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Colloids and Surfaces B: Biointerfaces 120 (2014) 160–167

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

ationic vesicles based on biocompatible diacyl glycerol-arginineurfactants: Physicochemical properties, antimicrobial activity,ncapsulation efficiency and drug release

. Tavanoa,b, A. Pinazoc, M. Abo-Riyad, M.R. Infantec, M.A. Manresae,. Muzzalupoa, L. Pérezc,∗

Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Edificio Polifunzionale, 87036 Arcavacata di Rende, ItalyDepartment of Engineering Modelling, University of Calabria, Via P. Bucci—Cubo 39C, 87036 Arcavacata di Rende, ItalyDepartment of Chemical and Surfactants Technology, IQAC—CSIC, Jordi Girona 18-26, 08034 Barcelona, SpainChemistry Department, Faculty of Science, Benha University, Benha, EgyptDepartment of Microbiology, University of Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain

r t i c l e i n f o

rticle history:eceived 24 October 2013eceived in revised form 14 April 2014ccepted 15 April 2014vailable online 22 May 2014

eywords:iacyl glycerol-arginine surfactantsesiclesiprofloxacin-Fluorouracil

a b s t r a c t

Physicochemical characteristics of cationic vesicular systems prepared from biocompatible diacylglycerol-arginine surfactants are investigated. These systems form stable cationic vesicles by themselvesand the average diameter of the vesicles decreases as the alkyl chain length of the surfactant increases.The addition of DPPC also modifies the physicochemical properties of these vesicles. Among the drugsthese cationic formulations can encapsulate, we have considered Ciprofloxacin and 5-Fluorouracil (5-FU).We show that the percentage of encapsulated drug depends on both the physicochemical properties ofthe carrier and the type of drug. The capacity of these systems to carry different molecules was evaluatedperforming in vitro drug release studies. Finally, the antimicrobial activity of empty and Ciprofloxacin-loaded vesicles against Gram-positive and Gram-negative bacteria has been determined. Three bacteriawere tested: Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The in vitro drug release

eleasentimicrobial activity

from all formulations was effectively delayed. Empty cationic vesicles showed antimicrobial activity andCiprofloxacin-loaded vesicles showed similar or higher antimicrobial activity than the free drug solution.These results suggest that our formulations represent a great innovation in the pharmaceutical field, dueto their dual pharmacological function: one related to the nature of the vehiculated drug and the otherrelated to the innate antibacterial properties of the surfactant-based carriers.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Vesicles are one of the most widely used carriers in the develop-ent of controlled drug release formulations [1,2]. They enhance

he pharmacological activity and pharmacokinetic properties ofrugs and reduce toxic side effects during therapy [3–8]. Vesiclesave been shown to be a promising delivery system for a largeumber of antibiotics and anticancer drugs [9,10]. One of the most

mportant advantages of vesicles is the possibility of a gradual andustained release of drugs when circulating in the body [11]. This

llows to maintain the proper drug concentration for a relativelyong period of time, protecting it against the hydrolytic activity ofnzymes and chemical and immunological deactivation.

∗ Corresponding author. Tel.: +0034934006164.E-mail address: lpmste@cid.csic.es (L. Pérez).

ttp://dx.doi.org/10.1016/j.colsurfb.2014.04.009927-7765/© 2014 Elsevier B.V. All rights reserved.

Cationic vesicles can act by themselves as anti-infective agentsand are very promising systems in the transfection of DNA into thecells. Moreover, vesicles tend to selectively accumulate in angio-genic endothelial cells of tumours [12]. Several studies showedthat uptake of cationic liposomes by endothelial cells in angiogenicblood vessels was considerably greater and strongly selective com-pared to anionic, neutral, or sterically stabilized neutral liposomes[13]. In addition, the use of cationic carriers to deliver anticancerdrugs to solid tumours has recently been recognized as a promisingtherapeutic strategy to improve the effectiveness of conventionalchemotherapy [14].

To form cationic vesicles, cationic diacyl glycerol arginine-basedsurfactants could become a valid alternative to the conventional

cationic surfactants. Cationic diacyl glycerol arginine-basedsurfactants belong to a class of biocompatible lipoaminoacidsurfactants which share properties with glycerides and phospho-lipids and are particularly attractive because of their biological

L. Tavano et al. / Colloids and Surfaces B:

aachala

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2

2

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s

Fig. 1. Molecular structure of 1010R and 1414R surfactants.

ctivity. These compounds consist of a glycerol backbone esterifiedt positions 1 and 2 with aliphatic acid chains and with thearboxylic group of an arginine residue at position 3 as a polaread, [15]. Because of their strong growth-inhibitory activitygainst pathogenic bacteria and yeasts shown in vitro tests, theseipids have been used as design templates for the development ofnti-infectious therapeutics [16,17].

In this work, cationic vesicles composed of pH-sensitive diacyllycerol arginine-based surfactants with alkyl chains of differ-nt length (1,2 didecanoyl-3-O-(N-l-arginyl) rac-glycerol (1010R)nd 1,2 dimiristoyl-3-O-(N-l-arginyl) rac-glycerol (1414R)) (Fig. 1),ere prepared starting from pure surfactant solutions or mixtures

f surfactant and dipalmitoyl phosphatidyl choline (DPPC). Theffect of the bilayer composition on the physicochemical proper-ies of the formulations was investigated in terms of particle size,article size distribution, zeta-potential and Ciprofloxacin or 5-FUncapsulation efficiencies. The in vitro drug release profiles werevaluated and antimicrobial activity was also studied for emptynd Ciprofloxacin loaded formulations against Gram-positive andram-negative bacteria.

. Materials and methods

.1. Materials

DPPC, Ciprofloxacin and 5-FU were purchased from Sigmahemical Company (St. Louis, USA). Spectra/Pore dialysis mem-ranes (12,000–14,000 molecular weight cut-off) were purchasedrom Spectrum Laboratories Inc. (Houston, USA). All solutions wererepared using deionized water from Milli-Q Millipore system with

total organic carbon value of less than 15 ppb and a resistivity of

8 M� cm. Mueller-Hinton broth (MHB) was purchased from Difcoaboratories (Detroit, USA).

The arginine based surfactants were synthesized using a three-teps procedure (Scheme 1) [15] (a) condensation of one hydroxyl

Scheme 1. Synthetic procedure to prepare the d

Biointerfaces 120 (2014) 160–167 161

group of the glycerol with the acid group of the protectedN�-carbobenzyloxy-arginine (Z-arginine) to produce 00R(Z), (b)acylation of the two free hydroxyl groups of the previously pre-pared 00R(Z) using the corresponding acyl chloride and (c) catalytichydrogenation to remove the Z protecting group. More details onthe synthesis are reported in the literature [15].

The purity, higher than 99%, was checked by elemental analysisand high-performance liquid chromatography (HPLC) (Table 1).

Synthesized arginine-based surfactants have two positivecharges, one on the protonated guanidine group and one on theprotonated �-amino group of the arginine. The protonation of thesegroups depends on both the pH conditions and the pKa valuesof the surfactants. The pKa values were determined by titrationwith NaOH in aqueous solutions. The pKa values correspondingto the �-amino group and to the guanidine group of the argi-nine are, respectively, 9.6 and 5.2 for the 1010R, and 8.8 and 4.7for the 1414R. The introduction of hydrophobic groups drasticallydecreases the pKa values of the protonated basic functions (pKa val-ues of the arginine are 12.48 for the guanidine group and 8.99 forthe �-amino group). The same trend has been observed for cationicsurfactants from lysine [18]. These results indicate that these com-pounds are pH sensitive surfactants because the number of cationiccharges in the molecule depends on the pH of the medium.

2.2. Sample preparation

Vesicle formulations were prepared slightly modifying the filmhydration method described by Bangham [19]. Surfactants andphospholipids, adequately weighed to obtain a final lipid con-centration of 2.5 mM (Table 2), were dissolved in methanol. Theorganic solvent was vacuum evaporated and the resulting lipidfilm was hydrated under sonication (15 min at 50 ◦C) with 4 mLof deionized water (empty vesicles) or 4 mL of Ciprofloxacin or 5-FU solution (2.59 × 10−3 M). After preparation, the dispersion wasleft to equilibrate at room temperature overnight before use. Toremove non-encapsulated drugs, vesicle purification was carriedout by exhaustive dialysis for 4 h, using Visking tubing (20/30),manipulated before use according to Fenton’s method [20].

2.3. Size distribution analysis and zeta-potential

The zeta-potential of the formulations was measured with thelaser Doppler electrophoretic mobility measurements using theZetasizer 2000 (Malvern Instruments Ltd., Malvern, U.K.), at 25 ◦C.Analysis were done in triplicate. Zeta-potential values and standarddeviations (±S.D.) were elaborated directly from the instrument.

Dynamic light scattering (DLS) was used to measure the size

distribution in all formulations. The DLS unit used to figure outvesicle sizes was a Malvern zeta nanosizer, working at 632.8 nmand 25 ◦C. The scattering intensity was measured at a 173◦ angleto the incident beam. This is known as backscatter detection and

iacyl glycerol arginine based surfactants.

162 L. Tavano et al. / Colloids and Surfaces B: Biointerfaces 120 (2014) 160–167

Table 1Analytical Data of 1,2-diacyl-3-O-(N-l-arginyl) rac-glycerol (XXR).

Compound Molecular formula molecularweight (g/mol)

HPLCa retention time (min) Elemental analysis calcd/found

%C %H %N %Cl

1010R C29H58N4O6Cl2 16.0 52.32 9.32 8.42 10.67629 52.80 9.48 8.72 10.51

1414R C37H74N4O6Cl2 19.3 57.7b 10.03b 7.20b 9.23b

741 57.41 9.86 6.84 9.23

a HPLC conditions: HPLC model Merck-Hitachi D-2500 using UV–vis detector L-4250 at 215 nm and Lichrospher 100 CN (propylcyano) 5-�m, 250 × 4 mm column. Ag 5/25 (w in H

assd

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radient elution profile was employed from the initial solvent composition of A/B 7as 0.1% (vol/vol) trifluoro acetic acid (TFA) in H2O and solvent B was 0.085% of TFAb Calculated with 2 mol H2O.

llows measurements in turbid dispersions, minimizing multiplecattering effects [21]. At least 10 runs were performed for eachample. The measured correlation was used to calculate the sizeistribution.

The size distribution profile was determined with the CONTINlgorithm [22] which allows to deal with monomodal as well asultimodal distributions. Optimal parameters for CONTIN were

dentified using the software developed by Zetasizer v6.20.The time decay of the autocorrelation function of the concen-

ration fluctuations has a characteristic decay rate given by � = Dq2

here D is the mutual diffusion constant and q is the scatteringector which, in turn, is defined as:

= 4 �n

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�/2) (1)

ere n is the solvent refractive index and � is the scattering angle.he measured mutual diffusion constant and the average hydro-ynamic radius, Rh, of the particles are related according to theollowing equation:

= kT

6 ��Rh(2)

ere k is the Boltzmann constant, � the solvent viscosity, and T thebsolute temperature.

.4. Drug entrapment efficiency

Entrapment efficiency (E% in Table 3) was determined usinghe dialysis technique [23]. The dialyzed vesicular suspensionas obtained by placing 3 mL of drug-loaded vesicular disper-

ion within a dialysis bag which was then immersed magneticallytirred distilled water. The dialysis was considered completedhen drug in the medium was not detectable. To assess the encap-

ulation efficiency, two solutions were prepared with 0.1 mL ofialyzed and 0.1 mL of non-dialyzed vesicular suspension, respec-ively, in 4 mL of methanol. Then absorbances were recorded

rom clear solutions with UV–vis spectrometry (Cary 300Bio, Var-an) at Ciprofloxacin or 5-FU specific wavelengths, 278 nm and66 nm, respectively. Efficiencies were determined as the percent-ge of amount of drug entrapped by the dialyzed and non-dialyzed

able 2omposition of the vesicular systems, hydrodynamic diameter (nm), polydispersity inde

= 3).

Sample formulation Surfactant/DPPCmole ratio

Diamete

1010R 1 1/0 182 ± 3

1010R/DPPC 8:2 8/2 164 ± 6

1010R/DPPC 5:5 5/5 140 ± 2

1414R 1 1/0 228 ± 1

1414R/DPPC 8:2 8/2 190 ± 121414R/DPPC 5:5 5/5 114 ± 1

by volume), changing during 24 min to a final composition of 5/95 where solvent A2O/CH3CN 1:4. The flow-rate through the column was 1.0 mL/min.

vesicle suspensions. All the experiments were carried out threetimes and the results were expressed as mean ± standard deviation.

2.5. Drug release study

The in vitro release of drugs from vesicles was measured apply-ing dialysis with a Spectra/Pore dialysis membrane that assuresdrug release while retaining vesicular carriers [24]. An accuratelymeasured amount of drug vesicular suspension was placed in adialysis bag and suspended in 20 mL of phosphate buffer saline(pH 7.4), under gentle magnetic stirring. At predetermined timeintervals (1 up to 24 h), 2 mL of the medium were withdrawnand assayed spectrophotometrically for drug content at the cor-responding wavelengths. The volume of receptor compartmentwas maintained with an equal volume of fresh phosphate buffersaline. The drug release was determined as the ratio in percent-age of the amount of drug released at each time interval to theinitial amount of drug encapsulated in the formulation. Experi-ments were repeated three times and results were expressed asmean ± standard deviation. The free drug release was measured inthe same way.

2.6. Antimicrobial activity

Antimicrobial activities were determined in vitro on the basisof the minimum inhibitory concentration (MIC) values, defined asthe lowest concentration of antimicrobial agent that inhibits thedevelopment of visible growth after 24 h of incubation at 37 ◦C [25].We tested three different bacteria: Two of them are Gram-negative,Escherichia coli and Klebsiella pneumoniae, and one is Gram-positive,Staphylococcus aureus.

The vesicular systems studied were dissolved in MHB in con-centration ranging from 0.1 to 256 �g/mL. Then 10 �L of a nutrientbroth starter culture of each bacterial strain was added to achievefinal inoculums of ca. 5 × 10−4–5 × 10−5 colony forming units mL−1.The cultures were incubated overnight at 37 ◦C. Nutrient broth

medium without the compound served as control. The growth ofthe microorganisms was determined visually by assessing solutionturbidity after incubation for 24 h at 37 ◦C. Because a rise in tur-bidity reflects increases in both mass and cell number, the lowest

x (PI) and the zeta-potential (mV) of the empty formulation at 25.0 ◦C. (mean ± SE;

r (nm) PI Zeta-potential (mV)

0.53 ± 0.08 71 ± 20.51 ± 0.06 65 ± 10.48 ± 0.05 71 ± 20.61 ± 0.06 62 ± 2

0.56 ± 0.03 52 ± 10.45 ± 0.04 20 ± 4

L. Tavano et al. / Colloids and Surfaces B: Biointerfaces 120 (2014) 160–167 163

Table 3Hydrodynamic diameter (nm), polydispersity index (PI), zeta-potential (mV) and entrapment efficiency (E%) of Ciprofloxacin (C) and 5-FU loaded formulations at 25.0 ◦C.(mean ± SE; n = 3).

Sample formulation Diameter (nm) PI Zeta-potential (mV) E% Encapsulated drug (�g/mL)

1010R 5-FU 122 ± 1 0.48 ± 0.02 78 ± 1 10.3 ± 0.4 34.8 ± 1.31010R C 124 ± 1 0.40 ± 0.05 72 ± 1 8.9 ± 0.5 76.2 ± 4.21010R/DPPC 8:2 C 164 ± 1 0.41 ± 0.02 68 ± 4 11.0 ± 0.3 92.7 ± 2.51010R/DPPC 5:5 C 142 ± 1 0.46 ± 0.03 60 ± 1 12.9 ± 0.5 112.6 ± 4.21414R 5-FU 254 ± 3 0.53 ± 0.07 57 ± 3 16.0 ± 0.9 53.3 ± 3.11414R C 220 ± 5 0.61 ± 0.08 61 ± 6 15.1 ± 1.1 129.5 ± 9.4

43

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1414R/DPPC 8:2 C 178 ± 3 0.51 ± 0.04

1414R/DPPC 5:5 C 122 ± 6 0.48 ± 0.05

oncentration of antimicrobial agent at which no visible turbidityas observed was taken as the MIC.

. Results and discussion

.1. Characterization of diacyl glycerol-based vesicles

The goal of the current study was to prepare and to character-ze cationic vesicles based on pH sensitive cationic surfactants ando explore the possibility to encapsulate drugs in these colloidalystems.

Diacyl glycerol arginine-based surfactants can be considerednalogues of diglycerides and phospholipids. These arginine basedurfactants exhibit similar properties of both along with improvedater solubility and antimicrobial activity [15].

The cytotoxicity of these compounds is significantly lower thanhose of classical cationic surfactants from quaternary ammonium.ased on the concentration that produces 50% haemolytic activityHC50) and haemoglobin denaturation index (DI), 1010R and 1414Rompounds can be classified as mildly irritating surfactants, whilehe hexadecyl trimethyl ammonium bromide (HTAB) is consid-red highly irritating [26]. The cytotoxicity against the 3T6 mousebroblasts has been also reported: the doses required to kill 50% ofhe cells (IC50) of 1010R and 1414R are74.8 �g/mL and 63.7 �g/mL,espectively, while the IC50 of the HTAB for the same cellular lines 0.5 �g/mL [26]. These values suggest that 1010R and 14141Rurfactants have a great potential in pharmaceutical fields as bio-ompatible lipids to prepare cationic carriers with antimicrobialroperties.

It has been reported that the toxicity of cationic lipids can beeduced using vesicular systems. In fact, vesicles are commonlysed in cosmetics and pharmaceutics as carriers for active agents.ationic lipid vesicles formed by pure cationic lipids can be toxic,ut the toxicity can be reduced adding another compound to theesicular systems. For example, cetyl trimethyl ammonium bro-ide (CTAB) is a cytotoxic surfactant (IC50 lower than 10 �g/mL

or different cell lines) but when cationic vesicles were formed byTAB–sodium dodecyl sulfate, cell survival was higher than 60% for

concentration of 75 �M [27]. Moreover, for some cationic lipidsuch as dioctadecyl–dimethyl–ammonium bromide cytotoxicityowards human cells is drastically reduced when this surfactants included in cationic vesicles formed by cholesterol and phos-hatidylcholine [28]. In addition, factors present in human serumeduce the cytotoxicity of active compounds in some human cells29].

With the aim of assessing how the bilayer composition affectshe physicochemical and biological properties of the formulations,n this study vesicles were obtained starting from pure surfactantsr surfactant/DPPC mixtures at two different molar ratios.

The diacyl glycerol-arginine surfactants formed vesicles in pres-nce or absence of DPPC as membrane additive. This is due to theirigh surface activity that lead them to form a great variety of struc-ures in solution [30]. Both 1010R and 1414R surfactants were able

± 2 15.7 ± 0.7 132.4 ± 6.0± 1 13.0 ± 0.6 112.2 ± 5.1

to form lamellar liquid crystal phases at room temperature whichcould became vesicles at 40 ◦C. Our formulations were preparedat 40 ◦C because, in these conditions, vesicles are easily obtained.Hydrodynamic diameter and the zeta-potential of the empty for-mulations are collected in Table 2.

A single population was obtained for almost all formula-tions with a polydispersity index (PI) within the range 0.4–0.6.These PI values indicate the presence of large size distributionswhich are in accordance with those reported in the literaturefor non-extruded cationic vesicles or micelles [14,31]. Resultsrevealed that vesicles obtained using 1414R were larger than thosebased on 1010R. This behaviour can be ascribed to the lengthof the saturated alkyl chain of the 1414R. According to resultsreported in [32] the longer the alkyl chain the larger the vesiclesize.

The introduction of DPPC into the vesicles slightly decreasestheir size when the surfactant is 1010R. The presence of DPPCresults in a reduction of the average vesicle diameter when the sur-factant is 1414R. It has been described that the use of phospholipidsoften helps in forming self-closed bilayers, leading to appropri-ate molecular geometry and hydrophobicity for vesicle formation[33,34]. The results obtained in this work also suggest that theincorporation of DPPC into the cationic vesicles improves the stabil-ity of the formulations, as revealed by the reduction of the averageaggregate size. These findings could be ascribed to the decrease ofpositive charged molecules in the vesicles, as confirmed by zeta-potential values (except in the case of 1010R/DPPC 5:5). Moreover,it is expected that the higher the charge carried by the molecules thelower the repulsions between adjacent positive groups. In addition,the formation of hydrogen bonds among the functional groups ofsurfactants and phospholipid moieties should be taken into consid-eration [35]. These interactions may favor the formation of vesicles,stabilizing the structures and decreasing vesicle diameter [36,37].This behaviour was more relevant in the case of the 1414R sur-factant, for which the diameter ranges from 228 nm to 190 nm for1414R/DPPC 8:2 and from 228 nm to 114 nm for 1414R/DPPC 5:5formulations.

Zeta-potential is a measure of the electrical charge close tothe surface of the vesicles thus zeta-potential measurements area convenient way to characterize electrostatic properties of vesi-cles. Formulations investigated in this work have large positivezeta-potential values ranging from 20 to 70 mV and presentedhigh colloidal stability, phase separation and precipitation were notobserved after 2 months formulations preparation. This behaviourcan be ascribed to the stabilizing effect produced by the repulsiveinteractions among aggregates with positive surface charges as wellas to the small average size of the aggregates. The positive chargedensity of the vesicles seems to be large enough to ensure that nocoagulation occurs.

Formulations based on 1010R showed higher zeta-potential val-ues than those based on 1414R analogues. The pKa of 1010R ishigher than the pKa of 1414R, hence, for a given pH, the per-centage of protonated groups in the 1010R based vesicles should

1 ces B:

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64 L. Tavano et al. / Colloids and Surfa

e higher than in those based on 1414R. Consequently, 1010Rormulations show superior zeta-potential values. When the sur-actant/DPPC ratio was reduced, zeta-potential values decreasedecause the number of cationic surfactant present in the compo-ition is reduced. This behaviour has not been observed for the010R/DPPC 5:5 formulation. The zeta-potential of cationic vesiclesepends on the number of cationic surfactants on the vesicle sur-ace and on the association degree of the counterions to the vesicle.y reducing the concentration of cationic surfactants (surfac-ant/DPPC ratio 5:5) the electrostatic repulsions between positiveharges also decrease. In this situation, we can expect that to stabi-ize the vesicle the required number of counterions decrease whilencreasing the zeta-potential [38]. A similar behaviour has beenbserved for cationic vesicles prepared with DMPC and cationicurfactants based on quaternary ammonium groups [39].

.2. Characterization of drug loaded diacyl glycerol-based vesicles

Encapsulating drugs in colloidal systems is a technique thatllows to control pharmacokinetic and pharmacodynamic prop-rties of drug release [40]. The ability of cationic vesicles, undertudy, to encapsulate different molecules was investigated usingiprofloxacin and 5-FU as model drugs. With the aim of assessingow these physicochemical properties changed with the encap-ulation of different compounds, the hydrodynamic diameters asell as the zeta-potential of loaded vesicles have been evaluated.iprofloxacin is active against a broad spectrum of Gram-positivend Gram-negative bacteria. 5-FU is one of the most effectiveolecules for the treatment of metastatic tumours. 5-FU oral

bsorption is incomplete; therefore it is parentally administeredlthough the biological half-life is short mainly due to its fastetabolism [41].Research described in the literature shows the therapeutic

fficacy of fluoroquinolones and 5-FU encapsulated in liposomes42,43]. Ciprofloxacin has two pKa values (approximately 6.3 and.8) and an isoelectric point at pH 7.2 [44]. For formulations pre-ared at a pH of 6 the drug may be partially positively ionized.his property may have an influence on drug distribution and onhe affinity of drugs for lipidic environments, particularly bilayers45]. 5-FU has a pKa value of 8.0, hence, it is not ionized in thexperimental conditions used in this work.

Table 3 collects the hydrodynamic diameter, PI, zeta-potentialnd entrapment efficiency values measured for Ciprofloxacin and-FU-loaded formulations. Values in the Table 3 show that therug presence within vesicles slightly affects the vesicle diam-ter. For 1010R-based formulation the diameter decreases from82 nm shown by empty vesicles down to 124 nm in the case ofiprofloxacin and to 122 nm in the case of 5-FU. The variations inerms of hydrodynamic diameter were very similar for both drugs.his means that while vesicles size is strongly related to the naturend composition of the bilayer, the chemical structure of the encap-ulated molecules does not have a relevant effect.

It has been reported that the diameter of the vesicles diminisheshen a hydrophilic drug is encapsulated. This behaviour is ascribed

o electrostatic attractions between the drug and the vesicularilayer, leading to an increase in vesicular cohesion. Moreover, theresence of positively charged drugs in cationic systems can pro-ote an increase in vesicle size, because of the repulsions between

he charged moieties [23].Values in Table 3 suggest that the presence of drug has a

ittle effect on the vesicle size and zeta-potential. Although repul-ions between the hydrophilic drugs and surfactants head could

esult in the increase of particle size, formation of hydrogen bondsinking functional groups of surfactants and drugs could takelace, increasing the bilayer cohesion [5]. These could explain the

imited variation of vesicles size when Ciprofloxacin and 5-FU are

Biointerfaces 120 (2014) 160–167

present. In addition, physicochemical properties of drug loaded-surfactant/DPPC vesicles do not change with the incorporation ofthese drugs. The amount of DPPC present does not affect the vesiclesize.

3.3. Drug entrapment efficiency

Usually, the nature, chemical structure of drugs and the pres-ence of charged moieties strongly affect the drug entrapmentefficiency (E%) [46]. Table 3 gives E% values and the total amountof encapsulated drug expressed in �g/mL. E% was evaluated asdescribed in the experimental section. It is known that lipids in highdoses can be toxic and compromise the pharmacokinetics of drugs[47]. In general, the obtained E% is moderate but these formulationscontain a therapeutic amount of drug and a reasonable amount oflipids. This relatively low encapsulation efficiency can be due to theencapsulation process. In fact, encapsulation efficiency using thethin method is usually low. As reported in the literature [47], E% ofdifferent antibiotics like Ciprofloxacin, gentamicin and meropenemin cationic vesicles formulated with PC/DOPE/DOTAP was in therange of 4.5–5.7. Using an active drug encapsulation method the E%of Ciprofloxacin increased up to a 67% [48]. Reverse-phase evapora-tion yields a high-efficient encapsulation however the presence ofresidual toxic solvents precludes its use in pharmaceutical appli-cations. The low E% obtained in this work can also be explainedby electrostatic repulsions between positively charged vesicles anddrug molecules, especially in the case of Ciprofloxacin which haspositive charges. It has been found that the gentamicin policationicdrug trapped in cationic vesicles showed an E% of about 3% whilethe efficiency of the encapsulation of this antibiotic in anionic vesi-cles increased up to 8–9% [49]. Moreover, hydrophilic drugs such asCiprofloxacin and 5-FU are located exclusively in the aqueous com-partment of liposomes and in general present low entrapment. Alarge number of drug molecules have been structurally modified torender them more lipophilic character in order to increase theirentrapment efficiency. The percentage of 5-FU retained in lipo-somes is 0.03% while for the modified lipophilic 5-FU derivativeis 99% [50].

To improve the entrapment, many drugs have been structurallymodified; however, our cationic formulations can encapsulatehydrophilic drugs without hydrophobic modifications and containtherapeutic amounts of drug. Moreover, their E% is higher thanthose reported for other liposomal formulations using similar drugsand the same encapsulating procedure. E% for 1010R C and 1414R Cwere 8.90% and 15.11%, respectively, what means that E% dependson the alkyl chain length. These results suggest that 1414R pro-motes a large space between adjacent bilayers and consequentlymore quantity of drug can be encapsulated. A similar trend wasdescribed in [51,52]. In [51] authors reported that the alkyl chainlength affects the permeability of vesicles and that a long alkylchain in the surfactants resulted in high drug encapsulation. In [52]Ciprofloxacin showed the highest E% value for the biggest cationicvesicles. These results suggest that the longer the alkyl chain thehigher the drug encapsulation. It can be also observed that the high-est entrapment efficiency was obtained for 5-FU, probably due tothe absence of charged groups on this drug. In this case, the E% alsodepends on the alkyl chain length.

By increasing the DPPC percentage in the 1010-based formula-tions the E% slightly increases. These data suggest that the cationiccharge on the surface affects the encapsulation efficiency. It hasbeen reported that DPPC may hinder the condensation of thebilayer membrane [36] thus both the permeability of the vesi-

cles membrane and drug encapsulation enhance [53]. For the1414R surfactant, the highest E% was 15.7% corresponding to the1414R/DPPC 8:2 C formulation. An increase of the DPPC concen-tration (1414R/DPPC 5:5 C), decreased the E% to 13.0%. Notice that

L. Tavano et al. / Colloids and Surfaces B: Biointerfaces 120 (2014) 160–167 165

F 414R1

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ig. 2. (a) In vitro Ciprofloxacin release profile at pH 7.4: (• drug; (�) 1010R 1C; (�) 1 5-FU. (mean ± SE; n = 3).

ompared to the other formulations, the 1414R/DPPC 5:5 C has veryow zeta-potential and diameter size.

These results suggest that the E% of loaded Ciprofloxacin isffected by several factors, among which the repulsions betweenhe similarly charged moieties seem to be the most important [54].he presence of DPPC results in a reduction of surface cationicharge, as confirmed by the zeta-potential values. This leads to

reduction of the potential repulsions between surfactant andrug, which could be encapsulated more easily. In addition, theembrane additive nature of DPPC determines the bilayer fluid

haracter.

.4. Drugs release studies

Experimental profiles of in vitro release of Ciprofloxacin and 5-U from the vesicles is show in Figs. 2 and 3. The release of freeiprofloxacin and 5-FU was used as control. The pH of the releaseedium was 7.4, therefore, Ciprofloxacin was present in its zwit-

erionic form.Fig. 2a shows the release profile of Ciprofloxacin from 1010R

and 1414R C samples. The total amount of Ciprofloxacin fromhe control solution was released in 3 h. As expected, the releaseates of drug from vesicles are lower than those obtained by freerug solution. The in vitro release results are consistent with the E%alue. The vesicular system based on 1414R C showed the highestntrapment efficiency, 15.1, and the lowest drug release percentagefter 24 h, 69%, while for the 1010R C sample with an efficiency of.9, the percentage of drug released after 24 h was 87%. The kineticsf Ciprofloxacin release is biphasic, with a fast release followed by

slow release.Fig. 2b shows the release profile of 5-FU. The free drug solution

otally releases the 5-FU within 2 h while the release of 1010R and

414R based vesicles within 6 h is of about 75% and 60%, respec-ively. It can be also observed that the higher the E% the slower theelease. In the case of 5-FU loaded vesicles an initial burst release isbserved. The burst release takes about 1 h and the amount of 5-FU

ig. 3. (a) In vitro Ciprofloxacin release profile at pH 7.4: (• drug; (�) 1010R/DPPC 8:2 C; (010R/DPPC 5:5 C; (�) 1414R/DPPC 5:5 C. (mean ± SE; n = 3).

1C; (b). In vitro 5-FU release profile at pH 7.4: (• drug; (�) 1010R 1 5-FU; (�) 1414R

released within this time is 50% for 1010R-5FU and 40% 1414R-5FU.The presence of this burst suggests that some 5-FU is bound weaklyon the surface of the vesicles. After the burst the 5-FU release kinet-ics is biphasic. Research described in the literature indicates thatliposomal formulations containing 5-FU show similar release pat-terns [55]. This behaviour is typical of compounds that are notsimply entrapped within the aqueous compartments of a vesicu-lar carrier, but also have the ability to interact with the vesicularcarrier bilayer. This ability enhances the vesicle loading power andprovides drug release profiles which result from the drug perme-ating through the bilayers and the drug retained in the bilayers[56].

Profiles of drug release measured for surfactants/DPPC sampleare given in Fig. 3 where trends similar to those showed by 1010Rand 1414R can be observed. The highest drug release profile (99%)was achieved for the sample that showed the lowest entrapmentefficiency (1010R/DPPC 8:2, 11.0%) and the lowest Ciprofloxacinrelease (50%) was achieved by the sample with the highest value ofE% (1414R/DPPC 8:2, 15.7%) (Fig. 3a). Moreover, the release profilesalso follow a biphasic release pattern.

These results agree with those previously published in the liter-ature for liposomes, aggregates of which can control the releaseof drugs [9]. Besides, our studies indicate that the release ofCiprofloxacin can be sustained and controlled by encapsulating thedrug in vesicles allowing a new delayed delivery systems. In ourstudies we found that an increase in the surfactants alkyl chainlength resulted in a reduction in drug release. This result agreeswith the fact that bilayer rigidity increases with the alkyl chainlength giving bilayers with superior stability and low permeability.Hence, superior drug retention and affinity can be observed [57].

3.5. Antimicrobial activity

The vesicle encapsulation of Ciprofloxacin has been demon-strated to greatly improve its pharmacological efficacy [9]. In thiswork, the antimicrobial activity of diacyl glycerol arginine-based

�) 1414R/DPPC 8:2 C; (b) In vitro ciprofloxacin release profile at pH 7.4: (• drug; (�)

1 ces B: Biointerfaces 120 (2014) 160–167

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esicles against S. aureus, E. coli and K. pneumoniae strains haseen studied. The MIC of empty and Ciprofloxacin-loaded vesi-les was evaluated with the tested bacteria after 24 h interactionime. The MIC of Ciprofloxacin was evaluated and taken as con-rol. Ciprofloxacin shows, MIC values of 0.5, <0.25 and <0.25 �g/mLgainst the three bacteria tested. These results agree with thoseeported by Gubernator et al. [48].

Table 4 includes the MIC values of the empty and Ciprofloxacin-oaded vesicles based on 1010R and 1414R surfactants, as pureomponents or mixed with DPPC.

It is worth noting that values in Table 4 show that vesicles basedn the 1010R surfactant have by themselves good activity against. coli and moderate activity against S. aureus and K. pneumoniae.his means that these systems can act as carriers and antimicro-ial agents simultaneously. In general, the antimicrobial activity ofhese liposomal formulations is lower than those shown by micel-ar solutions also based on arginine-surfactants [58]. It seems that

onomers or micelles can interact with biological membranes ofacteria better than vesicles do. The incorporation of DPPC into theilayer results in an important increase of MIC values. The trendf antimicrobial activity was maintained for the 1010R/DPPC 8:2:. coli < S. aureus < K. pneumoniae. By increasing the DPPC ratio up to0%, the formulation became inactive against S. aureus and K. pneu-oniae and less active against E. coli. Direct interactions between

esicles and bacterial cells depend on the cationic charge on theesicles surface and the zeta-potential of these vesicles decreases ashe DPPC ratio increases. In fact, it has been described that the cyto-oxicity of cationic surfactants can be reduced using mixed cationicipid/neutral lipid vesicles [28].

Ciprofloxacin was very active against Gram-positive and Gram-egative bacteria, hence introducing this drug strongly affects thentibacterial activity of the formulations. MIC of loaded vesicleseems to be governed by the Ciprofloxacin concentration. It isoteworthy that the antibiotic encapsulated in these cationic vesi-les maintains its high antimicrobial activity against Gram-positivend Gram-negative bacteria. All the formulations are very activegainst E. coli, in fact the MIC values are lower than the lowestoncentration used to determine the MICs. The 1010R/DPPC 5:5 Cample showed the greatest antimicrobial activity. This unexpectedehaviour could be related to the different physicochemical prop-rties shown by the 1010R/DPPC 5:5 C formulation which showshe highest E% and the slowest antibiotic release. These propertiesan promote the diffusion of a large and constant number of drugolecules into the cell.In general, formulations based on 1414R present lower antimi-

robial activity than their 1010R homologues and the introductionf DPPC in these vesicles dramatically reduces the antimicrobialctivity. This behaviour can be explained by the fact that 1414Resicles present bigger size and lower zeta-potential than the cor-esponding 1010R ones. Previous studies also showed that thentimicrobial activity of diacyl glycerol arginine based surfactantsecreases as the alkyl chain length increases [16].

As expected, with the presence of Ciprofloxacin the antimicro-ial activity clearly increases. MIC is governed by the antibioticoncentration. The formulations studied are active against thehree bacteria considered. For the 1414R, the highest activity wasxhibited by the 1414R/DPPC 8:2 C formulation (sample with theighest E%). As the percentage of DPPC increases up to 50%, theeta-potential decreases. Perhaps the limited cationic charge of414R/DPPC 8:2 contributes to the slight reduction of the antimi-robial efficiency of this formulation.

To summarize, we can conclude that the antibacterial activity of

ur formulations depend on the composition of the drug carriers, itslobal surface charge and the hydrophobic properties. In general,010R-based formulations are more active than the 1414R-based,

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y the surfactant alkyl chain length used for their preparation.he antibacterial activity of the formulations also are influencedy the vesicles permeability to the drug. The Ciprofloxacin releaserom 1010R-based samples was always higher than that obtainedrom the corresponding formulation based on the 1414R surfac-ant. All these facts may explain the highest antibacterial activityssociated with the 1010R-based formulations. Finally, it haseen found that the presence of DPPC as a membrane additiveeduces the antimicrobial efficiency of the cationic vesicles withoutrug, but improves the antimicrobial activity vesicles loaded withiprofloxacin.

. Conclusions

Surfactants from arginine can form stable cationic vesicularystems by themselves as well as in the presence of DPPC as a mem-rane additive. The physicochemical properties of these vesicles areodulated by the alkyl chain length of the surfactant and by the

urfactant/DPPC ratio: the mean diameter of the vesicles increasesith the alkyl chain length of the cationic lipid, and diminishes with

he introduction of DPPC. Ciprofloxacin and 5-FU can be loaded ontohese cationic systems and their in vitro release from all formu-ations was effectively delayed with respect to the correspondingree drug solutions. The E% as well as the permeability of the vesi-les seems to depend on the physicochemical properties of bothesicles and drug.

Our systems naturally have antimicrobial properties againsthe three bacterial strains tested. The antimicrobial efficacy ofhe vesicles based only on cationic lipids is strongly affectedy the hydrophobicity of lipids and the zeta-potential values ofhe dispersions. The introduction of DPPC strongly decreases thentimicrobial activity of these systems. On the other hand, theiprofloxacin encapsulated in these vesicles preserves its antimi-robial activity.

The results of this work suggest that formulations based on vesi-les prepared with these pH sensitive surfactants and containingiprofloxacin or 5-FU, represent a great innovation in the pharma-eutical field, because of their dual pharmacological function: oneelated to the nature of the encapsulated drug and the other relatedo the innate antibacterial properties of the surfactant-based carri-rs.

cknowledgments

The authors would like to thank the financial support frompanish Plan National I+D+I MAT2012-38047-C02-02, AGAUR 2009RG 246 and CTQ2010-14897. Moreover, the project has been co-unded with support from the Commission European Social Fundnd Region of Calabria (Italy) and the contract Estancia de Jóvenesoctores Extranjeros en Espana, MEC, SB2010-0129.

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