Fig. 1. Chemical structure of bolaform surfactants. The addition of a lipophilic long chain alkyl group to ahydrophiliccrownetherresultsintheformationofacrownether-based surfactant, known as bolaform, that can form micellesor more complex supramolecular structures in water [21,22],similarly to the common non-ionic surfactants [23].The production of new compounds that possess, in the samemolecular structure, a hydrophilic and a lipophilic part, or atleast two regions with different hydrophilicity, is of great inter-estforscientificandindustrialpurposes.Inparticularabolaformsurfactant is defined as a molecule, in which two or morehydrophilic groups are connected by a hydrophobic bridgingchain [24]. Most of these compounds have been studied for their polymorphic behaviour in water as a function of theirstructures [25–27]. When dispersed in aqueous medium, upon sonication, these molecules often self-organize into monolayervesicles (monolayer lipid membranes), which are stable overa long period of time to variations of temperature or to ionicstrength changes and are used as membrane models [28,29].In addition, the above mentioned properties of crown ethers tocomplex cations could play an interesting role in retaining somedrugs in vesicular systems made with bolaform lipids.In this work we have synthesized and characterized a newbolaform surfactant, 1,16-hexadecanoyl-bis-(2-aminomethyl)-18-crown-6, containing two identical crown ether units aspolar heads, called Bola A-16. We have evaluated itsability to form vesicles and the influence of a mem-brane additive, such as cholesterol (CH). These niosomeshave been compared with those formed with other knownbolaforms [30] containing two identical azacrown ether unitshave been the (4,7,10,13-pentaoxa-16-aza-cyclooctadecane)-hexadecanedioc acid diamide (Bola D-16) and   ,  -(4,7,10,13-pentaoxa-16-aza-cyclooctadecane)-hexadecane (Bola C-16).The surfactants chemical structures are reported in Fig. 1. A different hydrophobic spacer can allow a modulation in themolecular conformation in these bolaform lipids.The vesicular systems have been utilized for the deliveryof 5-fluorouracil (5-FU), which is a molecule effective againsttreatment of metastatic carcinomas of breast, gastrointestinaltract, pancreas, head, neck and ovary [31]. Its oral absorption is incompleteandunpredictable,thereforeitisparenterallyadmin-istered, but it manifests a short biological half-life due to its fastmetabolism. The formulation of 5-FU in niosomes [32] could optimizetheoralabsorptionorincreasethebiologicalhalf-lifeinthecaseofparenteraladministration,reducinginawaythetoxicside effects. Consequently, the encapsulation efficiencies of 5-FU in water and its in vitro release rates have been preliminarystudied for some formulations. 2. Materials and methods 2.1. Chemical and instruments All reagents have been purchased from Aldrich (Milano,Italy) and used without further purifications. All solvents havebeen high performance liquid chromatography grade. UV–visspectra have been recorded with a JASCO V-530 spectrometerusing1cmquartzcells.IRspectrahavebeenperformedwithFT-IR Jasco 4200 spectrometer and  1 H NMR have been recordedwith a Bruker 300 ACP NMR spectrometer.   R. Muzzalupo et al. / Colloids and Surfaces B: Biointerfaces 58 (2007) 197–202  199Fig. 2. Synthesis scheme of Bola A-16. 2.2. Materials The 1,16-hexadecanoyl-bis-(2-aminomethyl)-18-crown-6,Bola A-16, has been synthesized by following the sameprocedure used for the synthesis of Bola D-16 and Bola C-16(Fig. 2) and reported in literature [30,33]. The  ,  -hexadecanedioicacid(1.70mmol,0.488g)hasbeentransformed in the corresponding diacyl chloride by SOCl 2  inexcess (5ml), under stirring in refluxing conditions for 12h.The diacyl chloride has been evaporated, dried, and dissolvedin toluene. The product obtained has been 0.55g (yield 91.4%).IR  ν  (KBr): 2928, 1795, 1211.Diacylchloridetoluenesolution(1.55mmol,0.55g)hasbeenadded drop wise to a solution of 2-aminomethyl-18-crown-6(3.42mmol, 1.00g) in toluene, with triethylamine as a buffer.The diamide has been extracted with dichloromethane, chro-matographicallypurified,anddried.Thetotalfinalproductyieldhas been 78.2%. NMR  δ  values (ppm): 6.40 (s, 2 protons, NH),3.23–3.69 (m, 32 oxyethylene chain protons), 2.78 (s, 8 pro-tons), 2.80 (s, 2 protons), 2.03–2.08 (t, 4 protons), 1.49 (t, 4protons), 1.12–1.46 (m, 20 aliphatic protons). IR  ν  (film): 3033,2882,1124–1049cm − 1 . 2.3. Preparation of niosomes Vesicles have been prepared by using the conventionalchloroform film method [34]. The non-ionic surfactants and cholesterol have been dissolved in chloroform, which has beenthen evaporated at 60 ◦ C under vacuum by a rotary-evaporator.The resulting surfactant films have been dried over night ina desiccator under vacuum at 25 ± 2 ◦ C. The obtained filmshave been hydrated with 10ml of a 5-FU water solution undermechanical agitation for 1h. In order to prepare vesicles at atemperature above the gel–liquid transition temperature of theused amphiphiles, we have worked at 25 ◦ C with Bola D-16samples, at 80 ◦ C with Bola C-16 samples, and at 60 ◦ C withBola A-16 samples.The vesicle forming ability of surfactant Bola A-16 has beeninvestigated as a function of different molar ratio of cholesterol.The composition of tested samples is reported in Table 1.The resulting niosome dispersions have been then sonicatedin an ultrasound bath for 45min (15 cycles of 3min each). Thedispersions have been then left to stand at 25 ◦ C overnight. Theunentrapped (free) drug has been removed by exhaustive dialy-sis. The vesicle dispersions have been transferred into Visking Table 1Composition of vesicle system and mean hydrodynamic diameterFormulation code Surfactant (mg) CH (mg) Molar ratio, surfactant:CH  d   (nm)A 1  Bola A-16, 560 128 1:1 604  ±  35A 2  Bola A-16, 420 200 2:1 624  ±  25A 3  Bola A-16, 840 – 1:0 692  ±  50A 4  Bola C-16, 501 127 2:1 432  ±  30A 5  Bola D-16, 518 128 2:1 712  ±  15Total lipid concentration was 1 × 10 − 2 mol/l.  200  R. Muzzalupo et al. / Colloids and Surfaces B: Biointerfaces 58 (2007) 197–202 tubes (20/30), treated, according to Fenton method before use[35] and dialyzed against distilled water for 24h. The vesicleformation has been confirmed by microscopy analysis, whilethe ability of the vesicles to entrap 5-FU was examined by UVspectrophotometry. 2.4. Size distribution analysis Theniosomehydrodynamicdiameterhasbeendeterminedbydynamic light scattering using a 90 Plus Particle Size Analyzer(Brookhaven Instruments Corporation, New York, USA). Thehydrodynamic diameter (i.e. the core diameter plus the doublelayer thickness) is defined as the diameter that a set of iden-tical sphere should have in order to diffuse light at the samerate of the particles being measured. The hydrodynamic diam-eter of niosome dispersions has been determined at 25 ◦ C bymeasuring the autocorrelation function at 90 ◦ scattering angle.Cellshavebeenfilledwith100  lofsamplesolutionanddilutedto 4ml with filtered (0.22  m) water. Six separate measure-ments have been made to derive average. Data have been fittedby the method of inverse “Laplace transformation” and Contin[36,37]. 2.5. Transmission electron microscopy Morphology analysis of niosomes has been carried out usingtransmission electron microscopy. A drop of the niosome col-loidal suspension has been placed on a carbon-coated coppergrid and left for 1min thus allowing niosomes to adhere tothe grid. The excess of the niosome suspension has been thendrawn off by a piece of filter paper (Whatman Inc., Clifton,NJ, USA). A drop of negative stain solution, 1% (w/v) phos-photungstic acid solution, has been placed on the carbon gridthus staining the niosomes. After 3min, the excess stainingagent has been removed by adsorbing the drop with the tipof a filter paper and the sample has been then air-dried. Nio-some samples have been examined by using a ZEISS EM 900electron microscope operating at an accelerating voltage of 80kV. 2.6. Entrapment efficiency and drug release study Entrapmentefficiencies(  E  %)havebeenexpressedastheper-centageof5-FUtrappedinniosomesreferredtothetotalamountof 5-FU used. The entrapment efficiency has been determinedby dissolving 1ml of the niosomes (obtained from dialysis) in9ml of methanol, and measuring the absorbance of the clearsolution at 266nm.The release of 5-FU from niosomes in vitro has beenperformed according to literature [34,38]. Each dialysis tube containing 2ml of 5-FU loaded niosome dispersion has beenplaced into a flask containing 100ml of PBS (pH 7.4), whichhas been constantly stirred at 37.0 ± 0.5 ◦ C. At regular timeintervals, 2ml of the dialyzed solution have been taken andreplenished immediately with an equal volume of fresh PBS.The drug release has been quantified spectrophotometrically at266nm.Alldissolutiontestshavebeenrunintriplicateandmean Fig. 3. (A) Typical vesicle size distribution, sample A2. (B) Negative-staintransmission electron micrographs, sample A2. values have been reported. The release of free drug has beeninvestigated in the same way. 3. Results and discussion Various niosome formulations with different surfactants andratios of cholesterol have been prepared at the same total lipidconcentration (Table 1) to evaluate the influence of surfactant structure as well as their ability to form vesicles. Samples A4and A5 have been prepared by using Bola C-16 and Bola D-16 surfactants, respectively, mixing with cholesterol in molarratio surfactant:cholesterol=2:1. Such ratio is known to allowthe best entrapment efficiency [30].Fig. 3A shows a typical light scattering histogram (sam-ple A2) as a function of particle diameter which results rathermonodispersed.Fig.3BreportsatypicalTEMmicrophotograph ofniosomes(sampleA2)andconfirmsthesizehomogeneityandthe spherical shape of niosomes.The niosome average diameter of different formulations isreported in Table 1. The formulations with cholesterol loading (A1andA2)showareductionintheirdimensionscomparedwithsample prepared with pure surfactant (A3). Both the Bola A-16and Bola D-16 surfactants give rise to the unilamellar vesiclewith diameters (between 600 and 700nm) larger those obtained   R. Muzzalupo et al. / Colloids and Surfaces B: Biointerfaces 58 (2007) 197–202  201Table 2Surfactant structure on niosomes entrapment efficiencyFormulation code Surfactant  E  %A 1  Bola A-16 40.00  ±  0.66A 2  Bola A-16 27.00  ±  0.23A 3  Bola A-16 4.00  ±  0.12A 4  Bola C-16 19.00  ±  0.21A 5  Bola D-16 66.00  ±  0.67The initial concentration of 5-FU is 2 × 10 − 4 mol/l. with Bola C-16 (432nm), this is due to their similar structureaffinity.It is important to observe that the three-amphiphilic molec-ulesarecharacterizedbydifferenthydrophilicities,whichcanbealso modulated by cholesterol addition. Such a parameter influ-ences the average diameter of niosomes. In fact, depending onthe head polarity the molecular conformation of the surfactantsmay be less or more compressed at the same cholesterol content[39].Itisexpectedthatthehighertheheadpolarity,thelargertheaverage radius because a modulation of packing density in thebilayer results in a significant change in the structure curvature(i.e. in the vesicle size).The amount of drug (5-FU) entrapped in niosomes has beenfound to be dependent both on the cholesterol content and thetype of used surfactants. Experimental results for entrapmentefficiency are reported in Table 2.The niosomes obtained by Bola A-16 show a sensible rise inthe encapsulation efficiency, while increasing cholesterol molefraction. In particular, the efficiency of sample A3, with nocholesterol, is one tenth of that of sample A1, whose surfac-tant:cholesterol ratio is 1:1. It is important to note that 5-FU is arather small molecule characterized by a large mobility and lowassociation within the lipid bilayer, in fact, in our samples theentrapmentefficiencyisgenerallylow,ifnocholesterolisaddedto formulations. The inclusion of cholesterol markedly reducestheeffluxof5-FU,asaconsequenceofitsmembranestabilizingability.Studies of 5-FU release from the prepared formulations havebeen followed  in vitro  using dialysis, through hydrophilic mem-brane of regenerated cellulose, within a period of 6h.The  in vitro  release rate profiles are shown in Fig. 4, except the formulation A3 due to very low entrapment efficiency (4%).The release rates of 5-FU from niosomes are significantly lowerthan that of free solution. In fact, the free drug solution totallyreleases5-FUwithin2h(insetFig.4),whilethevesiclesrelease 20%, 44%, 80% and 10% for samples A1, A2, A4 and A5,respectively, within 6h. Efflux of 5-FU from niosomes has beenbiphasic with an initial faster release in about 2h followed by aperiodofslowrelease.Thebiphasicreleasepatternof5-FUdrugseems to be a general characteristic of bilayer vesicles; this hasbeen already reported for some liposomes [40] and niosomes[32]. The Table 2 and Fig. 4 show that samples, characterized by the highest values of entrapment efficiency, have the lowestrelease profiles. Consequently, the best compromise betweenentrapment efficiency and release percentage is reached withthe A2 and A4 formulations. Fig.4. Effectoftypeofbolaformsurfactantonthereleaserateof5-FUinvesiclesystems. The release experiments clearly indicate that the amountof drug released from all niosome formulations is effectivelyretarded, so it is expected that the toxic side effects of 5-FU canbe reduced by using these drug delivery systems. 4. Conclusions In this study a novel crown ether amphiphile has beensynthesized with the aim of developing a long time stablecontrolled release system and its performance compared withthe vesicular systems prepared with other synthetic bolaformsurfactants. The applicability of these vesicles as drug deliv-ery systems has been evaluated by loading an antineoplasticdrug, 5-fluorouracil.  In  v itro  studies have shown that the releaseof drug was sustained on encapsulation in niosomes com-paring with free solution. In particular, we have found thesamples characterized by the highest values of entrapment effi-ciency show the lowest release percentages. Having in mindthat niosomes bilayer acts as a rate-limiting membrane barrierfor encapsulated hydrophilic drug substances, higher entrap-ment of 5-FU into niosomes results in slower dissolution. Thebest compromise between good entrapment efficiency and anacceptable release is reached with the A2 and A4 formulations.The biphasic release pattern suggests that niosomes can act asreservoir systems for a continuous drug delivery consequently,these preparations could be successfully used in anticancertherapy. Acknowledgment MIUR, the Italian Ministry for University, is acknowledgedfor financial supports (Grants No EX 60%, PRIN 2005). References [1] G. Betageri, M. Habib, Pharm. Eng. 14 (1994) 76.[2] H. Schreier, J. Bouwstra, J. Control. Rel. 30 (1994) 1.
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