Biomed Microdevices (2007) 9:421–433DOI 10.1007/s10544-007-9046-6

In vitro and in vivo evaluation of Bola-surfactant containingniosomes for transdermal deliveryDonatella Paolino · Rita Muzzalupo ·Antonio Ricciardi · Christian Celia · Nevio Picci ·Massimo Fresta

Published online: 25 January 2007C© Springer Science + Business Media, LLC 2007

Abstract A novel niosome formulation is proposed fortopical drug delivery of ammonium glycyrrhizinate, a nat-ural compound with an efficacious anti-inflammatory ac-tivity. Niosomes were made up of a new non ionicsurfactant, α,ω-hexadecyl-bis-(1-aza-18-crown-6) (Bola-surfactant)-Span 80-cholesterol (2:3:1 molar ratio). Nio-some vesicles were prepared with the thin layer evapo-ration method and were physico-chemically characterized.The tolerability of Bola-surfactant both as free moleculesor assembled ion niosome vesicles was evaluated in vitroon cultured of human keratinocyte cells (NCTC2544). Hu-man tolerability was evaluated on volunteers. The ability ofBola-niosomes to promote intracellular delivery was evalu-ated by confocal laser scanning microscopy (CLSM) studies.Human stratum corneum and epidermis (SCE) membraneswere used in vitro to investigate the percutaneous perme-ation. The anti-inflammatory activity of ammonium gly-cyrrhizinate was evaluated in vivo on human volunteers witha chemically induced erythema. Experimental data show thatBola-niosomes are characterized by a mean size of ∼ 400 nmand are able to provide an encapsulation efficiency of 40%with respect to the drug amount used during preparation.

Part of this research was presented at the 33rd Annual Meeting of theControlled Release Society in Vienna, Austria, July 22–26, 2006.

D. Paolino · C. Celia · M. Fresta (�)Department of Pharmacobiological Sciences, Faculty ofPharmacy, University “Magna Græcia” of Catanzaro, CampusUniversitario, Building of Biosciences,Viale Europa, I-88100 Germaneto (CZ), Italye-mail: fresta@unicz.it

R. Muzzalupo · A. Ricciardi · N. PicciDepartment of Pharmaceutical Sciences, University of Calabria(UNICAL), area Polifunzionale,I-87036 Arcavacata di Rende (CS), Italy

CLSM showed that Bola-niosomes were able to promotethe intracellular uptake of the delivered substances. Bola-niosomes were also able to significantly improve (p < 0.001)the percutaneous permeation of ammonium glycyrrhizinatewith respect to both the aqueous drug solution and a physicalmixture between unloaded Bola-niosomes and the aqueousdrug solution. Bola-niosomes showed a suitable tolerabil-ity both in vitro and in vivo. Ammonium glycyrrhizinate-loaded Bola-niosomes determined a significant (p < 0.001)and noticeable improvement of the in vivo anti-inflammatoryactivity of the drug. An effective example of conjugatinginnovative colloidal carriers, coming from pharmaceuticalnanotechnology, and therapeutically effective natural com-pounds, coming from traditional medicine, was reported.

Keywords Niosomes . Bola surfactant . Skin delivery .

Ammonium glycirrhizinate . Human skin percutaneouspermeation . Human anti-inflammatory in vivo evaluation

1 Introduction

The self assembly of non-ionic surfactants into vesiclesrepresents an interesting opportunity to achieve vesicularcolloidal drug carriers (the so-called niosomes), whichresemble liposomes in their architecture and can be used asan effective alternative to liposomal drug carriers (Uchegbuand Vyas, 1998).

Niosomes may be made up of a variety of amphiphilesbearing sugar, polyoxyethylene, polyglycerol, crownether and amino acid hydrophilic head groups and theseamphiphiles typically possess one to two hydrophobic alkyl,perfluoroalkyl or steroidal groups (Arunothayanun et al.,2000; Manconi et al., 2002). Among various components,amphiphiles bearing crown ethers were synthesized in an

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422 Biomed Microdevices (2007) 9:421–433

attempt to obtain new surfactants with biocompatible polarheadgroups for particular technological applications. Thelatest generation of this kind of surfactants is representedby amphiphiles having two polar crown ether headgroupslinked to both ends of a long alkyl chain (Muzzalupoet al., 1996; Caponetti et al., 2004). These amphiphiles aregenerally referred to as bolaform surfactants and can beanionic, cationic, zwitterionic or nonionic as a function ofthe kind of ion, which can be complexed by the two crownether headgroups (McKenzie et al., 1987; Muzzalupo et al.,1996). Bolaforms have recently been used as componentsfor the preparation of niosomes (Muzzalupo et al., 2005).

Niosomes have been proposed for a number of poten-tial therapeutic applications, i.e. as immunological adjuvants(Jain et al., 2005), anti-cancer and anti-infective drug target-ing agents (Balasubramaniam et al., 2002; Gude et al., 2002),carriers of anti-inflammatory drugs (Shahiwala and Misra,2002) and as diagnostic imaging agents (Uchegbu and Vyas,1998). In addition, niosomes are versatile carrier systemsthat can be administered through various routes. Particu-lar efforts have been aimed at using niosomes as effectivetransdermal drug delivery systems (Barry, 2001; Choi andMaibach, 2005).

In fact, skin represents the principal natural barrier to drugpermeation and hence the principal limit to percutaneoustherapy. The aim of this work was the preparation of avesicle formulation to increase the activity of ammoniumglycyrrhizinate, a natural active compound with well-knownanti-inflammatory effects (Baltina, 2003; Matsui et al.,2004), following skin application. In a recent paper (Paolinoet al., 2005), we proposed ethosomes as a successfultopical carrier of ammonium glycyrrhizinate. In this studythe potential dermal application of innovative niosomesmade up of a novel non ionic surfactant, which has thecharacteristic of assembling in a bilayer form and alsobehaving as an ion complexing agent, was investigated.Therefore, a polyfunctional carrier is achieved.

In this study, free surfactant and niosome toxicity werefirst evaluated to assess formulation safety. The percutaneouspermeation of free or niosome-encapsulated ammonium gly-cyrrhizinate through excised human skin was evaluated. Am-monium glycyrrhizinate-loaded niosomes were then tested invivo on human volunteers to determine the effective percu-taneous absorption and the anti-inflammatory activity withrespect to the free drug.

2 Material and methods

2.1 Chemicals

α,ω-hexadecanedioic acid, 1-aza-18-crown-6 thionyl chlo-ride, toluene, lithium aluminum hydride, tetra-hydro-

furane (THF), cholesterol (Chol), amphotericin B so-lution (250 µg/ml), trypan bleu solution (0.4% v/v),3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bro-mide (MTT) dye test (TLC purity ≥ 97.5%), am-monium glycyrrhizinate and phosphate buffer solu-tion (PBS) were purchased from Sigma Chemi-cals Co. (St. Louis, USA). Sorbitan mono-oleate80 (Span 80) was obtained from ACEF Spa (Pia-cenza, Italy). N-(fluorescein-5-thiocarbamoyl)-1,2-dihexa-decanoyl-sn-glycero-3-phosphoethanolamine, tryethylam-monium salt (Fluorescein DHPE) was purchased fromMolecular Probe (Eugene, Oregon, USA). Double distilledpyrogen-free water from Sifra S.p.A. (Verona, Italy) wasused in the experiments. Sterile saline solution (Freke-nius Kabi Potenza S.r.l., Verona, Italy) was used to ob-tain noisomes. Human keratinocyte cells (NCTC2544)were obtained from Istituto Zooprofilattico della Lombar-dia e dell’Emilio Romagna (Brescia, Italy). DMEM culturemedium, fetal bovine serum (FBS), penicillin-streptomycinsolution and Trypsin/EDTA (1×) solution were obtainedfrom GIBCO (Invitrogen Corporation, Giuliano Milanese(Mi), Italy). All other materials and organic solvents were ofanalytical grade (Carlo Erba, Milan, Italy).

2.2 Synthesis of aza crown ether surfactant

α,ω-hexadecyl-bis-(1-aza-18-crown-6) (Bola C16) was syn-thesized from N-aza-18-crown-6 and α,ω-hexadecanedioicacid by using the experimental procedure reported in aprevious work (Muzzalupo et al., 1996). Briefly, α,ω-hexadecanedioic acid in thionyl chloride reacted thus form-ing a diacyl chloride derivate. The product obtained wasmixed stoichiometrically with N-aza-18-crown-6 in the pres-ence of thriethylamine thus forming an aza-crown-ether sur-factant compound. Carboxylic groups of surfactant derivatewere then reduced by using lithium aluminum hydride in thepresence of dry tetrahydrofuran (THF) reagent. Organic sol-vent was evaporated by using an Heidolph Laboratory Dig-ital 4010 rotary evaporator (Heidolph Instruments GmbH& Co., Schwabach, Germany) and α,ω-hexadecyl-bis-(1-aza-18-crown-6) was extracted and crystallized by using acold ether solution. Synthesized Bola surfactant was char-acterized by 1H-NMR, FT-IR spectroscopy melting point,and presented physicochemical properties in agreement withpreviously reported investigations (Muzzalupo et al., 1996;Caponetti et al., 2004).

2.3 Niosome preparation

Niosomes containing Bola-surfactant were prepared fol-lowing the film hydration method normally used forphospholipid-based vesicle preparation (Arunothayanunet al., 2000; Manconi et al., 2003). A mixture (20 mg) of

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Bola surfactant, Span r© 80 and Chol (2:3:1 molar ratio) wasdissolved with 3 ml of chloroform in a round-bottomed flask.Organic solvent was removed by using a Heidolph Labora-tory Digital 4010 rotary evaporator (Heidolph InstrumentsGmbH & Co., Schwabach, Germany) thus obtaining a thinfilm. Fluorescein-DHPE at 0.1% molar concentration wasadded to surfactants and Chol to achieve labeled niosomes.Surfactants and Chol films were kept over night under vac-uum at room temperature using a Buchi T51 glass dryingoven connected with a vacuum pump to remove any tracesof the organic solvent. Thin films were hydrated with 2 mlof a pH 7.4 isotonic aqueous solution of ammonium glycir-rhizinate (3 mg/ml) under mechanical stirring (700 rpm) ata temperature of 70◦C, above the gel-liquid transition tem-perature of Span r© 80. Niosome colloidal suspensions thusobtained were maintained at room temperature for 12 h toachieve stable colloidal vesicles. When necessary empty nio-somes were prepared by hydrating films with a sterile salinesolution.

2.4 Loading capacity of niosomes

Ammonium glycyrrhizinate associated with niosomes wasseparated from the untrapped drug using a centrifugation-purification method. Niosomes were centrifuged with aBeckman AvantiTM 30 centrifuge equipped with a F1202fixed angle rotor (Beckman Coulter Inc., Fullerton, CA,USA) at 58000 × g for 1 h at a controlled temperatureof 4◦C. Supernatant containing untrapped ammonium gly-cyrrhizinate was withdrawn, filtered through a 0.22 µm poresize nylon membrane (Whatman Inc., Clifton, NJ, USA) andsubmitted to HPLC analysis. The amount of ammonium gly-cyrrhizinate associated with niosomes was calculated as thedifference between the amount of drug added during prepa-ration and the amount of drug determined in the supernatant.The loading capacity was expressed as the percentage ofammonium glycyrrhizinate that became niosome-associated.Niosome loading capacity data were obtained from five dif-ferent experiments and the results are expressed as the meanvalue ± standard deviation.

2.5 Physicochemical characterization of niosomes

Mean size and polidispersity index of niosome colloidalvesicles were measured at 25◦C with a laser light scatter-ing technique using a Nicomp mod. 370 submicron parti-cle sizer, equipped with a 4.5 mW laser diode light sourceset at 670 nm. Experiments were carried out at a scatter-ing angle of 90◦. Before analysis, all samples were suitablydiluted with filtered sterile saline solution to avoid multi-scattering phenomena and then poured into quartz cuvettes.The polidispersity index of niosomes was performed as ameasurement of the size distribution of the colloidal system.

A third-order cumulant fitting correlation function was ap-plied using a Nicomp mod. 370 submicron particle sizer toachieve mean size and polidispersity index values of variousniosome samples. Polydispersity index values ≤ 0.2 wereconsidered as evidence of a homogeneous size distributionof the colloidal vesicles, while values ≥ 0.5 showed a het-erogeneous distribution of colloidal vesicles. Mean size andpolidispersity index values were the result of five differentexperiments (ten measurements per experiment) and wereexpressed as the average values ± standard deviation.

2.6 Transmission electron microscopy (TEM)

Morphology analysis of niosomes was carried out usingtransmission electron microscopy. A drop of the niosomecolloidal suspension was placed on a carbon-coated coppergrid and left for 1 min thus allowing niosomes to adhereto the grid. The excess of the niosome suspension was 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, was placed on the carbon gridthus staining the niosomes. After 3 min, the excess stainingagent was removed by adsorbing the drop with the tip ofa filter paper and the sample was then air-dried. Niosomesamples were examined by using a ZEISS EM 900 electronmicroscope operating at an accelerating voltage of 80 kV.

2.7 Ammonium glycirrhizinate release from niosomes

The drug release from niosome suspensions was evalu-ated by using a Franz diffusion cell. A synthetic membrane(molecular cut-off 10000 dalton) was placed between donorand receptor compartment. An ammonium glycyrrhizinate-loaded niosome suspension (200 µl) was placed in the donorcompartment with a drug concentration of 3 mg/ml. Ster-ile saline solution was used as the receptor phase, whichwas constantly stirred throughout the release experimentsand warmed (GR 150 thermostat, Grant Instruments Ltd,Cambridge, UK) to 37 ± 0.1◦C. Sink conditions were ob-served. At predetermined time intervals (every hour over 24 hof incubation) 1 ml of the receptor compartment fluid waswithdrawn and then immediately replaced with the same vol-ume of fresh fluid. Release samples were submitted to HPLCanalysis to determine the amount of ammonium glycyrrhiz-inate. The drug release was expressed as the percentage ofdrug released with respect to the amount associated withniosomes.

2.8 Percutaneous permeation of ammoniumglycyrrhizinate-loaded niosomes

Continuous flow Franz diffusion cells were used to evalu-ate in vitro the percutaneous permeation of free and nioso-

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mally entrapped ammonium glycyrrhizinate through humanstratum corneum and viable epidermis (SCE) membranes.SCE membranes were prepared using fresh abdominal hu-man skin obtained from plastic surgery. The stratum corneumand viable epidermis were separated from subcutaneous fattissue according to the method proposed by Kligman andChristophers (1963). All experiments were performed within24 h of surgical removal of the skin. Continuous flow Franzdiffusion cells were characterized by a 0.75 cm2 diffusionsurface area and a nominal receptor volume of 4.75 ml. Thereceptor chamber was filled with a sterile saline solution andstirred throughout permeation experiments at 600 rpm witha small magnetic anchor to ensure a continuous and constantshaking of the compartment. SCE membranes were placedbetween the donor and receptor compartment with the stra-tum corneum side up and this system was allowed to equili-brate for 6 h. The donor compartment was filled with 200 µlof free or niosomally entrapped ammonium glycyrrhizinate.A steady state condition was maintained during experimentalinvestigations. The experiments were carried out in non oc-clusive conditions for 24 h at a thermostated temperature of35 ± 1◦C. A minimum of three diffusion cells were contem-porarily used for each formulation and 1 ml of each samplewas withdrawn every 1 h up to 24 h of incubation by usingan FC 204 fraction collector (Gilson Italia S.r.l., CiniselloBalsamo (MI), Italy) connected to a Minipuls 3 peristalticpump (Gilson Italia S.r.l., Cinisello Balsamo (MI), Italy).The volume withdrawn was replaced by the same volume offresh receptor phase. Samples collected from the receivingcompartment was analyzed by HPLC. Six different experi-ments were carried out for each formulation and the resultsare expresses as the mean value ± standard deviation.

2.9 HPLC determination of ammonium glycirrhizinate

A Hewlett-Packard model 251 liquid chromatographic sys-tem (Hewlett-Packard, Milan, Italy) equipped with a 20 µlRheodyne model 7125 injection valve (Rheodyne, Cotati,CA) was used for HPLC determination of ammoniumglycyrrhizinate. A reverse phase SUPELCO C-18 column(3 µm, 150 × 4.6 mm i.d.) was used for the analysis. TheHPLC apparatus was connected to a UV detector set at awavelength of 254 nm (λmax of ammonium glycyrrhizinate).HPLC determination was carried out at room temperature.The mobile phase was acetonitrile/water/0.1 N phosphoricacid (79.95/20/0.05 v/v/v) and was pumped at a flow rateof 0.5 ml/min. Ammonium glycyrrhizinate had a retentiontime of 3.97 min. The analytical determination of ammo-nium glycyrrhizinate was carried out by using the followingcalibration curve

y = 3 · 107x + 57678 (1)

where y is the peak area and x the drug concentration (mM),with an r2 of 0.9998.

2.10 NCTC 2544 cell cultures

Human keratinocyte cells (NCTC 2544) were transferredfrom cryovial tubes to plastic culture dishes (100 mm di-ameter) and seeded (5% CO2) for three days at 37◦C usingDMEM supplemented with penicillin (100 UI/ml), strepto-mycin (100 µg/ml), amphotericin B solution (1% v/v) andFBS solution (10% v/v) as the culture medium throughout allin vitro experiments. After the incubation time, NCTC 2544cells reached a confluence of 80% and were treated with2 ml of trypsin solution, washed with 2 ml PBS, transferredinto 10 ml plastic centrifuge tubes and harvested. Four mlof culture medium were added to human keratinocytes thusachieving a final volume of 8 ml. Samples thus obtainedwere centrifuged using a Megafuge 1.0 (Heraeus Sepatech)at 1200 rpm for 10 min at room temperature. Pellets were sep-arated from the supernatant and resuspended with 6 ml of cul-ture medium to obtain 1 × 106 cells/ml. The cell suspensionwas diluted up to a final concentration of 3 × 104 cells/mland 1 ml of the diluted suspension was seeded into 12 wellplastic culture dishes before in vitro investigations.

2.11 In vitro evaluation of toxicity

Cytotoxic effects of pure Bola-surfactant and Bola-niosomeson NCTC 2544 cells were evaluated with the trypan bluedye exclusion assay (cell mortality) and the MTT dyetest (cell viability). NCTC 2544 were seeded at a den-sity of 3 × 104 cells/ml in 12-well plastic culture dishesfor the trypan blue dye exclusion test and at a density of1.8 × 103 cells/100 µl in 96-well tissue culture plates for theMTT test. After 24 h of incubation the culture medium wasreplaced for each well by fresh culture medium and pureBola-surfactant or Bola-niosomes were added at final scalardilutions (0.01, 0.1, 1, 10 µM in surfactant concentration).The toxicity experiments were carried out at three differentincubation times, i.e. 24, 48 and 72 h. Untreated NCTC2544cells and empty niosome vesicles were used as the controland the blank sample in various experiments, respectively.

To perform the trypan blue dye exclusion assay,NCTC2544 were harvested using trypsin/EDTA (1×) solu-tion (2 ml), washed twofold with phosphate buffer solution(2 ml) and transferred into 5 ml plastic centrifuge tubes.Culture medium (4 ml) was added to plastic centrifuge tubesup to a final volume of 8 ml. Samples were then centrifugedwith a Megafuge 1.0 (Heraeus Sepatech) at 1200 rpm atroom temperature for 10 min. The supernatant was with-drawn and the pellet was suspended in 200 µl of trypan bluebuffer, re-suspended for 30 s and the amount of dead cells(blue stained cells) was observed with a hematocytometer

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chamber using an optical microscope (Labophot-2, Nikon,Japan). The percentage of cell mortality was calculatedusing the following equation:

% Mortality = (CD/CT ) × 100 (2)

where CD is the number of dead cells and CT the total num-ber of cells. Cell viability assay was carried out followingthe MTT dye test. NCTC2544 cells were seeded at a densityof 1.8 × 103 cells/100 µl in a 96-well culture plates for 24 hat 37◦C and 5% CO2 to allow the adhesion of culture cells.After 24 h of incubation, the culture medium was removedand substituted with fresh medium, human keratinocyte cellswere then treated with Bola-surfactant or Bola-niosomes.10 µl of MTT tetrazolium salt (5 mg/ml dissolved in PBSbuffer) were added to each well at the end of incubationtime and NCTC2544 cells were incubated for an additional3 h, thus allowing the formation of violet formazan crystals.A mixture (200 µl) of dimethylsulfoxide/ethanol (1:1 v/v)was used to dissolve the formazan crystals and the 96-wellculture plates were gently shaken at 230 rpm (KS 130 Con-trol, IKA r© WERKE GMBH & Co., Staufen, Germany) for20 min. Cell viability of different samples was determinedby an ELISA microplate reader (Labsystem mod. MultiskanMS, Midland, ON, Canada) set at a wavelength of 570 nm inabsorption and 670 nm in emission. The percentage of viablecells, directly proportional to the amount of formazan crys-tals formed, was calculated using the following equation:

% Cell viability = (AbsT /AbsU ) × 100 (3)

where AbsT is the absorbance of treated cells and AbsU isthe absorbance of untreated cells. Values of cell mortalityand cell viability are expressed as the mean of six differentexperiments ± standard deviation.

2.12 Confocal laser scanning microscopy (CLSM)

The interaction between NCTC 2544 cells and Bola-niosomes was evaluated by means of confocal laser scan-ning microscopy. Cells were seeded in 6-well plastic culturedishes at 3 × 105 cells/ml. After 24 h, NCTC 2544 weretreated with a niosome formulation labeled with fluorescein-DHPE for the different incubation times from 3 h up to 24 h.NCTC 2544 cells were then washed twofold with 2 ml of pH7.4 PBS and fixed for 3 min on the glass cover slides by using1 ml of an ethanol solution (70% v/v). Samples thus obtainedwere placed on a glass holder and treated with a cooled glyc-erol solution (90% v/v) for 5 min. The glass holder was sealedby using a transparent glue to avoid air inclusion in the sam-ples. Fluorescent observations were performed using a LeikaTCS SP2 MP confocal laser scanning microscope equippedwith an argon ion laser beam set at 496 nm for the excitation

input and 519 nm for the emission output. A scan resolutionup to 4096 × 4096 pixels with an Ar/Kr laser beam of 75 mW,equipped with a fluorescein analyzer filter, was used for ex-perimental investigations. Samples were acquired by a macrodeveloper software package having multi-dimensional seriesacquisition and direct-access digital control knobs.

2.13 In vivo evaluation of skin tolerability

Skin tolerability of Bola-surfactant and Bola-niosomes wasevaluated on human volunteers by means of the non invasivetechnique of reflectance spectrophotometry. The reflectancespectrophotometer, SP60 (X-Rite Incorporated, USA), cor-related at a 0◦ illumination and viewing angle, was calibratedwith the supplied white standard traceable to the NationalBureau of Standard’s perfect white diffuser. The spectropho-tometer was connected to a personal computer, which per-formed different color determinations from the spectral datausing the X-Rite acquisition and evaluation software. Re-flectance spectra were observed over a wavelength range400–700 nm using an illuminant C and 2◦ standard observer.The investigation was performed on twelve healthy volun-teers who gave their written informed consent before enteringthe study after receiving full details of the procedure and whatwas involved. Subjects did not suffer from any ailments andthey were not under any pharmacological therapy for at least1 week before this experiment. Human volunteers rested for30 min at room temperature (22 ± 2◦C and 40 ± 5% rela-tive humidity) before the experiment. Six sites on the ventralsurface of each forearm were defined using a circular tem-plate (1 cm2). The distance between sites was at least 2 cmin order to avoid any possible interference. Erythema index(EI) baseline values were recorded at each designated sitebefore application of formulations and then Bola-niosomesor Bola-surfactant were applied on two sites using Hill Topchambers (Hill Top Research, Inc., Cincinnati, Ohio, USA).Before reflectance spectrophotometric readings, the cham-bers were removed and the skin surface was gently washedwith water to remove the applied formulation and the skinwas allowed to dry for 15 min. The possible induced ery-thema was monitored at various times, 6, 24, and 48 h andwas calculated according to the following equation (Paolinoet al., 2005):

E.I. = 100

[log

1

R560+ 1, 5

(log

1

R540+ log

1

R580

)

−2

(log

1

R510+ log

1

R610

) ](4)

where 1/R is the inverse reflectance at a specific wavelength(510, 540, 560, 580, 610). The results are expressed as differ-ences between the EI values measured after the application

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of various formulations and the EI baseline values measuredat the beginning of the experiment (�EI).

2.14 In vivo evaluation of anti-inflammatory activity

The efficacy of ammonium glycyrrhizinate-associated Bola-niosomes in reducing the chemically induced erythema incomparison with aqueous solution of ammonium glycyrrhiz-inate was evaluated in vivo on healthy volunteers. Six siteson the ventral surface of each forearm were randomly de-fined using a circular template (1 cm2) and demarcated withpermanent ink. The distance between sites was at least 2 cmto avoid any possible interference. For each volunteer (a totalof six), all the sites were treated with an aqueous solution(100 µl) of methyl-nicotinate (0.2% w/v) for 15 min usingHill Top Chambers. At the end of this period the chamberswere removed and the skin surface was gently washed withwater to remove the applied formulation and then 200 µl ofthe Bola-niosomes or the simple aqueous solution contain-ing ammonium glycyrrhizinate (0.3% w/v) were applied.A saline solution (NaCl 0.9% w/v) (200 µl) was used asthe blank. The induced erythema was monitored until itsdisappearance. The reduction of erythema was directly pro-portional to the amount of ammonium glycyrrhizinate per-meated through the skin from the examined formulations(Paolino et al., 2005).

2.15 Statistical analysis

Data analysis of experimental investigations was performedusing a one-way ANOVA test. Statistically significant dif-ferences were calculated using a Bonferroni t-test to checkthe ANOVA test. A p ≤ 0.05 obtained from different analy-ses was considered as the minimal level of significance. Allvalues are reported as the mean ± standard deviation.

3 Results and discussion

3.1 Physicochemical characterization of niosomes

Bola-surfactant is not able per se to form stable bilayerstructures, such as vesicles, but micelles and rod-like mi-celles are formed when Bola-surfactant is dispersed in water(Eggers et al., 2003; Muzzalupo et al., 2006). In a previ-ous investigation (Muzzalupo et al., 2005), it was shownthat vesicles were formed when Bola-surfactant was mixedwith Chol, thus achieving niosome colloidal suspensions.In this paper, the efficacy of the Bola-surfactant to form acolloidal formulation with improved physicochemical prop-erties as a drug delivery system was investigated. In thisattempt a ternary lipid/surfactant system was used to pre-pare Bola-niosomes, i.e. niosomes were made up of Bola-surfactant:Span r© 80:Chol (2:3:1 molar ratio). In particular,

the presence of cholesterol promoted the self-assembly ofsynthetic surfactant in a vesicle dispersion with or withoutammonium glycyrrhizinate. While, the presence of Span r©

80 produced a more stable and compact bilayer struc-ture, thus improving the self-assembling behavior of Bola-surfactant in colloidal vesicles. Moreover, the hydration oflipid/surfactant films with a sterile saline solution favoredthe formation of spherical niosomes and prevented the for-mation of a colloidal aggregate (Muzzalupo et al., 2005), asdemonstrated by the physicochemical characterization of thevesicle.

Bola-niosomes had a mean size of ∼ 500 nm and a ho-mogenous side distribution as confirmed by polydispersityindex values. The encapsulation of ammonium glycyrrhiz-inate in the niosome formulation caused a reduction of meansize and a slight increase of the polidispersity index value(Table 1). This effect was probably due to the interactionof the encapsulated drug with the Bola-niosome matrix. Inparticular, the mean size reduction could be due to the inter-action between the steroid ring of ammonium glycyrrhizinateand the acyl chains of the synthetic surfactant, a certain con-tribution could also come from a slightly ionic interactioncaused by carboxylic and oxydrilic groups of the drug at thelevel of the outer side of the Bola-niosome bilayer struc-ture. This hypothesis is in agreement with the fact that theability of a drug to influence the mean size and size distri-bution of a vesicular drug carrier is correlated to a certaininteraction of the molecule with the bilayer structure (Frestaet al., 2000, 2002). Therefore, ammonium glycyrrhizinatewas not simply entrapped in the aqueous compartments ofBola-niosomes but it was also associated, to a certain extent,with the bilayer structure of the Bola-niosomes.

TEM analysis showed that the Bola-surfactant was able toform niosome vesicles and that the presence of ammoniumglycyrrhizinate did not modify the spherical shape and thevesicular structure of the Bola-niosomes (Fig. 1).

Fig. 1 Transmission electron micrographs of ammoniumglycyrrhizinate-loaded Bola-niosomes. Unloaded Bola-niosomesshowed a similar morphology (data not reported). The bar is 0.33 µm

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Table 1 Physicochemical parameters and loading capacity of niosomecolloidal vesicles made up of Bola-surfactant/Chol/Span r© 80 (2:1:3molar ratio)a

Sample Size (nm) P.I.c LC (%)d

UnloadedBola-niosomes

456.2 ± 3.27 0.212 ± 0.021 –

Loaded Bola-niosomesb

325.5 ± 5.74 0.304 ± 0.049 39.7 ± 3.1

aEach value represented the average of three different experiments ±standard deviation.bBola-niosomes prepared in the presence of ammonium glycyrrhizinatein the hydrating aqueous phase.cPolidispersity index of niosome colloidal vesicles. Results are theaverage values of five different experiments (ten measurements perexperiment) ± standard deviation.dLoading capacity of colloidal vesicles expressed as the percentage ofthe drug that became niosome-associated. Data are the mean values offive different experiments ± standard deviation.

The ability of colloidal drug delivery devices to entrap andretain an active agent is very important to evaluate the poten-tial therapeutic use of a certain drug carrier, particularly if thedrug carrier has to be used for topical applications (Dayanand Touitou, 2000; Touitou et al., 2000; Alsarra et al., 2005;Manconi et al., 2006). Therefore, an important parameter tobe evaluated for the Bola-niosome formulation was its load-ing capacity. As shown in Table 1, Bola-niosomes presenteda loading towards ammonium glycyrrhizinate of ∼ 40% withrespect to the amount of drug added during preparation. Inthis case, it is noteworthy that the amount of ammonium gly-cyrrhizinate encapsulated in Bola-niosomes was higher thanthat expected by using a conventional vesicular carrier, suchas liposomes (Furneri et al., 2000). As mentioned above, thisfinding was probably due, to a certain extent, to the ammo-nium glycyrrhizinate association with the bilayer matrix ofBola-niosomes.

The release of ammonium glycyrrhizinate from niosomeswas also investigated. Figure 2 shows that a rapid releaseof ammonium glycyrrhizinate from niosomes was observedduring the first hour. This finding was probably due to a rapiddesorption of the untrapped ammonium glycyrrhizinate ad-sorbed on the surface of the niosomal colloidal system. After1 h, the release profile was characterized by a constant drugrelease following a zero order kinetic, which is typical forcolloidal systems with a drug reservoir architecture. Theamount of ammonium glycyrrhizinate released over 24 hwas ∼ 50% of the entrapped amount. The slow release ofthis drug could be elicited by the strong interaction occurringbetween the Bola moieties along the surface of niosomal bi-layers, which were able to complex cations (Muzzalupo et al.,1996), i.e. Na+ and NH4

+, thus assuming a positive charge,and the negatively charged molecule of glycyrrhizinate. Theformation of a kind of ion pair between the glycyrrhizinate

Fig. 2 Release profile of ammonium glycyrrhizinate from Bola-niosomes. Experiments were carried out at room temperature. Valuesrepresent the average of six different experiments ± standard deviation

molecule and the Bola-surfactant therefore hampered an easypassage of the drug through the vesicular structure of Bola-niosomes thus providing a sustained release over 24 h.

3.2 In vitro human skin percutaneous permeation ofammonium glycyrrhizinate

The percutaneous permeation of ammonium glycyrrhizinatethrough the stratum corneum was investigated by using anin vitro Franz diffusion cell. This parameter is very impor-tant in evaluating the potential use of a drug delivery systemas a topical device (Date et al., 2001; Fang et al., 2001;Paolino et al., 2005). Figure 3 shows that the penetrationof ammonium glycyrrhizinate from the solution through thestratum corneum was lower than that obtained from Bola-niosomes. Percutaneous permeation profiles showed that theamount of ammonium glycyrrhizinate permeated throughthe stratum corneum after 24 h was ∼ 3.5 folds higherfor Bola-niosomes than that observed for the free drug, i.e.989.28 ± 77.8 µg/cm2 .h−1 vs 258.23 ± 41.7 µg/cm2 .h−1,respectively. This effect was probably due to the fact that thebilayer structures of Bola-niosomes are mainly made up ofsurfactant agents that can behave as permeation enhancersthus allowing a temporary change in the packing order ofstratum corneum lipids and hence an improvement of thedrug passage through the skin (Blume et al., 1993; Fang et al.,2001; El Maghraby et al., 2004). In addition, according toliterature [32–34], the presence of surfactant agents can actas edge activators with respect to colloidal niosome vesicles,thus allowing niosome vesicle deformation and promotingthe passage of niosomes through human skin. In this case,

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Fig. 3 In vitro percutaneous permeation through an SCE membraneof various formulations containing ammonium glycyrrhizinate at aconcentration of 0.3% (w/v). Legend keys: �, ammonium glycyrrhiz-inate aqueous solution;•, ammonium glycyrrhizinate-associated Bola-niosomes; �, physical mixture between unloaded Bola-niosomes andan ammonium glycyrrhizinate aqueous solution. Each value is the av-erage of six different experiments ± standard deviation

niosomes can further promote the percutaneous passage of ahydrophilic drug such as ammonium glycyrrhizinate.

To evaluate the contribution of the Bola-niosomedeformability and of the penetration enhancer effect of sur-factans, the percutaneous permeation of a physical mixtureof an ammonium glycirrhizinate solution with unloadedBola-niosomes through human SCE membranes was alsoinvestigated (Fig. 3). The physical mixture improved thepercutaneous passage of ammonium glycyrhizinate with re-spect to the simple aqueous solution of the drug, but the totalamount permeated after 24 h was significantly (p < 0.001)lower than that achieved by means of the drug-associatedBola-niosomes. This finding showed that the noticeableimprovement of the drug percutaneous permeation must beattributed to a certain extent to the encapsulation of the drugin the Bola-niosomes. In particular, the percutaneous perme-ation profiles of various samples showed that both effects,i.e. penetration enhancement effect and edge activator action,played a significant role in human skin permeation of thehydrophilic drug ammonium glycyrrhizinate. In agreementwith percutaneous experimental data and to the literature(Van Kuijk-Meuwissen et al., 1998; El Maghraby et al., 2000;Date et al., 2006), it was possible to hypothesize that theammonium glycyrrhizinate percutaneous passage could bepromoted by (i) the adsorption and fusion of Bola-niosomal

vesicles on the surface of the skin, and (ii) the deformationand passage of the carrier through the stratum corneum.

3.3 Interaction of Bola-niosomes with NCTC 2544 humankeratinocyte cells

In this paper Bola-niosomes have been proposed as a col-loidal delivery system for the topical administration of am-monium glycyrrhizinate. Considering that Bola-niosomeswere able to improve the percutaneous permeation of ammo-nium glycyrrhizinate similar to vesicles showing a certain bi-layer flexibility, i.e. ethosomes and transferosomes (Touitouet al., 2000; Cevc and Blume, 2001; Cevc and Gebauer, 2003;Godin and Touitou, 2004; Alsarra et al., 2005; Paolino et al.,2005), the potential interaction of these vesicles with NCTC2544 human keratinocyte cells was investigated. In partic-ular, to evaluate the mechanism involved in the internaliza-tion and intracellular delivery mediated by Bola-niosomesCLSM experiments were carried out to follow the cellularuptake of Bola-niosomes labelled with fluorescein-DHPEin the hydrophobic zone of the niosomal bilayers. Figure4 shows the findings from CLSM experiments followingincubation of NCTC 2544 cells with fluorescent labelledBola-niosomes at different times. CLSM micrographs showthat Bola-niosomes were able to deliver the highly lipophiliclabelling agent both to the cellular membrane and to the cy-toplasm, which showed a homogenous green-fluorescencedistribution. It is possible to observe (Fig. 4) that NCTC2544 cells appear highly fluorescent after 6 h of incubationand then the cellular fluorescence gradually increases up to24 h of incubation. The green fluorescence inside the cellwas caused by the presence of lipophilic fluorescein-DHPEand not by the water soluble product of cleavage, i.e. fluo-rescein. In fact, the labelling compound fluorescein-DHPEwas stable in the incubation medium of NCTC 2544 cellsup to 24 h, as demonstrated by HPLC analysis (data notshown).

A quantitative evaluation of CLSM experiments (Fig. 5)showed a time-dependent cellular penetration of the probe-loaded Bola-niosomes. While the fluorescent intensity ofNCTC 2544 cells due to incubation with the aqueous sus-pension of fluorescein-DHPE was significantly and notice-ably lower than that observed for the labelled Bola-niosomesand was not affected by the incubation time. The finding re-garding the intracellular delivery of fluorescein-DHPE wasproof of the capacity of Bola-niosomes to penetrate the cellmembrane.

3.4 In vitro and in vivo evaluation of Bola-surfactant andBola-niosome tolerability

The safety of innovative synthetic material is an importantfactor to be evaluated in a drug delivery system. This char-

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Fig. 4 Confocal laser scanningmicrographs showing theBola-niosomes—NCTC 2544human keratinocyte cellinteraction and the intracellularlocalization of the labellingcompound fluorescein-DHPE asa function of the incubationtime: panel a, 3 h; panel b, 6 h;panel c, 12 h; panel d, 24 h.Photomicrograph of untreatedNCTC 2544 human keratinocytecell (control) achieved intransmission mode (panel e). Nosignificant cellular fluorescencewas observed coming from theautofluorescence phenomenacaused by the cellular proteins

acteristic is able to influence the therapeutic application,the bio-mimetic properties of the derived drug delivery sys-tem and, as a consequence, potential clinical use (Fruijtier-Polloth, 2005; Zhong et al., 2005). To verify the compatibilityof Bola-surfactant to be proposed as a component of vesic-ular colloidal drug delivery systems for topical application,the in vitro cytotoxic effect of Bola-surfactant both as a freemolecule or assembled in niosome vesicles was evaluatedon NCTC2544 cells by using the trypan blue dye exclusion

assay and MTT viability test. The Bola-surfactant was as-sayed in vitro in terms of both dose-dependent effect andincubation-time effect.

As shown in Fig. 6, both free Bola-surfactant and Bola-niosomes had a dose-dependent and an exposition time ef-fect on viability and mortality of NCTC2544 cells. In par-ticular, free Bola-surfactant showed no significant effect onNCTC2544 cell mortality with respect to the control (un-treated cells) for concentrations up to 0.1 µM at exposition

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Fig. 5 Quantitative analysis of cellular penetration as total fluo-rescence intensity (coming from CLSM localization experiments) offluorescein-DHPE–labelled Bola-niosomes ( • ) or a simple aqueousdispersion of fluorescein-DHPE (�) in cultured NCTC 2544 humankeratinocyte cells as a function of time. Results are the mean of fivedifferent experiments ± standard deviation

times of 24 h and 48 h. The findings regarding cell mortalitywere supported by viability data (Fig. 6), even if the MTT testshowed a certain sensitivity of NCTC2544 cells due to thepresence of the synthetic surfactant, which caused a slightdecrease of the percentage of cellular viability. Followingan incubation time of 72 h, free Bola-surfactant caused aslight but significant (p < 0.001) increase of cellular mortal-ity. Also in this case cellular mortality data were supportedby cellular viability findings.

At the highest concentrations investigated in this pa-per, particularly at the concentration of 10 µM, free Bola-surfactant elicited a noticeable cytotoxic effect on humankeratinocyte NCTC2544 cells at all the investigated exposi-tion times. Namely, a cellular mortality higher than 85% wasobserved for incubation times ≥ 48 h (Fig. 6). The cellularviability data were in good agreement with mortality dataconfirming the noticeable cytotoxic effect.

The in vitro data on NCTC2544 cellular cytoxicity showthat free Bola-surfactant was well tolerated for expositiontimes up to 48 h at concentrations up to 1 µM. Similarfindings were observed for other synthetic surfactants (Leeet al., 2000; Lanigan and Yamarik, 2002; Sanchez et al.,2004), i.e. Span series and Tween series, that are commonlyused as ingredients in topical formulations both for cosmeticand pharmaceutical use.

The assembling of Bola-surfactant in niosomes caused adrastic reduction of the cytotoxic effect of the free Bola-

surfactant on human keratinocyte NCTC2544 cells. In par-ticular, no significant difference of cellular mortality levelswith respect to control (untreated cells) was observed onhuman keratinocytes NCTC2544 cells treated with Bola-surfactant assembled in niosome vesicles at Bola-surfactantconcentrations ranging from 0.01 µM to 1 µM for all theexposition time investigated in this paper (24–72 h) (Fig. 6).The Bola-niosome effect of NCTC2544 cellular cytotoxicityreduction was much more evident for the highest investigatedsurfactant concentration, i.e. 10 µM. In this case, it was veryinteresting to note that Bola-niosomes showed mortality val-ues of 10.8%, 10.7% and 32.5% with respect to untreatedNCTC2544 cells following 24, 48 and 72 h of exposition,respectively.

Only the mortality level observed after 72 h of NCTC2544cell exposition with Bola-niosomes was significantly higher(p < 0.001) than the control value. Bola-niosomes at a con-centration of 10 µM showed a noticeable and significant(p < 0.001) reduction of NCTC2544 cell mortality with re-spect to free Bola-surfactant at all the investigated expositiontimes. Namely, Bola-surfactant assembled in niosome vesi-cles produced up to an eight fold reduction of NCTC2544cell mortality levels (for 48 h exposition) with respect to thefree molecule.

The MTT assay (cellular viability test) on human ker-atinocyte NCTC2544 cells treated with different amounts ofBola-niosomes for the various exposition times supported thedata of cellular mortality, thus showing a much lower viabil-ity reduction than that observed for the free Bola-surfactant.

The reduced cytotoxicity of the Bola-surfactant onNCTC2544 cells when assembled in niosome vesicles wasproof that the cellular cytotoxic effect was mostly mediatedby the free molecules rather than the assembled colloidalcarrier. The cytotoxicity reduction and hence the increasedtolerability showed by Bola-niosomes were therefore due tothe fact that the free Bola-surfactant self-assembled in vesic-ular structures during niosome preparation led to a drasticreduction of the free molecules in solution and hence to adrastic reduction of their cytotoxic action. This mechanism istypical of surfactant molecules (Sanchez et al., 2006), whichare toxic in their free form, particularly when their concen-tration is equal or higher their critical micellar concentration.In fact, surfactants, as well as Bola-molecules, can exert theircellular cytotoxic action by perturbing cellular membranesand hence altering cellular permeability and cellular home-ostasis (Tsujino et al., 1999; Courrier et al., 2003). If micellesare present, a solubilizing effect on cellular membranes oc-curs with the formation of mixed micelles between surfactantmolecules and phospholipids or other lipid components ofcellular membranes (Xia and Onyuksel, 2000; Simoes et al.,2005).

The potential irritancy effect of both the free Bola-surfactant and of empty Bola-niosomes was also evaluated on

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Fig. 6 In vitro tolerability on human keratinocyte NCTC2544 cellsof free Bola-surfactant (circle) or Bola-surfactant assembled in nio-some vesicles (square) as a function of surfactant concentration andexposition times, i.e. 24 h (panel a), 48 h (panel b), 72 h (panel c).The NCTC2544 cell tolerability is reported both as percentage of cellmortality (filled symbols and solid line) and percentage of cellular vi-

ability (hollow symbols and dashed line). Cell mortality was evaluatedby trypan blue dye exclusion assay, while cell viability was evaluatedby MTT test. Control (Ctrl) is untreated cells. Error bars, if not shown,are within symbols. Results are presented as the mean of six differentexperiments ± standard deviation

human volunteers with a non-invasive technique, reflectancespectrophotometry. Taking into consideration in vitro ex-periments on human keratinocytes, human volunteers weresubmitted to topical treatment with formulations presentinga Bola-surfactant concentration of 7.5 mg/ml, up to a skin ex-position of 24 h. This concentration corresponds to a possibleconcentration to be proposed and used for topical formula-tions in a potential clinical setting (Fiume, 2001; Laniganand Yamarik, 2002).

The results of skin tolerability are expressed as the vari-ation of the erythema index (�EI) with respect to baselinevalue. The effect of the Bola-surfactant as a free molecule orassembled in niosome vesicles on human skin was compared

with that induced by a 0.9% (w/v) NaCl solution (Fig. 7). �EIdata show that unloaded Bola-niosomes elicited no signifi-cant erythema induction with respect to the control (aqueoussaline solution) at all the exposition times investigated inthis paper (6–24 h). Whereas, the free Bola-surfactant aque-ous solution determined a slight but significant (p < 0.01)increase of �EI values with respect to both unloaded Bola-niosomes and the control. These data were in good agree-ment with in vitro toxicity experiments, thus showing thatBola-surfactant molecules are very well tolerated also in vivoin human volunteers when assembled in vesicular colloidalstructures, such as niosomes. These findings represent a goodstarting point for the topical application of Bola-niosomes.

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Fig. 7 In vivo skin tolerability in human volunteers of Bola–surfactantas free molecules or assembled in niosome vesicles as a function of theexposition time, i.e. 6 h, 12 h and 24. Results are expressed as thevariation of the erythema index with respect to the baseline values(untreated human volunteer skin) and are the mean value of �EI ±standard deviation of six different volunteers

3.5 Anti-inflammatory activity of ammoniumglycyrrhizinate-associated Bola-niosomes

The potentialities of Bola-niosomes for topical applicationof ammonium glycyrrhizinate to improve the topical anti-inflammatory activity of this natural compound were stud-ied in vivo on human volunteers. Different skin sites ofeach volunteer were pre-treated with a solution of methyl-nicotinate to induce an erythema, the formulations contain-ing ammonium glycyrrhizinate at a concentration of 0.3%(w/v) were then applied. The chemical-induced erythemafollowed different profiles as a function of the applied anti-inflammatory formulation. As shown in Fig. 8, the ammo-nium glycyrrhizinate-associated niosome formulation wasable to reduce the erythema much more rapidly and effica-ciously than the aqueous solution of ammonium glycyrrhiz-inate. In particular, 2 h after the topical application, thechemically induced erythema observed in the sites treatedwith ammonium glycyrrhizinate-associated niosomes wasreduced by 2.3 fold with respect to the aqueous solutionof the drug, which showed a slight reduction with respectto the sites treated with the saline solution (control). Fol-lowing 3 h of treatment with ammonium glycyrrhizinate-associated niosomes, the disappearance of the chemicallyinduced erythema was observed with an improvement of theanti-inflammatory activity of the niosome delivered drug of10 fold with respect to the simple drug aqueous solution. As

Fig. 8 In vivo anti-inflammatory activity as a function of the time inhuman volunteers of various formulations containing ammonium gly-cyrrhizinate (0.3% w/v) assayed as the ability to reduce a chemicallyinduced skin erythema by a pre-treatment with an aqueous solution(100 µl) of methyl-nicotinate (0.2% w/v). Symbol keys:•, inflamedskin sites treated with ammonium glycyrrhizinate-associared niosomes;�, inflamed skin sites treated with an aqueous solution of ammoniumglycyrrhizinate; �, inflamed skin sites treated with a saline aqueous so-lution (control). Results are expressed as �EI mean values (six differentvolunteers) ± standard deviation

shown in Fig. 8, it was still possible to observe an intenseerythema in all skin sites treated with ammonium glycyrrhiz-inate aqueous solution up to 5 h.

The in vivo findings on the improvement of ammoniumglycyrrhizinate anti-inflammatory activity when deliveredin a Bola-niosome formulation can be justified by the invitro experiments regarding the drug permeation throughhuman SCE membranes. In fact, ammonium glycirrhizinate-associated niosomes provided a noticeable increase of thepercutaneous permeation of the natural anti-inflammatorydrug, thus allowing an improvement of the drug therapeu-tic activity, which is strictly related to the amount of theavailable drug at the inflammation site.

4 Conclusion

Experimental findings show that the Bola-surfactantachieved, together with cholesterol and Span, colloidal vesi-cles with suitable physicochemical characteristics to be usedas a topical drug delivery device. It is worth noting thatBola-niosomes presented a certain safety and tolerabilityboth in vitro on human keratinocyte NCTC2544 cells up toan incubation time of 72 h for the different concentrations

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investigated (0.01, 0.1, 1 and 10 µM) and in vivo on hu-man volunteers that showed no skin erythema when topi-cally treated with the unloaded Bola-niosome formulation.The Bola-niosomes were also able to improve the intracel-lular delivery as demonstrated by CLSM experiments. Thecolloidal system based on Bola-niosomes showed certaintherapeutic potentialities being able to promote the in vitroand in vivo delivery of ammonium glycirrhizinate throughthe skin, thus increasing its permeation and hence its anti-inflammatory activity.

These findings represent one of the few examples of howit is possible to combine innovative colloidal carriers andtherapeutically effective natural compounds coming fromtraditional medicine, thus showing the possibility of unitingthe traditional world of medicinal herbs and the innovationof pharmaceutical nanotechnology. Therefore, these experi-mental findings are very encouraging and show that the Bola-surfactant may represent a good synthetic biomaterial to beused in topical colloidal carriers for the delivery of activeagents. The encouraging results are prompting further inves-tigations on the poly-functional role that the Bola-surfactantcan confer to colloidal carriers.

Acknowledgments The authors would like to thank Maria GraziaCalvagno Ph.D. (Department of Pharmacobiological Sciences, Univer-sity of Catanzaro) for her excellent and valuable support in the in vitroexperiments (particularly concerning CLSM experiments) and for herhelpful discussion and suggestions throughout this paper. The authorsalso thank Dr. Antony Bridgewood for his revision of the languageof this paper. This research was partially supported by the Faculty ofPharmacy of the University “Magna Græcia” of Catanzaro. The authorsacknowledge the financial support (PRIN 2005) from MIUR.

References

I.A. Alsarra, A.A. Bosela, S.M. Ahmed, and G.M. Mahrous, Eur. J.Pharm. Biopharm. 59, 485 (2005).

P. Arunothayanun, M.S. Bernard, D.Q. Craig, I.F. Uchegbu, and A.T.Florence, Int. J. Pharm. 201, 7 (2000).

A. Balasubramaniam, V.A. Kumar, and K.S. Pillai, Drug Dev. Ind.Pharm. 28, 1181 (2002).

L.A. Baltina, Curr. Med. Chem. 10, 155 (2003).B.W. Barry, Eur. J. Pharm. Sci. 14, 101 (2001).A. Blume, M. Jansen, M. Ghyczy, and J. Gareis, Int. J. Pharm. 99, 219

(1993).E. Caponetti, D. Chillura-Martino, C. La Mesa, R. Muzzalupo, and L.

Pedone, J. Phys. Chem. B 108, 1214 (2004).G. Cevc and G. Blume, Biochim. Biophys. Acta 1514, 191 (2001).G. Cevc and D. Gebauer, Biophys. J. 84, 1010 (2003).M.J. Choi and H.I. Maibach, Skin Pharmacol. Physiol. 18, 209 (2005).H.M. Courrier, M.P. Krafft, N. Butz, C. Porte, N. Frossard, A. Remy-

Kristensen, Y. Mely, F. Pons, and T.F. Vandamme, Biomaterials24, 689 (2003).

A.A. Date, B. Naik, and M.S. Nagarsenker, Skin Pharmacol. Physiol.19, 2 (2006).

N. Dayan and E. Touitou, Biomaterials 21, 1879 (2000).

P.K. Eggers, T.M. Fyles, K.D. Mitchell, and T. Sutherland, J. Org.Chem. 68, 1050 (2003).

G.M.M. El Maghraby, A.C. Williams, and B.W. Barry, Int. J. Pharm.276, 143 (2004).

G.M.M. El Maghraby, A.C. Williams, and B.W. Barry, Int. J. Pharm.196, 63 (2000).

J.Y. Fang, C.T. Hong, W.T. Chiu, and Y.Y. Wang, Int. J. Pharm. 219, 61(2001).

Z. Fiume, Int. J. Toxicol. 20, 21 (2001).M. Fresta, S. Guccione, A.R. Beccari, P.M. Furneri, and G. Puglisi,

Bioorg. Med. Chem. 10, 3871 (2002).M. Fresta, M. Ricci, C. Rossi, P.M. Furieri, and G. Pugliesi, J. Coll

Interf Sci. 226, 222 (2000).C. Fruijtier-Polloth, Toxicology 214, 1 (2005).P.M. Furneri, M. Fresta, G. Pugliesi, and G. Tempera, Antimicrob.

Agents Chemother. 44, 2458 (2000).B. Godin and E. Touitou, J. Control. Rel. 94, 365 (2004).R.P. Gude, M.G. Jadhav, S.G. Rao, and A.G. Jagtap, Cancer Biother.

Radiopharm. 17, 183 (2002).S. Jain, P. Singh, V. Mishra, and S.P. Vyas, Immunol. Lett. 101, 41

(2005).A.M. Kligman and E. Christophers, Arch. Dermatol. 88, 702 (1963).R.S. Lanigan and T.A. Yamarik, Int. J. Toxicol. 21, 93 (2002).J.K. Lee, D.B. Kim, J.I. Kim, and P.Y. Kim, Toxicol. In Vitro 14, 345

(2000).M. Manconi, C. Sinico, D. Valenti, F. Lai, and A.M. Fadda, Int. J.

Pharm. 311, 11 (2006).M. Manconi, C. Sinico, D. Valenti, G. Loy, and A.M. Fadda, Int. J.

Pharm. 234, 237 (2002).M. Manconi, D. Valenti, C. Sinico, F. Lai, G. Loy, and A.M. Fadda, Int.

J. Pharm. 260, 261 (2003).S. Matsui, H. Matsumoto, Y. Sonoda, K. Ando, E. Aizu-Yokota, T. Sato,

and T. Kasahara, Int. Immunopharmacol. 4, 1633 (2004).D.C. McKenzie, C.A. Bunton, D.F. Nicoli, and G. Savelli, J. Phys.

Chem. 91, 5709 (1987).R. Muzzalupo, G. Gente, C. La Mesa, E. Caponetti, D. Chillura-

Martino, L. Pedone, and M.L. Saladino, Langmuir 22, 6001(2006).

R. Muzzalupo, G.A. Ranieri, and C. La Mesa, Langmuir 12, 3157(1996).

R. Muzzalupo, S. Trombino, F. Iemma, F. Puoci, C. La Mesa, and N.Picci, Colloid Surf. B: Biointerfaces 46, 78 (2005).

D. Paolino, G. Lucania, D. Mardente, F. Alhaique, and M. Fresta, J.Control. Rel. 106, 99 (2005).

L. Sanchez, M. Mitjans, M.R. Infante, and M.P. Vinardell, Pharm. Res.21, 1637 (2004).

L. Sanchez, M. Mitjans, M.R. Infante, and M.P. Vinardell, Toxicol.Lett. 161, 53 (2006).

H. Schreier and J. Bouwstra, J. Control. Rel. 30, 1 (1994).A. Shahiwala and A. Misra, J. Pharm. Sci. 5, 220 (2002).S.I. Simoes, J.M. Tapadas, C.M. Marques, M.E. Cruz, M.B. Martins,

and G. Cevc, Eur. J. Pharm. Sci. 26, 307 (2005).E. Touitou, N. Dayan, L. Bergelson, B. Godin, and M. Eliaz, J. Control.

Rel. 65, 403 (2000).I. Tsujino, T. Yamazaki, M. Masutani, U. Sawada, and T. Horie, Cancer

Chemother. Pharmacol. 43, 29 (1999).I.F. Uchegbu and S.P. Vyas, Int. J. Pharm. 172, 33 (1998).M.E. Van Kuijk-Meuwissen, L. Mougin, H.E. Junginger, and J.A.

Bouwstra, J. Control. Rel. 56, 189 (1998).B. Vora, A.J. Khopade, and N.K. Jain, J. Control. Rel. 54, 149 (1998).W.J. Xia and H. Onyuksel, Pharm. Res. 17, 612 (2000).Z. Zhong, J. Feijen, M.C. Lok, W.E. Hennink, L.V. Christensen,

J.W. Yockman, Y.H. Kim, and S.W. Kim, Biomacromol. 6, 3440(2005).

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