liic

bDepartment of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt

a r t i c l e i n f o

Article history:Received 8 May 2013Accepted in revised form 9 September 2013Available online 18 September 2013

Keywords:

delivery systems [1].Glyceryl monoolein (GMO) is a long chain unsaturated mono-

glyceride that is able to form lyotropic liquid crystalline cubicphases in water [2]. This polar lipid is essentially insoluble inwater, but self-associates, and may depending on the water con-

cubic phases arehases, indhe polar me formed

and the low-cost raw material, which makes them desirable tobe used as consumer products and in the pharmaceutical industryapplications [4]. Cubosomes are discrete, submicron, nanostruc-tured particles of bicontinuous cubic liquid crystalline phase,which are able to incorporate large amounts of drugs, and can belocalized in body cavities, on the skin or different mucosal surfaces[5]. This structure offers two separate lipid and water domains.This compartmentalization can be used to introduce guest mole-cules that are either hydrophilic, lipophilic or amphiphilic in

Corresponding author. Faculty of Pharmacy, Cairo University, Kasr El-AinyStreet 11562, Egypt. Tel.: +20 225311260; fax: +20 223628426.

European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

Contents lists availab

European Journal of Pharmace

.eE-mail address: ihabrasmy@gmail.com (E.R. Bendas).lation and administration of drugs, and the formulation of novel monoglyceride, temperature stability, high internal surface areaIn the recent years, self-assembled lyotropic liquid crystallinephases of lipids and water have gained increasing interest. Thatis due to their potential in different application elds, as encapsu-

ous cubic phase (C) as visualized in Fig. 1. Theoften referred to as reversed or inverse cubic pthe curvature of the consistent bilayer toward t[3]. The interesting properties of the cubic phas0939-6411/$ - see front matter 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejpb.2013.09.008icatingediumby thisand upper lip area and overall improvement in skin color and texture in most volunteers. There wereno instances of irritation, peeling or other apparent adverse side effects.

2013 Elsevier B.V. All rights reserved.

1. Introduction tent form a reversed micellar (L2), a lamellar (La), or a bicontinu-Liquid crystalsDrug releaseAntioxidantAlpha lipoic acidGlyceryl monooleateCubosomesPoloxamer gelCosmeceutical applicationClinical studya b s t r a c t

Topical 5% alpha lipoic acid (ALA) has shown efcacy in treatment of photo-damaged skin. The aim of thiswork was to evaluate the potential of poloxamer (P407) gel as a vehicle for the novel lipid base particu-late system (cubosome dispersions) of ALA. Cubosome dispersions were formulated by two differentapproaches, emulsication of glyceryl monoolein (GMO) and poloxamer (P407) in water followed byultrasonication, and the dilution method using a hydrotrope. Three different concentrations of GMO wereused to formulate the cubosome dispersions using the rst method, 5% (D1), 10% (D2) and 15% w/w (D3).In the second technique an isotropic liquid was produced by combining GMO with ethanol, and this iso-tropic liquid was then diluted with a P407 solution (D4). The dispersions were characterized by zetapotential, light scattering techniques, optical and transmission electron microscopy, encapsulation ef-ciency and in vitro drug release. Results showed that D4 was not a uniform dispersion and that D1, D2and D3 were uniform dispersions, in which by increasing the GMO content in the dispersion, the sizeof the cubosomes decreased, zeta potential became more negative, encapsulation efciency increasedup to 86.48% and the drug release rate was slower. P407 gels were prepared using the cold method.Two concentrations of P407 gel were fabricated, 20 and 30% w/w. P407 gels were loaded with eitherALA or dispersions containing ALA cubosomes. P407 gels were characterized by critical gelation temper-ature, rheological measurements and in vitro drug release studies. Results suggested that by increasingP407 concentration, the gelation temperature decreases and viscosity increases. Drug release in bothcases was found to follow the Higuchi square root model. Gel loaded with ALA cubosomes provided a sig-nicantly lower release rate than the gel loaded with the un-encapsulated ALA. A double blinded placebocontrolled clinical study was conducted, aiming to evaluate the efcacy as an anti-wrinkle agent andvolunteers satisfaction upon application of topical 30% P407 gel loaded with ALA cubosomes. Resultsindicated reduction in facial lines, almost complete resolution of ne lines in the periorbital regionSaly Sherif , Ehab R. Bendas , Sabry BadawyaDepartment of Pharmaceutical Technology, Faculty of Pharmacy, Misr International University, Cairo, EgyptResearch paper

The clinical efcacy of cosmeceutical appnanostructured dispersions of alpha lipo

a b, a

journal homepage: wwwcation of liquid crystallineacid as anti-wrinkle

le at ScienceDirect

utics and Biopharmaceutics

l sevier .com/locate /e jpb

nature [6]. GMO, as the major structure lipid of cubosomes, hasbeen reported to be an effective transdermal enhancer [7], whichmakes GMO dispersions more desirable to be used in the cosme-ceutical eld. This effect might be due to the structural organiza-tion of cubosomes, which is similar to that found ofbiomembranes [8,9].

Poloxamer 407 (P407) is a triblock copolymer with a centralhydrophobic chain of polyoxypropylene (PPO) and two identicallateral hydrophilic chains of polyoxyethylene (PEO) [10]. The wide-spread application of P407 gel in topical delivery systems is due tothe reversible solgel property that allows cool solution to owonto the skin and permit good contact with skin on formation of

of GMO/P407 mixtures in water followed by ultrasonication. Threeformulations (D1, D2 and D3) were prepared by weighing appro-priate amounts of GMO and P407. The mixture was gently melted

C

Fig. 2. Structure of ALA.

252 S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259a non-occlusive gel at body temperature. The gel could also be eas-ily removed by washing with cold water [11]. Increasing the P407concentration increases the viscosity of the gel, which can changethe releasing process of additives (as ALA or ALA cubosomes) fromthe gel. The presence of drugs can also change some rheologicalcharacters of these gels [10].

ALA is a natural occurring fatty acid with potent antioxidantactivity which exists in the mitochondria of all kinds of prokaryoticand eukaryotic cells [12,13]. Structure of ALA is illustrated in Fig. 2.ALA is known as a network antioxidant due to its ability to regen-erate/recycle itself, as well as other antioxidants such as vitamins Cand E, so that they can continue destroying free radicals [12]. Sinceavailable data of formulations containing 5% ALA were successfulto produce a dramatic reduction in facial lines in cases associatedwith photo-aging, this compound has gained the attention of cos-metologists and dermatologists.

The objective of the present work was to explore the potentialof P407 solution to be used as a vehicle for the novel lipid baseparticulate system (cubosome dispersions), to prepare a cosmeti-cally acceptable preparation that could stabilize and sustain thedelivery of ALA when used as anti-wrinkle agent.

2. Materials and methods

2.1. Materials

Myverol 1899 K (Myverol) was used as the source of GMOand was kindly supplied as a gift from Kerry Bioscience (Norwich,NY, USA). Poloxamer 407 (P407), absolute ethanol and palladium(II) chloride were purchased from SigmaAldrich (St. Louis, MO,USA). Alpha lipoic acid (ALA) was kindly supplied by EVA pharma(Cairo, Egypt). Milli-Q puried water was used for all experiments.Other reagents were of analytical grade.

2.2. Preparation of cubosome dispersions

Cubosome dispersions were fabricated using two differentmethods. The rst conventional method involved emulsication

L2 LIncreasing wa

Fig. 1. Transformation of mesophases from L2 (reversed micellar) to La (lamin a water bath (Clifton, England) at 70 C. This was injected intopreheated water at 70 C and maintained under mechanicalstirring at 1500 rpm. Dispersions were cooled to room temperaturethen ultrasonicated at maximum power of 130 kW (Elma, Trans-sonic, Germany) for 1 min according to the method published byEsposito et al. [14]. Fifty milligrams of ALA was added to waterprior to the addition of GMO and P407. The second methodadopted was dilution of an isotropic liquid by a hydrotrope. Anisotropic liquid was prepared by combining GMO with ethanol.This isotropic liquid was diluted with P407 solution as describedby Spicer and Hayden [15]. Fifty milligrams of ALA was added toethanol prior to the dilution (D4). The detailed compositions ofthe dispersions are listed in Table 1.

2.3. Preparation of P407 gels

P407 gels were prepared using the cold method [16]. Concen-trations of P407 and ALA were expressed by weight percent (%w/w). Appropriate amounts of poloxamer 407 were slowly addedto cold distilled water at 5 C to yield 20% and 30% gels and con-stant stirring was maintained [17]. The poloxamer solution waskept refrigerated until a clear solution was obtained (612 h). Forthe gel to be formed, the solution should be kept at 30 C. Theappropriate amounts of ALA or ALA cubosome dispersions werethen added to the gels to yield 5%.

2.4. Assay of alpha lipoic acid

Stock solution of ALA having a concentration of 1 103 M wasfreshly prepared in a 1:1 mixture of ethanol and water. To ensurecomplete drug dissolution, the solution was sonicated at 60 kW for2 min. Palladium (II) chloride standard solution (1 102 M) wasprepared by dissolving palladium (II) chloride in water (to which0.1 ml of concentrated hydrochloric acid has been added). The mix-ture was then warmed in a water bath to ensure complete dissolu-tion. The ionic strength (l) of the nal solution for determinationwas kept constant at 0.2 M, by the addition of a 2 M potassiumchloride solution. Britton Robinson buffer was used to adjust theter content

ellar) to C (bicontinuous cubic phase), by increasing the water content.

spectrophotometrically at kmax 250 nm. Concentration obtained

bottom of the dissolution vessel. The membrane was used to retain

Method [20]. Briey, two glass vials, one containing 1 g of the

euticpH of the nal solution for determination at 2.2. This was preparedby using a ratio of 1:1:1 of 0.04 M boric acid, 0.04 M phosphoricacid and 0.04 M acetic acid, and the pH was adjusted by using0.2 M sodium hydroxide solution. Serial dilutions were preparedby adding known aliquots from the stock solution, followed by5 ml of Britton Robinson buffer, then 1 ml of potassium chloridesolution and nally 0.2 ml of palladium (II) chloride solution. Thevolume was then diluted to 10 ml by addition of distilled water.The mixture was mixed well then left to stand for at least10 min. The absorbance was then measured spectrophotometri-cally at the predetermined kmax 250 nm against a reagent blank.All measurements were done at a temperature of 25 1 C. Baseline correction was carried out to delete any absorbance readingof the blank. The assay method was validated.

2.5. Characterization of cubosomes

2.5.1. Particle size and zeta potential measurementsThe particle size and zeta potential of cubosomes were deter-

mined by dynamic light scattering (DLS) (Zetasizer, MalvernInstruments, UK). Each sample was diluted with deionized water,adjusted to a suitable light scattering intensity (300 Hz) and mea-sured at 25 C in triplicate. Following the particle size analysis ofthe cubosomes, the mode was switched from Size to Zeta,and three measurements of zeta potential were recorded.

2.5.2. Light microscopySamples of the prepared cubosome dispersions were suitably

diluted with deionized water and examined under a Leica micro-scope (Leica image analyzer, Model Q 550IW equipped with LeicaDMLB microscope Cambridge, England. Connected to Camera,Model TK-C1380 JVC, Victor Company, Japan) calibrated with amicrometer slide using polarized light or differential interferencecontrast at magnications between 10 and 100.

2.5.3. Transmission electron microscopy (TEM)The samples were prepared by placing 5 ll droplet of the

cubosome dispersion onto a 300 mesh carbon-coated copper grid,and letting the cubosomes settle for 35 min. Excess uid wasremoved by wicking it off with an absorbent paper. The sampleswere then viewed on a JEOL Model (JEM 1400, USA) 120KV trans-mission electron microscope.

Table 1Composition of dispersions of ALA cubosomes D1, D2, D3 and D4.

Dispersion Content (% w/w)

GMO P407 Ethanol Water

D1 5.0 1.0 94.0D2 10.0 1.0 89.0D3 15.0 1.0 84.0D4 68.0 0.3 5.0 26.7

S. Sherif et al. / European Journal of Pharmac2.5.4. Entrapment efciencyA sample of each dispersions containing 50 mg of ALA was pre-

pared as explained previously. In order to determine the amount ofdrug that was successfully entrapped inside the cubosomes (EE%),it was rst mandated to separate the cubosomes from the resultingdispersion. Then the amount of free drug in the dispersion wasthen analyzed spectrophotometrically at kmax 250 nm, which wasthen subtracted from the total amount of drug initially added.

A volume of 1 ml from each of the dispersions was diluted with4 ml of deionized water. A volume of 1 ml from this diluted disper-sion was further diluted with another 4 ml of deionized water. Theresulting diluted dispersion was then passed through a syringeP407 solution and the other 1 g of water, were placed in a waterbath. The temperature was slowly increased and the temperatureat which the solution stopped owing upon tilting was noted asthe gelation temperature (t1). Similarly, the temperature of thewater bath was lowered and the temperature, at which the gelstarted owing, was noted (t2). The thermo-couple of a digital ther-mometer (Fluke, USA) was placed in the water tube. The mean SDof t1 and t2 is reported as the critical gelation temperature (CGT).Each measurement was repeated three times.

2.6.2. Rheological measurementsthe formula inside the disk. The dissolution medium used was700 ml of hydro-alcoholic solution (1:1) to ensure sink condition.The apparatus was equilibrated to 32 0.5 C and the stirrer paddlespeed was set at 50 rpm. Aliquots were withdrawn at appropriatetime intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and ltered through a syr-inge lter having a pore size of 0.1 lm then analyzed spectropho-tometrically at wavelength of 250 nm (according to the method ofdrug assay). The amount of drug released was calculated from thestandard curve. This procedure was performed in triplicates foreach formulation. Cumulative % drug released were calculatedout and plotted against time. ALA release from cubosomes in gelshas been shown to be primarily controlled by diffusion throughthe matrix [18] and consequently can be described by the Higuchidiffusion equation given by:

Q DmCd2A Cdt 1=2 2

where Q is the mass of ALA released at time t, and is proportional tothe apparent diffusion coefcient of the drug in the matrix, Dm, theinitial amount of ALA in the matrix, A and the solubility of the drugin the matrix Cd [19]. The slope of the linear t to the data from thisplot is proportional to the apparent diffusion coefcient for the drugin the matrix, and permits rstly, assessment of diffusion as the pri-mary means of drug release from the correlation coefcient for thelinear t, and second, a means to compare the diffusion of a drugfrom the different matrices into the release medium.

2.6. Characterization of P407 gels

2.6.1. Gelation temperatureThe gelation temperatures of the examined formulations of

P407 solutions were determined using the Visual Tube Inversionwas multiplied by the total volume of the dispersion produced,considering the dilution factor. This represented the concentrationof free drug (Cf, namely that not entrapped in cubosomes). Thiswas then subtracted from the total drug concentration (Ct) in theformulation to give the amount of drug that was successfully en-trapped inside the cubosomes. Each experiment was repeatedthree times.

EE %of cubosomes Ct Cf=Ct 100 1

2.5.5. In vitro drug release from cubosomesA modied stainless steel disk assembly (USP Apparatus 5, pad-

dle over disk assembly), was used for the assessment of the releaseof the drug from the dispersions. A sample containing 50 mg of ALAwas placed in a disk and covered by a membrane then placed at thelter having a pore size of 0.1 lm. The ltrate was analyzed

s and Biopharmaceutics 86 (2014) 251259 253P407 gels were assayed by a continuous shear method using aRheotest 2.1 viscometer with concentric cylinders, designed foruse with preparations having viscosities between 20,000 and

2.6.3. In vitro drug release from the gels

the preceding 12 months. Exclusion criteria also comprehended for

appropriate. At the start of the study volunteers were subjected to

3.2. Clinical assessment of response to treatment

made above the zygomatic bone. The following parameters weremeasured: epidermis and dermis thickness and dermis density.Ultrasonic images were taken three times at each time for eachsubject.

3.3. Data analysis

The differences between the percent increase in the thickness ofthe epidermis and dermis layers, for both groups (Treatment and

Table 2Classication of volunteers according to the Glogau scale.

Volunteer number Age (yr) Glogau scale

1 38 I2 45 II3 41 III4 42 II5 48 III6 42 III

Table 3Patient satisfaction scoring criteria.

Score Grade Description

0 Dissatisfaction Patient feels worse than before/the same asbefore

1 Slightly satised Patient feels slightly better but still not worth it2 Moderately Patient feels good with need for slight

utics and Biopharmaceutics 86 (2014) 2512593.2.1. Clinical photographyan assessment of wrinkles using Glogau Photo-aging Classicationdened as follows; type I no wrinkles, type II winkles in mo-tion, type III wrinkles at rest and type IV only wrinkles as de-scribed in Table 2. Volunteers were instructed to apply theprepared formulation on the right side of their faces twice daily,for a duration treatment of 3 months. Volunteers were followedup on a weekly basis till the end of the treatment plan.patients with active inammation, infection, cancerous or precan-cerous lesions and unhealed wounds of the face region. Patientswith collagen related diseases, alteration of blood clotting (e.g.hemophilia or on anticoagulant treatment), diabetes as well asthose treated with systemic retinoids within 2 years were ex-cluded. Volunteers eligibility for study participation was deter-mined on the rst visit through a thorough history taking and adetailed clinical examination. Pregnancy test was done only whenThe same procedure that was used for the evaluation of ALArelease from cubosomes was adopted for evaluating release fromP407 gels loaded with ALA or ALA cubosomes Aliquots were with-drawn at appropriate time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h).Percentage ALA released from each of the two concentrations ofP407 gel loaded with 50 mg ALA or ALA cubosomes (D3) has beenplotted against the time.

2.7. Statistical analysis

All experiments were performed in replicate for validity of sta-tistical analysis. Results were expressed as mean SD. One-wayanalysis of variance (ANOVA) was used to assess statistical signif-icance where required. Differences were considered signicant forP-values < 0.05.

3. In vivo evaluation of skin rejuvenation effect in volunteers

3.1. Study design

The study was designed as a double blinded placebo controlledstudy. Ethical approval was granted by Research Ethics Committeeof the Faculty of Pharmacy, Cairo University and the protocol com-plies with the declarations of Helsinki and Tokyo for humans. Thenature and purpose of the study were fully explained to volunteersand written informed consent was obtained from all of them.Twelve healthy female volunteers within the age of 3864 yearswere chosen to participate in our study. They are divided randomlyinto two groups; Group I: consisting of eight volunteers, receivedP407 gels loaded with ALA cubosomes (Treatment). Group II:consisting of four volunteers, received P407 gels loaded withcubosomes free from the drug (Placebo). The volunteers couldnot be pregnant, lactating or smokers nor received any cosmeticprocedures in the face including botulinum toxin treatment ofany serotype, dermal llers, injection lipolysis, laser treatment,dermabrasion or anti-aging treatments (creams and tablets) during380,000 cP. Samples were subjected to shear rates between 0 and165 s1. The full scale torque was 5500 dyne cm2.

254 S. Sherif et al. / European Journal of PharmaceHigh resolution digital photographs were obtained for bothsides of the face, at each visit, by a xed digital camera, set at axed distance and constant settings for standardization.Clinical efcacy was assessed subjectively using a 5-gradeGlobal Aesthetic Improvement Scale (GAIS). The volunteers weregraded the overall esthetic change (worse 1, no change 0,somewhat improved 1, moderately improved 2 or very muchimproved 3) by comparing the patients visual appearance atfollow-up against an archival photograph taken prior to treatment.

3.2.2. Patient satisfaction levelThe volunteers were allowed to independently grade their

satisfaction level (PS) at each assessment time according to the cri-teria in Table 3. Meticulous reporting of any adverse skin reactionssuch as erythema, itching or swelling, if any, was also recorded.

3.2.3. Ultrasound biomicroscopy (UBM)Ultrasound measurements were performed using paradigm

ultrasound biomicroscope plus Model P45 (UBM plus), which is amicroprocessor-based digital instrument that uses very high fre-quency ultrasound (50 MHz) to produce a two dimensional sectionview of the examined tissue in B-scan (brightness) mode for diag-nostic evaluation, and A-scan mode that measures the axial lengthwith depth of penetration up to 5 mm. The ultrasound scan imagesacquired by the UBM transducer are viewed on a high resolutioncolor monitor in real-time. The reected ultrasound waves are con-trolled and assembled by the computer and magnied to provide ahigh resolution B-scan image. The cross-sectional picture can befurther enhanced by digital lters to improve image contrast orbrightness to identify interesting ndings without altering theoriginal scanned le image. Skin thickness measurements were

7 51 III8 64 III9a 39 II

10a 47 III11a 43 III12a 42 I

a Placebo group.satised improvement3 Satised Patient feels optimal cosmetic result

Placebo) were assessed by a students t-test using the statisticalpackage SPSS version 16.0. Differences were considered statisti-cally signicant at P-values less than 0.05.

4. Results and discussion

Table 4 illustrates the EE % of each of the 3 dispersions. This maybe attributed to the high solubility of the drug in the GMO.

4.6. Gelation temperature and rheological measurements

The gelation of poloxamer solution is known to be a reversibleprocess, i.e. gels revert to free-owing solutions when the temper-ature drops below the critical gel temperature (CGT). If the gelationtemperature is higher than 36 C, the formulation will remain li-quid at body temperature, and thus does not control the releaseof ALA. It would be convenient to have a gelation temperature be-tween 30 and 36 C, to be liquid at room temperature, and thuscould be spread efciently on the skin, and form a gel instantlyupon contact with the skin [24]. The CGTs of 20 and 30% w/w of

Table 4

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.0 D1 D2 D3

Zeta

Pot

entia

l (mV)

Formulation

Fig. 3. Chart illustrating zeta potential measurements of ALA cubosome dispersionsD1, D2 and D3 (n = 3). (For interpretation of the references to color in this gurelegend, the reader is referred to the web version of this article.).

S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259 255Particle size distribution and encapsulation efciency (EE) of ALA cubosomes D1, D2and D3 (n = 3).

Dispersion Particle size (nm) SD EE (%) SD

D1 148 0.9 76.60 0.624.1. Preparation of ALA cubosomes

Formulations, D1, D2 and D3 produced uniform dispersions ofcubosomes. D4 was found to be non-uniform. Dispersions D1, D2and D3 which were prepared by emulsication of GMO and P407in water followed by ultrasonication were chosen for furthercharacterization whereas D4 was rejected, as there were manyaggregates produced and less cubic structures were found.

4.2. Particle size and zeta potential

The particle size distribution for dispersions D1, D2 and D3 istabulated in Table 4. It can be observed that by increasing the con-centration of GMO in the formulation the particle size becomessmaller.

Zeta potential is an important parameter for the stability andbio-distribution of colloidal dispersions [21]. Generally, high sur-face charges lead to electrical repulsion between particles and thusprevent their aggregation [22]. As demonstrated in Fig. 3, resultsshowed that by increasing the GMO concentration used in the for-mulation the negative charge increases. This could be attributed tothe presence of free oleic acid with the GMO (as the purity of thecommercially available GMO is 95%). As it is lipophilic, free oleicacid should be absorbed onto the surface of the cubosomes, someof which might ionize to carry a negative charge. Therefore, thenegative charge on the surface of cubosomes was mainly due tothe ionization of the carboxylic end group of oleic acid. Althoughthe zeta potential in this system was not high enough to provideeffective electric repulsion to avoid the aggregation of particles,however, P407 acting as a steric stabilizer, would not only stabilizethe cubic phase dispersions efciently but also preserve the innercubic structure of the particles [23].

4.3. Optical microscopy

The outer cubic structure of the cubosomes was clearly obviousin each of the three dispersions as viewed in Fig. 4.

4.4. Transmission electron microscope

When the samples were viewed under the electron microscope,discrete particles with an outer cubic structure could be viewed aspresented in Fig. 5.

4.5. Encapsulation efciency

D3 showed an entrapment efciency of 86.48% which is consid-ered as the highest encapsulation efciency amongst the 3 formu-lations tested. The EE % increased as the GMO content increased.D2 123 0.8 83.85 1.39D3 101 0.7 86.48 0.68Fig. 4. Optical micrograph of ALA cubosome dispersions D1, D2 and D3. (Forinterpretation of the references to color in this gure legend, the reader is referredto the web version of this article.).

05

10

15

20

25

30

20 30

Gel

atio

n te

mpe

ratu

re (o

C)

P407 gel concentration (% w/w)

P407 placebo solution

P407 solution loaded with ALA

P407 solution loaded with ALA cubosomes

Fig. 6. Chart illustrating changes in the critical gelation temperature using theVisual Tube Inversion Method as function of loading 20 and 30% w/w P407solution with ALA or ALA cubosomes, in comparison with P407 placebo solution(n = 3). (For interpretation of the references to color in this gure legend, the readeris referred to the web version of this article.).

0

100

200

300

400

500

600

20 30

App

aren

t visc

osity

(Pois

e)

P407 gel concentration (% w/w)

P407 placebo gelP407 gel loaded with ALAP4O7 gel loaded with ALA cubosomes

Fig. 7. Apparent viscosities of 20 and 30% w/w poloxamer gels as function of

utics and Biopharmaceutics 86 (2014) 251259P407 solution loaded with ALA or ALA cubosomes compared withP407 placebo solution are shown in Fig. 6. Results indicate thatby increasing the concentration of P407 in aqueous solutions, thegelation temperature is lowered [24]. The inuence of loadingP407 gel with ALA or ALA cubosomes on the gelation temperatureis a further lowering the CGT. The cubosomes utilized in this studyare dispersions of GMO bulk cubic phase. The cubosomes formedare stabilized by the addition of P407, where the hydrophobicPPO portion is presumed to adsorb to the surface of cubosomeswhile the hydrophilic PEO chains extend out into the aqueous envi-ronment to provide steric shielding [25].

The presence of P407 on the surface of the cubosomes increasesthe apparent concentration of polymer in the solutions and therebyincreasing the viscosity of the gel as demonstrated in Fig. 7 andlowering the CGT [17]. Similar effects were previously reportedwhen introducing inorganic salts to P407 systems, which havebeen described as salting-out effects. This phenomenon has beenascribed to the ability of the salts to reduce the water activity, byhydrogen bonding to the water and thereby increasing the effec-tive aqueous concentration of the polymer [19,26]. Similarly, theaddition of cubosomes might also result in decreased water activ-ity leading to an increased effective concentration of P407 andFig. 5. Transmission electron micrograph of ALA cubosomes dispersion D3.

256 S. Sherif et al. / European Journal of Pharmacethereby lowering of the CGT of the system [17].

4.7. In vitro drug release from cubosomes

In vitro release prole of ALA from cubosomes was performed inhydro-alcoholic solution using the paddle over disk method. Forcubosomes of D1, 97.54% of the drug was released within 6 h. Forcubosomes of D2, 94.18% of the drug was released within 6 h.For cubosomes of D3, 90.04% of the drug was released within 6 h(Fig. 8). The results indicated a trend toward a decrease in releaserate with increasing GMO concentration, although this was not sta-tistically signicant (p > 0.05). This may be attributed to the highsolubility of ALA in GMO. The release rate constants and correla-tion coefcients were calculated after tting the release data tosquare root Higuchi model and are summarized in Table 5. The cor-relation coefcients (R) calculated for each formulation were above0.99, which indicated that in vitro drug release proles of ALAcubosomes t well with the square root Higuchi model. Hence for-mulation D3 was selected as the optimized formulation by virtueof slowest drug release and maximum entrapment efciency.

The release proles of ALA from 20 and 30% w/w P407 gel asillustrated in Fig. 9, display a trend of a decrease in the release rateas P407 concentration increases [10], in which 98.9% and 98.02% of

loading gel with ALA or ALA cubosomes, in comparison with P407 placebo gel, at25 C (n = 3). (For interpretation of the references to color in this gure legend, thereader is referred to the web version of this article.).

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

% A

LA re

leas

ed

Time (hrs)

D1

D2

D3

Fig. 8. In vitro ALA release from dispersions of cubosomes with different concen-trations of GMO, 5% w/w (D1), 10% w/w (D2) and 15% w/w (D3) using Paddle overDisk Assembly method in (1:1) hydro-alcoholic solution at 32 0.5 C (n = 3).

100

Time (h)Fig. 10. Time dependent effect of P407 concentration on ALA release from P407 gelloaded with ALA cubosomes using Paddle over Disk Assembly method in (1:1)hydro-alcoholic solution at 32 0.5 C (n = 3).

Table 7Release rate constants and correlation coefcients of 20 and 30% w/w P407 gel loadedwith ALA cubosomes calculated in accordance with the release proles obtained usingsquare root Higuchi model.

euticALA were released within 8 h respectively and the difference wasnot statistically signicant (p > 0.05). The release rate constantsand correlation coefcients were calculated after tting the releasedata to square root Higuchi model and are summarized in Table 6.

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8

% A

LA re

leas

ed

Time, hr

20% W/W P407 gel with ALA

30% W/W P407 gel with ALA

Fig. 9. Time dependent effect of P407 concentration on ALA release from P407 gelloaded with 50 mg ALA, using Paddle over Disk Assembly method in (1:1) hydro-alcoholic solution at 32 0.5 C (n = 3).Table 5Release rate constants and correlation coefcients of ALA cubosome dispersions D1,D2 and D3, calculated in accordance with the release proles obtained using squareroot Higuchi model.

Dispersion Rate constant R Equation

D1 20.933 0.9902 y = 20.933t1/2 0.277D2 20.086 0.9923 y = 20.086t1/2 0.2414D3 18.195 0.9941 y = 18.195t1/2 0.0149

S. Sherif et al. / European Journal of PharmacThe correlation coefcients (R) calculated for each gel concentra-tion were above 0.99, which indicated that in vitro drug releaseproles of ALA from P407 gel t well with the square root Higuchimodel.

The release prole of ALA from 20 and 30% w/w P407 gel loadedwith ALA cubosomes, showed that 65.34% and 55.52% of the drugwere released after 8 h respectively, as illustrated in Fig. 10. The re-lease rate constants and correlation coefcients were calculatedafter tting the release data to square root Higuchi model andare summarized in Table 7. The correlation coefcients (R) calcu-lated for each gel concentration were above 0.99, which indicatedthat in vitro drug release proles of ALA from P407 gel t well withthe square root Higuchi model. From these results it can bededuced that by increasing the P407 concentration of the gel, therelease rate of ALA was prolonged. This could be explained thatby increasing the concentration of P407, the viscosity of the gel in-creases as well, making it harder for the cubosomes to diffuse outof the gel matrix.

4.8. In vivo evaluation of skin rejuvenation effect in volunteers

Clinical outcome was evaluated based on the GAIS level ofimprovement. It was observed that no marked improvement wasseen after 1.5 months of treatment for group II (Placebo) but 75%of volunteers showed some sort of improvement for group I (Treat-ment). However, after 3 months of treatment moderate improve-ment was seen in about 58.33% of the volunteers treated by(Treatment) and 25% of volunteers of group I (Treatment) showedvery much improvement and satisfaction, while none of the volun-teers of group II showed such improvement, as presented in Fig. 11.

After 3 months of treatment, it was noted that there wasimpressive changes in the ne lines and also the pore sizes onTable 6Release rate constants and correlation coefcients of 20 and 30% w/w P407 gel loadedwith ALA calculated in accordance with the release proles obtained using squareroot Higuchi model.

P407 gel loadedwith ALA

Rate constant R Equation

20% w/w 21.058 0.9964 y = 21.058t1/2 5.266430% w/w 18.804 0.9985 y = 18.804t1/2 4.9405

0

10

20

30

40

50

60

70

0 2 4 6 8

% A

LA re

leas

ed

20%w/w P407 gel with ALA cubosomes30%w/w P407 gel with ALA cubosomes

s and Biopharmaceutics 86 (2014) 251259 257the face were diminished (Fig. 12). A visible reduction in the depthof ne periorbital lines and ne vertical lines on the upper lip wasalso observed (Fig. 13), deeper periorbital lines appeared shallowafter completing the treatment regimen (Fig. 14). This assessmentwas conrmed by comparison with photographs taken at thebeginning of the study. It is believed, however, that this effectwas due to increased collagen production by saturation of bro-blasts with ALA. Cellular membrane repair has been attributed toantioxidant administration to tissues [27]. In addition to the abovendings, the female volunteers also reported a noticeable differ-ence in skin tone of the face, which was described as a healthyglow (Fig. 14) [28]. Subjects reported no complaints regardingirritation from daily use of the gel. Two of the subjects reportedimprovement of facial scars, which was conrmed by comparisonwith photographs taken at the beginning of the study.

Fig. 15 shows the visible change in the dermis density by fol-lowing the 3 month treatment course which was conrmed byultrasound. The increase in dermal density implies an increase inthe amount of collagen. After the 3 month treatment course solarscars were treated and are not apparent in the ultrasound image.The activation of transcription factor AP-1, through production ofcollagenases, could remodel the damaged collagen, resulting inthe clinical improvement of solar scars [28].

Fig. 15, visualizes the progressive replenishment of lowechogenic, dark areas seen over treatment course of 3 months.

P407 gel loaded withALA cubosomes

Rate constant R Equation

20% w/w 14.401 0.9950 y = 14.401t1/2 8.381230% w/w 12.809 0.9924 y = 12.809t1/2 9.9124

utics and Biopharmaceutics 86 (2014) 25125950

60

70

80

unte

ers

1.5 months (Treatment)

1.5 months (Placebo)

3 months (Treatment)

3 months (Placebo)

258 S. Sherif et al. / European Journal of PharmaceThe results indicate an increase in the thickness of the epidermisand dermis layers after 3 months of treatment for the Treatmentgroup. The average % increase in the thickness of the epidermisdermis layer was 8.84 4.93% for the Treatment and was2.95 2.14% for the Placebo. Statistical analysis revealed a signif-icant difference in the % increase in the thickness of epidermisdermis layers between the Treatment and Placebo groups after3 months of treatment as (p-value = 0.048).

It is worthy to note that application of ALA cubosomes twicedaily for 3 months leads to satisfactory progress in treatment ofwrinkles and scars in the studied volunteers. The results conrmthat cubosomes are useful in topical drug delivery, particularlydue to their bioadhesive properties, their characteristics as

0

10

20

30

40

No change Somewhat improved

Moderately improved

Very much improved

% o

f Vol

GAIS Category

Fig. 11. Categorical outcomes of the Global Improvement Scale (GAIS) score at 1.5and 3 months post-treatment for both groups of volunteers (Treatment andPlacebo). (For interpretation of the references to color in this gure legend, thereader is referred to the web version of this article.).

Fig. 12. Photographic images depicting the facial area of a 45-years old volunteer(A) before treatment and (B) 3 months after treatment with 30% P407 gel containingALA cubosomes D3. (For interpretation of the references to color in this gurelegend, the reader is referred to the web version of this article.).

Fig. 13. Photographic images depicting the facial area of a 48-years old volunteer(A) before treatment and (B) 3 months after treatment with 30% P407 gel containingALA cubosomes D3. (For interpretation of the references to color in this gurelegend, the reader is referred to the web version of this article.).

Fig. 14. Photographic images depicting the facial area of a 51 years old volunteer(A) before treatment and (B) 3 months after treatment with 30% P407 gel containingALA cubosomes D3. (For interpretation of the references to color in this gurelegend, the reader is referred to the web version of this article.).

References

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259 259sustained-release delivery systems, and their ability to protect theencapsulated drug from degradation. It is also important to men-tion that Cubosomes show a structural organization identical tothat found in biomembranes which make them biocompatibleand of fewer side effects.

5. Conclusions

From the results, it could be concluded that ALA cubosomescould be formulated using the conventional emulsication/ultra-sonication technique, whereas the dilution method was notsuccessful in producing uniform dispersions. By increasing theGMO content in the dispersion, the cubosomes size decreases,the encapsulation efciency increases, zeta potential becomesmore negative. From the in vitro study, it was demonstrated thatcubosomes can incorporate and deliver the potent antioxidantALA, with a release prole that ts with the square root Higuchi

Fig. 15. High resolution ultrasonic images of pre- and post-treatment (A and B) fortwo representative volunteers.model. It was also apparent that 90.04% of the drug was releasedfrom formula D3 within 6 h. When ALA cubosome dispersionswere loaded into 30% P407 gels, formulation signicantlydecreased the drug release rate. In summary, preliminary clinicalevidence indicates that 30% w/w P407 gel loaded with ALA cubo-somes is safe and effective in creating esthetic correction of the fa-cial region when used for at least 3 months in the great majority ofsubjects. Further clinical studies will be conducted on largenumber of volunteers to conrm the clinical efciency of ALAcubosomes as anti-wrinkle.

Acknowledgment

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The clinical efficacy of cosmeceutical application of liquid crystalline nanostructured dispersions of alpha lipoic acid as anti-wrinkle1 Introduction2 Materials and methods2.1 Materials2.2 Preparation of cubosome dispersions2.3 Preparation of P407 gels2.4 Assay of alpha lipoic acid2.5 Characterization of cubosomes2.5.1 Particle size and zeta potential measurements2.5.2 Light microscopy2.5.3 Transmission electron microscopy (TEM)2.5.4 Entrapment efficiency2.5.5 In vitro drug release from cubosomes

2.6 Characterization of P407 gels2.6.1 Gelation temperature2.6.2 Rheological measurements2.6.3 In vitro drug release from the gels

2.7 Statistical analysis

3 In vivo evaluation of skin rejuvenation effect in volunteers3.1 Study design3.2 Clinical assessment of response to treatment3.2.1 Clinical photography3.2.2 Patient satisfaction level3.2.3 Ultrasound biomicroscopy (UBM)

3.3 Data analysis

4 Results and discussion4.1 Preparation of ALA cubosomes4.2 Particle size and zeta potential4.3 Optical microscopy4.4 Transmission electron microscope4.5 Encapsulation efficiency4.6 Gelation temperature and rheological measurements4.7 In vitro drug release from cubosomes4.8 In vivo evaluation of skin rejuvenation effect in volunteers

5 ConclusionsAcknowledgmentReferences