J Liposome Research

Journal of Liposome Research, 2013; 23(1): 47–53 © 2013 Informa Healthcare USA, Inc. ISSN 0898-2104 print/ISSN 1532-2394 online DOI: 10.3109/08982104.2012.738329 RESEARCH ARTICLE Physicochemical characterization of surfactant incorporating vesicles that incorporate colloidal magnetite Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. Raquel de Melo Barbosa1, Christine L. Luna Finkler2, Maria Vitória L. B. Bentley3, and Maria Helena A. Santana1 1 Biotechnological Process Department, College of Chemical Engineering, State University of Campinas, Campinas, São Paulo, Brazil, 2Federal University of Pernambuco, Academic Center of Vitória, Pernambuco, Brazil, and 3 Pharmaceutical Sciences Faculty of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Abstract Drug administration through the transdermal route has optimized for the comfort of patients and easy application. However, the main limitation of transdermal drug delivery is the impermeability of the human skin. Recent advances on improvement of drug transport through the skin include elastic liposomes as a penetration enhancer. Entrapment of ferroluids in the core of liposomes produces magnetoliposomes, which can be driven by a highgradient magnetic ield. The association of both strategies could enhance the penetration of elastic liposomes. This work relies on the preparation and characterization of elastic-magnetic liposomes designed to permeate through the skin. The incorporation of colloidal magnetite and the elastic component, octaethylene glycol laurate (PEG8-L), in the structure of liposomes were evaluated. The capability of the elastic magnetoliposomes for permeation through nanopores of two stacked polycarbonate membranes was compared to conventional and elastic liposomes. Magnetite incorporation was dependent on vesicle diameter and size distribution as well as PEG-8-L incorporation into liposomes, demonstrating the capability of the luid bilayer to accommodate the surfactant without disruption. On the contrary, PEG-8-L incorporation into magnetoliposomes promoted a decrease of average diameter and a lower PEG-8-L incorporation percentage as a result of reduction on the luidity of the bilayer imparted by iron incorporation into the lipid structure. Elastic liposomes demonstrated an enhancement of the deformation capability, as compared with conventional liposomes. Conventional and elastic magnetoliposomes presented a reduced capability for deformation and permeation. Keywords: Magnetoliposomes, magnetophoresis, conventional liposomes, elastic liposomes Introduction constitutes the outermost part of the epidermis. he skin barrier could be characterized as a complex nanoporous medium (20–30 nm pores), in which individual molecules migrate by difusion. he permeation of compounds is controlled mainly by permeant hydrophilicity, size, and hydrogen-bonding ability (Cecv and Vierl, 2010). Because of those characteristics, large molecules or colloid structures do not penetrate in the skin eiciently. Some mechanisms have been applied to modify the corneum stratum and to improve the transport of compounds in its free form, such as the use of chemical agents, and electric methods, such as sonophoresis and electroporation (Cevc et al., 1995) as well as he administration of drugs through noninvasive routes, such as oral, pulmonary, and transdermal routes, has been emphasized for the comfort of patients and easy application. In particular, the transdermal route has been considered as the most innovative and promising in the ield of the research in drug-controlled delivery. he main limitation on the transdermal route is the impermeability of the human skin. Basically, the skin consists of the dermis, a 250-µm-thick inner skin region, and epidermis, which is a 50-µm-thick outer skin region. he biological barrier is mainly because of the stratum corneum, a very thin layer (between 5 and 8 µm), which Address for Correspondence: Christine L. Luna Finkler, Universidade Federal de Pernambuco, Centro Acadêmico de Vitória, Rua Alto do Reservatório, s/n, Bela Vista, Vitória de Santo Antão 55608–680, Pernambuco, Brasil; Fax: +55 81 3523-3351. E-mail: [email protected] (Received 15 November 2011; revised 10 September 2012; accepted 06 October 2012) 47 Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. 48 R. de Melo Barbosa et al. iontophoresis and magnetophoresis (Barry, 2001) for ionic drugs. Besides those methods, liposomes also have been studied as an enhancer mechanism of transdermal transport. Liposomes are vesicles composed of lipid bilayers, which arrange themselves into concentric and closed membranes. Drugs may be entrapped either in the inner aqueous phase or in lipid bilayers (Gregoriades et al., 1993). he main advantage of these particles is the similarity with cell membranes in properties and functions. Research on liposome technology has progressed from conventional vesicles (“irst-generation liposomes”) to liposomes with modiied surfaces, obtained by modulating the lipid composition, size, and charge of the vesicle (Immordino et al., 2006). Elastic liposomes incorporate these concepts and represent the most recent formulation for drug delivery through the transdermal route (Cevc, 2004). he capability of deformation in liquid-state liposomes has been successfully generated by the addition of various edge activators, such as sodium cholate (Cevc et al., 1997; Cevc and Schätzlein, 2002; Cecv and Vierl, 2010), nonionic surfactants, such as polyglycerol alkyl ethers (Handjani-Vila et al., 1979; Baillie et al., 1986), or polyoxyethylene alkyl ethers (Bouwstra et al., 1992; van den Bergh et al., 2001). Bouwstra et al. (1999) investigated the efect of incorporating a single-chain nonionic pegylated surfactant on vesicle structure, and in vitro permeation through 50-nm polycarbonate membranes showed the superiority of octaethylene glycol laurate (PEG-8-L). hese characteristics, added to the better luidity of the bilayer imparted by the presence of the surfactant, promote the lowing of liposomes through nanopores, maintaining their integrity (Elsayed et al., 2007). Redelmeier and Kitson (1999) and Bouwstra et al. (2003) showed that difusion of elastic liposomes, when compared with conventional liposomes, led to increased drug concentration in the skin and subcutaneous tissues. Insigniicant levels of lipid monomers were detected only in the intercellular spaces of the skin when conventional liposomes were used. hese characteristics of elastic liposomes could be attributed to the high luidity and elasticity of the membrane, being capable of penetrating through the pores in the stratum corneum. he driving force for penetration into the skin is the “transdermal gradient” caused by the diference in water content between the relatively dehydrated skin surface (approximately 20% water) and the aqueous viable epidermis (close to 100%) (Benson, 2005). Magnetoliposomes are nanometer-sized iron oxide particles wrapped in a phospholipid bilayer. hese biocolloidal structures can be eiciently captured from solution under the action of a high-gradient magnetic ield (De Cuyper and Joniau, 1990). he lexibility of the phospholipid membrane to ancor hydrophilic or hydrophobic molecules as well as for surface modiications, extensively demonstrated in the literature for liposomes, has been also illustrated in magnetoliposomes (De Cuyper et al., 1999, 2002; De Cuyper and Valtonen, 2001). he combination of magnetic capture and lexibility of the phospholipids bilayer for chemical or physical modiication has motivated various applications of magnetoliposomes in the ield of modern biotechnology and biomedicine (Melbourne et al., 2007; Martins et al., 2008). For instance, the construction of an enzyme magnetically controlled bioreactor (De Cuyper and Joniau, 1990), adsorption of antibodies in ainity magnetoliposomes (Zollner et al., 2003; de Pinho et al., 2008), and fractionating of mixtures in studies of intermembrane changes (De Cuyper et al., 2004) have been investigated. Magnetophoresis has been studied as a novel approach in enhancing the delivery of diamagnetic drugs across biological barriers. Difusion of ionic drugs across rat abdominal skin was enhanced as a result of the inluence of the magnetic ield (Murthy, 1999). he combination of elastic and magnetic properties in magnetoliposomes could provide a strategy for transport of drugs through the skin under the action of a magnetic ield (Murthy et al., 2010). In this article, the incorporation of the surfactant, PEG8-L, in magnetoliposomes was studied, aiming for the production of elastic magnetoliposomes. Elastic efects of PEG-8-L in the bilayer of magnetoliposomes were evaluated by permeation through polycarbonate membranes at low pressure, simulating the transepidermal pressure gradient. Elastic, nonpegylated liposomes and magnetoliposomes were used as the control. Liposomes were considered elastic when they were able to permeate through two polycarbonate membranes (with a pore size of 50 nm), thus preserving vesicle contents and size characteristics. No magnetic ield was used in permeation to compare the permeation of liposomes under the gradient of pressure only. he inluence of the ferroluid on the permeation of pegylated magnetoliposomes was analyzed in comparison with conventional and elastic liposomes. Methods Synthetic phospholipid dimyristoylphosphatidylcholine (DMPC) and soy phosphatidylcholine (PC; Epikuron 200SH) were supplied by the Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA) and Lucas Meyer GmbH (Hamburg, Germany), respectively. Elastic magnetoliposomes were prepared using the surfactant, PEG-8-L (purchased from Lipo Chemicals, Inc., São Paulo, Brazil), as an elastic component. he lipid was hydrated using Hepes bufer (Sigma-Aldrich). FeCl2.4H2O (Acros Organics, Geel, Belgium), FeCl3.6H2O (Merck, Whitehouse Station, New Jersey, USA), ammonia hydroxide (Merck), and lauric acid (Merk) were used on the colloidal magnetite preparation. All other chemicals were of analytical grade. Membranes of 100 and 50 nm were supplied by Osmonics, Inc. (Minnetonka, Minnesota, USA). Preparation of colloidal magnetite Colloidal magnetite was synthesized, as described by Rheimers and Khalafalla (1976), by alkaline Journal of Liposome Research Vesicles that incorporate colloidal magnetite 49 coprecipitation of FeCl2.4H2O and FeCl3.6H2O in a concentrated ammonia hydroxide solution. he precipitate was washed with 100 mL of ammonia/distilled water (5:95, v/v), and 1 g of lauric acid was added during heating (at 90ºC for 4 minutes) for dispersion and stabilization of the colloidal magnetite, which was stored at room temperature (25°C). Colloidal magnetite was stable for 4 months, presenting a mean diameter of 7.7 nm, according to transmission electron micrographs (Martins, 1998). Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. Preparation of conventional and elastic-magnetic liposomes Conventional liposomes were prepared by the classic Bangham method (thin lipid ilm hydration) (New, 1990). Lipid suspension (at a 5-mM concentration) was prepared by mixing in an organic solvent, chloroform/ methanol (9:1, v/v). he solution was transferred to a glass vessel and homogenized during 5 minutes using a rotator evaporator (Fisaton Scientiic Equipment Ltd., São Paulo, Brazil). Further, a dry lipid ilm was deposited on the walls of the vessel under a vacuum (650 mmHg; 25°C; 1 hour) and hydrated in 10 mL of Hepes (10 mM, pH 7.4; Sigma-Aldrich). Elastic liposomes were prepared by adding PEG-8-L to conventional liposomes through surfactant incubation to preformed liposomes for 1 hour at a molar concentration of 40 mol%. To produce elasticmagnetic liposomes, the resulting thin lipid ilm was hydrated according to two procedures, initially by solubilization of PEG-8-L simultaneously with the magnetic luid used as the hydration solution. he other method consisted of hydration only with the magnetic luid and further incorporation of PEG-8-L by incubation during 1 hour, to obtain elastic-magnetic liposomes. In both cases, the phospholipid/Fe ratio was 1.3 mM/g, as described by Rocha et al. (2001). Magnetoliposomes were homogenized by sonication, and nonincorporated phospholipids were removed in a high-gradient magnetophoresis (HGM) system, as described below. In both procedures was maintained a molar phospholipid/surfactant ratio equal to 60:40 mol%. Liposome-size homogenization To obtain magnetoliposomes, dispersions were sonicated using a tip sonicator (VC-50, titanium micro-tip, Vibra Cell; Sonics & Materials, Inc., Danbury, Connecticut USA). he inluence of the amplitude and sonication time on average diameter and colloidal magnetite incorporation into magnetoliposomes was studied at 20, 40, and 60% of maximum amplitude (50 W) and sonication times of 10, 20, 30, and 40 minutes, in cycles of 30 seconds, at 25°C. Size homogenization of conventional liposomes was obtained by extrusion (extruder model T.001; Lipex Biomembranes, Inc., Vancouver, British Columbia, Canada) at 18 psi through compressed nitrogen gas and two polycarbonate membranes (100-nm pore diameter). To control the extrusion temperature at 37ºC (above of the gel-transition temperature), an external water bath was circulated through the extruder. © 2013 Informa Healthcare USA, Inc. HGM Retention of the magnetoliposomes and the remotion of the nonincorporated phospholipids were performed in an HGM system, constituted of two electromagnetic poles operating with a source of stabilized tension (5 A and 50 V) and projected to induce a magnetic ield of approximately 1.8 T. he distance between the poles was 3.5 mm, where there was placed a capillary containing 29.3 ± 7.1 mg of magnetic wool (Figure 1). his system is similar to that described by De Cuyper and Joniau (1988) and was used in an earlier work performed by Rocha et al. (2001). Initially, retention of the magnetoliposomes was performed at a low low rate (3 mL/hour) using 100-µL samples. After remotion of nonincorporated phospholipids, the magnetic ield was switched of and the magnetoliposomes were eluted using the same volume of Hepes bufer at a high low rate (1,000 mL/hour). Phospholipid and iron contents in the dispersion retained by the HGM system indicate the incorporation of the colloidal magnetite in the liposomes. Flow rate, elasticity, and deformation of liposomes Liposome elasticity was estimated from in vitro assays using two polycarbonate membranes with a pore size of 50 nm (Osmonics). Flow rate was determined by processing 5 mL of the liposome dispersion through the ilter using an extruder (Lipex Biomembranes) at 37°C (above the phase-transition temperature of lipids). Experiments were carried out under low pressure (2.5 psi), simulating the osmotic pressure of the skin during a maximum time of 10 minutes (van den Bergh et al., 2001). At the end of the process, the volume of the sample was measured. Flow rate was evaluated by Equation 1: J= Vp (mL) t (min) (1) where J is the low rate, Vp is the permeate volume, and t is the sonication time. he modulus of elasticity (E) was determined according to the equation proposed by Cevc and Blume (2001) (Equation 2): Figure 1. System of HGM operating at 5 A and 50 V, with a magnetic ield intensity between poles of approximately 1.8 T: (1) tension source; (2) electromagnetic poles; (3) capillary containing magnetic wool; (4) sample separated in the HGM system; (5) peristaltic pump; (6) original sample; and (7) velocity control pump. 50 R. de Melo Barbosa et al. d  E≅ J 1   dp  2 (2) where d1 is the liposome mean diameter before extrusion and dp is the pore diameter of the membrane. Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. Kinetics of surfactant incorporation into liposomes Kinetics of surfactant incorporation into liposomes was analyzed through changes in average diameter, absorbance (340 nm), and supericial tension values for the dispersion along the time. he percentage of PEG-8-L incorporation was determined through the changes of surface tension of medium related to its initial value, using a standard curve relating surface tension and surfactant concentration. Because the surfactant concentration was above the critical micellar concentration (CMC), changes of surface tension were determined using diluted samples to obtain values below the CMC. Measurements were performed at 25ºC using a tensiometer (Krüss K12; Krüss GmbH, Hamburg, Germany) with an immersion plate and through Wilhelmy’s method of type plate. Surface-tension values were evaluated by Equation 3: σ= Pw Lb.cosθ (3) where σ is the surface tension, Pw is the force, Lb is the liquid molhability, and θ is the contact angle. Calibration curve for empty liposomes Initially, the calibration curve for empty liposomes was plotted by a standard curve of surface tension versus surfactant molar concentration (in the range of 1–50 mol%) using empty liposomes at a PC concentration of 1 mM. Measurements were made in short time intervals (up to 5 minutes), so that there was no signiicant surfactant incorporation in the lipid bilayer. Experiments were performed in triplicate. he molar concentration of surfactant in the solution external to the liposomes, Ce, was determined by linear range of the calibration curve below the CMC. Determination of surfactant concentration incorporated into lipid bilayer Surface tension of elastic liposomes was measured after dilution of the solutions to reach the range below the CMC, and surfactant concentration was determined using the calibration curve previously constructed. From these data, molar surfactant concentration incorporated into the lipid bilayer, Ci, was determined by Equation 4: Ci = Ct − Ce (4) where Ct is the total molar concentration of surfactant in the solution. Surfactant-incorporated content (Ci*) was expressed as a percentage of the total concentration of surfactant in the solution (Equation 5):  Ci  Ci * =  1 −  × 100  Ce  (5) Liposome characterization Liposomes were characterized by their phospholipid and iron concentrations as well as by their average hydrodynamic diameter and size distribution. Iron concentration was determined by the methodology described by Yoe and Jones (1994), using 4,5-dihydroxyl-1,3-benzenedisulphonic acid (Tiron; Sigma-Aldrich) as the complexing reagent. Phospholipid concentration was quantiied through inorganic phosphate content, according to Chen et al. (1956). Average diameter and size distribution were determined by quasielastic laser light scattering (QELS), using a Malvern Autosizer (model 4700; Malvern Instruments Ltd., Malvern, UK) connected to an acquisition data system. Measurements were performed at a 90-degree scattering angle using He-Ne laser radiation (32 mW). Results and discussion Magnetite incorporation was not signiicant when PEG8-L was added to the hydration solution during liposome preparation. his efect was detected after sonication and separation in HGM. hus, the surfactant might be incorporated after magnetoliposome preparation. he efect of amplitude and sonication time on magnetoliposome average diameter and magnetite incorporation is shown in Table 1. Results show that as amplitude and ultrasound exposition time is increased, magnetoliposomes diameter is reduced. Magnetite incorporation was dependent on vesicle diameter and size distribution. A maximum incorporation (phospholipid/iron ratio of 1.3 mM/g) was observed for an amplitude of 20 V and a sonication time of 20 minutes. Rocha et al. (2001) reported that average diameter and size distribution in magnetoliposomes composed of cardiolipin/DMPC at equimolar concentrations were dependent on the inal phospholipid/magnetite ratio. Maximum iron incorporation (at the phospholipid/iron ratio of 1.3 mM/g) was determined by the investigators from adsorption isotherm for DMPC/DMPE and colloidal magnetite. he curve of surface tension versus surfactant molar concentration was in the range of 1–50 mol% using empty liposomes at a PC concentration of 1 mM, and the linear range was used to determine the surfactant concentration incorporated into the lipid bilayer. he CMC of PEG8-L was equal to 5 mol%. Figures 2 and 3 show PEG-8-L incorporation kinetics into liposomes and magnetoliposomes through changes in average diameter and absorbance for dispersions during this time. PEG-8-L incorporation into liposomes promoted a signiicant variation on average diameter, with a 90% size increment, as compared with its initial value (130 nm). Average diameter reached 239 nm after 300 minutes of incubation. Polydispersity values Journal of Liposome Research Vesicles that incorporate colloidal magnetite 51 Table 1. Average diameter (z-average), polydispersity indices, and colloidal magnetite incorporation as a function of amplitude and sonication time. Time Population percentage Average diameter mM phospholipid/g Amplitude (%) (minutes) (%) (nm) Polydispersity iron ratio 20 10 100 92.3 0.54 2.2 20 30 40 40 10 Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. 20 30 40 60 10 20 30 40 100 49.4 25.3 25.3 86.6 13.4 1.5 83.7 14.8 4.2 71.4 24.4 1 84 15 76.1 23.9 78.5 21.5 9 69 22 77.7 22.3 74.2 25.8 123.0 32.9 66.1 267.9 59.1 363.3 7.9 56.5 283 11.7 46.2 319 7.3 43.2 302.5 42.7 290.3 43.4 293 8.6 40.1 285.6 38.6 278.9 39.1 293.9 0.62 0.65 1.3 2.5 0.64 1.9 0.56 2.3 0.77 2.5 0.61 2.0 0.69 2.2 0.69 2.4 0.71 2.2 0.58 2.4 0.75 2.7 Figure 2. Kinetics proiles of PEG-8-L incorporation into conventional liposomes and changes in average diameter, polydispersity, and absorbance. Phospholipid (DMPC) concentration: 3 mM. Total lipid/surfactant ratio: 60:40 mol%. Temperature: 25ºC. Figure 3. Kinetics proiles of PEG-8-L incorporation into magnetoliposomes and changes in average diameter, polydispersity, and absorbance. Phospholipid (DMPC) concentration: 3 mM. Total phospholipidipid/surfactant ratio: 60:40 mol%. Total phospholipid/ iron ratio: 1.3 mM/g. Temperature: 25ºC. remained at approximately 0.13, indicating the physical stability of particles. Absorbance values showed a variation between 0.48 and 0.68, conirming the increment in size of liposomes containing PEG-8-L. Surfactant incorporation percentage reached 78% after 60 minutes. After this period, values become stable, with a maximum incorporation equal to 84% at 300 minutes of incubation. hese data demonstrate the capability of the luid bilayer of DMPC liposomes to accommodate surfactant without disruption. A diferent behavior was observed for results of PEG8-L incorporation into magnetoliposomes (Figure 3). A © 2013 Informa Healthcare USA, Inc. Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. 52 R. de Melo Barbosa et al. decrease of average diameter was observed in the irst 30 minutes, probably the result of lipid rearrangement in the presence of surfactant when magnetite was previously incorporated into liposomes. After 360 minutes, the inal diameter was 76 nm. Polydispersity values were approximately 0.4. PEG-8-L incorporation percentage was approximately 65% after 150 minutes, remaining constant during this time. his lower value, as compared with PEG-8-L incorporation into conventional liposomes, is the result of reduction in luidity of the bilayer imparted by iron incorporation into the lipid structure. Results of diameter variation, low rate, and phospholipid concentration for conventional and elastic liposomes and for conventional and elastic-magnetoliposomes (both with 100% encapsulated colloidal magnetite) are presented in Table 2. Conventional PC liposomes presented low concentrations of vesicles according to the amount of phosphate quantiied after the permeation test. (It was not possible to measure the particle diameter as a result of the low concentration of the sample.) his result was caused by the high transition temperature of the lipid phase (65°C) and therefore the permeation temperature (37°C) in the gel phase. Permeate low was measured in practice because of the passage of bufer solution through the membrane. Conventional DMPC liposomes had a low low rate (0.03 mL/min), approximately equal diameters before and after the permeation, and a phosphate content of 55%. On the other hand, the elastic liposome prepared with DMPC/PEG8L (60:40 mol%) had a high low rate (17.50 mL/min) and a signiicant reduction in diameter, indicating high elasticity (473.2 mL/min), but no disruption, inferred by the phosphate amount after permeation (99%). hese results show the enhancement of the deformation capability of liposomes, composed of the low transition-phase temperature lipid (DMPC) resulting from incorporation of PEG-8-L. van den Bergh et al. (2001) studied the physical properties of several liquid-state liposomes in which a single-chain surfactant was incorporated using the technique of electron spin resonance to investigate bilayer luidity. Membrane luidity was increased in the presence of 30 mol% PEG-8-L. For conventional DMPC magnetoliposomes, diameter decreased strongly (from 123 to 57 nm) after permeation, and both low rate (0.04 mL/min) and phosphate content in permeate (4%) were lower than conventional or elastic DMPC liposomes. he presence of magnetite seems to reduce the capability of liposomes for deformation and permeation, and only the smaller structures in dispersion were permeated. Although there was similarity in low rate (0.05 mL/min), elastic DMPC magnetoliposomes had lower elasticity (0.08 mL/min), as compared with conventional DMPC magnetoliposomes (0.24 mL/ min). his is a consequence of their smaller initial mean diameter and slighter capability for permeation of magnetic structures containing PEG-8-L. Assays using a reduced amount of magnetite incorporated into elastic DMPC magnetoliposomes allowed us to quantify magnetite in permeate dispersion using a phospholipid/iron ratio equal to 2.8 mM/g. In this condition, we obtained a ferroluid and phospholipid content in the permeate of 3 and 20%, respectively, and an elasticity of 1 mL/min. hese results show the potential of these nanomagnetic particles for applications in magnetophoresis. Optimization of the phospholipid/iron ratio could enhance its permeation characteristics. he action of a magnetic ield could provide representative permeation low rates for applications in drug delivery through the skin. Conclusions Preparation of elastic-magnetoliposomes incorporating PEG-8-L surfactant is feasible. Operational sonication conditions inluence the diameter of magnetoliposomes, as well as the incorporation of iron. he presence of colloidal magnetite caused a reduction in diameter of vesicles after the permeation test. Magnetoliposomes with a 1.3-mmol/g iron ratio (phospholipids/magnetite) had smaller elasticity and low rates than conventional or elastic liposomes. However, the 2-fold reduction in the amount of magnetite incorporated into liposomes increased the low and Table 2. Average diameter before (D1) and after (D2) permeation, low rate, and phospholipid concentration after permeation for liposomes and magnetoliposomes. Sample D1 (nm) D2 (nm) Flow rate (mL/min) PO43–(%)a Conventional PC liposomes 115.0 — 0.05 Conventional DMPC liposomes 127.0 121.0 0.03 132.4 118.8 5.60 Elastic DMPC liposomesb (80:20)d Elastic DMPC liposomesb (60:40)d 260.0 193.6 17.50 141.0 109.7 5.10 Elastic DMPC liposomesb (30:70)d Conventional DMPC magnetoliposomes 123.0 57.0 0.04 64.0 58.0 0.05 Elastic DMPC magnetoliposomesc,d Permeation at 2.5 psi through two stacked polycarbonate membranes (50 nm) at 37ºC. a Phospholipid percentage in the permeated sample in relation to initial phosphate concentration. b Elastic liposomes prepared by dry-ilm phospholipid hydration and further incubation with PEG-8-L. c Elastic-magnetic liposomes prepared by dry-ilm phospholipid hydration using magnetic luid at a phospholipid/iron ratio equal to 1.3 mmol/g and further incubation with PEG-8-L. d Molar phospholipid/surfactant ratio (mol%). 1.0 55.0 82.0 98.0 87.0 4.0 6.4 Journal of Liposome Research Vesicles that incorporate colloidal magnetite 53 permeation of elastic magnetoliposomes. Permeation of liposome-magnetic elastic into polycarbonate membrane, maintaining integrity, therefore depends on optimization of the concentration of magnetite in liposomes, which could be potentially important for transdermal delivery of drugs under the action of a magnetic ield. Journal of Liposome Research Downloaded from informahealthcare.com by 187.78.59.92 on 01/30/13 For personal use only. Acknowledgments he authors are grateful to the Brazilian agencies, Conselho Nacional de Desenvolvimento Cientíico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), for their inancial support and to Dr. Leandro Tessler from the State University of Campinas (Brazil) for the project and construction of the HGM system. Declaration of interest his research was supported by FAPESP (02/12637–9) and CNPq (150044/2004–6). References Baillie, A. J., Coombs, G. H., Dolan, T. F., Laurie, J. (1986). 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S., Ponec, M. (2003). Structure of the skin barrier and its modulation by vesicular formulations. Prog Lipid Res 42:1–36. Cevc, G. (2004). Lipid vesicles and other colloids as drug carriers on the skin. Adv Drug Deliv Rev 56:675–711. Cevc, G, Blume, G. (2001). New, highly eicient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes. Biochim Biophys Acta 1514:191–205. Cevc, G., Schätzlein, A. (2002). Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements. Biochim Biophys Acta 1564:21–30. Cevc, G., Schätzlein, A., Blume, G. (1995). Transdermal drug carries properties, optimization, and transfer eiciency in the case of epicutaneously applied peptides. J Control Release 36:3–16. Cevc, G., Schätzlein, A., Blume, G. (1997). 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