Formulation of a Novel Oxybenzone-Loaded Nanostructured Lipid Carriers (NLCs)

Research Article

Formulation of a Novel Oxybenzone-Loaded Nanostructured Lipid Carriers(NLCs)

Rania A. Sanad,1 Nevine Shawky AbdelMalak,2,3 Tahany S. elBayoomy,1 and Alia A. Badawi2

Received 7 September 2010; accepted 12 November 2010; published online 24 November 2010

Abstract. The objective of the current study was to formulate oxybenzone into nanostructured lipidcarriers (NLCs) to enhance its sunscreening efficacy and safety. NLCs of oxybenzone were prepared bythe solvent diffusion method. A complete 23 factorial design was used for the evaluation of the preparedoxybenzone NLCs. The study design involves the investigation of the effect of three independentvariables namely liquid lipid type (Miglyol 812 and oleic acid), liquid lipid concentration (15% and 30%),and oxybenzone concentration (5% and 10% with respect to total lipids) on the particle size (p.s.) , theentrapment efficiency (EE%) and the in vitro drug release after 8 h. The prepared NLCs were sphericalin overall shape and were below 0.8 μm. Miglyol 812 and 30% liquid lipid were found to significantlydecrease the p.s. and increase the EE% when compared to oleic acid and 15% liquid lipid. Increasingoxybenzone concentration increased significantly the p.s. but did not affect the EE%. NLCs preparedusing Miglyol 812, 15% liquid lipid, and 10% oxybenzone showed slower drug release when compared tothose prepared using oleic acid, 30% liquid lipid, and 5% oxybenzone, respectively. The candidateoxybenzone-loaded NLC dispersion was then formulated into gel. The incorporation of oxybenzone intoNLCs greatly increased the in vitro sun protection factor and erythemal UVA protection factor ofoxybenzone more than six- and eightfold, respectively, while providing the advantage of overcoming sideeffects of free oxybenzone as evidenced by very low irritation potential.

KEY WORDS: in vitro erythemal UVA protection factor; in vitro SPF; irritation test; nanostructuredlipid carriers (NLCs); oxybenzone; vitro-Skin™.

INTRODUCTION

Nowadays, the public awareness regarding the harmfuleffects of UV radiation combined with the problem of theozone layer is increasing. Therefore, the use of sunscreensbecomes necessary in daily life. There are two different waysof action for sunscreens: physical sunscreens and molecularsunscreens. Molecular sunscreens contain conjugated π-andn-electrons which are excited by certain wavelengths. Theabsorbed radiation is then re-emitted as lower energy rays,thus avoiding the skin harmful ultraviolet rays from reachingthe skin (1). Oxybenzone is a widely used lipophilic, broad-spectrum molecular sunscreen agent, which effectivelyabsorbs ultraviolet B (UVB) (290–320 nm), some ultravioletA (UVA; 320–360 nm), and some ultraviolet C (250–290) (2).However, oxybenzone is the most common cause of photo-allergic contact dermatitis (3). In addition, systemic absorp-tion of oxybenzone following its topical application to theskin has been reported (4).

Therefore, there is an urgent need for the developmentof safer sunscreen systems. This can be achieved by for-

mulations that penetrate less into the skin or by formulationswith a reduced amount of potentially dangerous molecularUV blocker while maintaining the sun protection factor byother means, e.g., carriers with sunblocking characteristics(5).

Nanostructured lipid carriers (NLCs) consisting of a lipidmatrix with a special nanostructure had been developed as anew, improved generation of lipid nanoparticles (6). NLCsadd additional benefits to the well-known opportunities ofconventional solid lipid nanoparticles (SLNs) and are suitedfor topical use (7). In contrast to SLNs being produced fromsolid lipids, the NLCs are produced using blends of solidlipids (long chain) and liquid lipids (short chain) (8).Compared to SLNs, NLCs possess lower melting point dueto their oil content, while maintaining their particulatecharacter and being solid at body temperature (6).

Literature lacks any previous attempts to formulateoxybenzone as sole sun screening agent in nanostructuredlipid carriers.

In the present study, we attempted to formulateoxybenzone-loaded NLCs. Then, a candidate formula withoptimum physicochemical characterization was selected.This formula was then formulated into gel. Skin irritationtest, in vitro sun protection factor (SPF), erythemal UVAprotection factor of free oxybenzone and the candidateformula before and after formulation into gel weremeasured using Vitro- Skin™.

1 Department of Pharmaceutics, National Organization of DrugControl and Research (NODCAR), Cairo, Egypt.

2 Department of Pharmaceutics, Faculty of Pharmacy Cairo University,Kasr El Ainy street11562, Cairo, Egypt.

3 To whom correspondence should be addressed. (e-mail: [email protected])

AAPS PharmSciTech, Vol. 11, No. 4, December 2010 (# 2010)DOI: 10.1208/s12249-010-9553-2

1530-9932/10/0400-1684/0 # 2010 American Association of Pharmaceutical Scientists 1684

MATERIALS AND METHODS

Materials

Oxybenzone, was obtained from International SpecialityProducts, ISP, USA; Glyceryl monostearate (GMS) which is amixture of 40–50% mono-, 30–45% di- and 5–15% triglycer-ides esters of stearic acid (C21) and palmitic acid (C19) withmelting point of 55–66°C was obtained from Carl RothGmbH, Germany; Miglyol 812 which is caprilic/capric trigly-cerides, was obtained from Goldschmidt GmbH, Germany;oleic acid was obtained from Riedel de-Haen, Germany;polyvinyl alcohol (PVA) of average molecular weight146,000–186,000 was obtained from Celanese, USA; ethanol95% and acetone were obtained from Riedel-de HaenGrambH, Germany; Carbopol 934 was obtained from Good-rich Chemical Co, USA. All other chemicals and solventswere of analytical grade.

Design of the Experiments

A complete 23 factorial design was used to study theeffect of the liquid lipid type, liquid lipid concentration, andoxybenzone concentration on the particle size, theentrapment efficiency and the percentage of oxybenzonereleased after 8 h. Table I summarizes the independentvariables along with their levels. The experimental resultswere analyzed using StatView Abacus Concept Version 4.57software. The concentration of the total lipid (10%), type ofsolid lipid (glyceryl monostearate), concentration of PVA(1%) as well as solvent ratio 1:1 (ethanol/acetone (v/v)) werekept constant and were selected based on preformulationstudies (data not shown).

Preparation of Oxybenzone Nanostructured Lipid Carriers

NLCs loaded with oxybenzone (5% or 10% w/w) wereprepared by the solvent diffusion method in an aqueoussystem (9) with slight modification. The amount of the drug tobe added (in grams) was calculated as a percentage of thelipid matrix as follows: 100 g of a 10% NLCs dispersionloaded with 5% drug contained 10 g solid consisting of 9.5 glipid mixture (liquid lipid 1.425 g and GMS 8.075 g) and 0.5 gdrug. For NLCs dispersion loaded with 10% drug, the properamounts of the formulation ingredients were used.

The lipid mixture (GMS with Miglyol 812 or oleic acid)and oxybenzone were completely dissolved in a 12-mlmixture of ethanol and acetone 1:1 (v/v) in a water bath at50°C. The resultant lipid solution was poured into 240 ml ofan aqueous phase containing PVA (1% w/v) under mechan-

ical agitation using mechanical stirrer (Falc Instruments,Italy) at 400 rpm in a water bath at 70°C for 5 min. Theobtained dispersion was allowed to cool to room temperaturewhile stirring by a magnetic stirrer to get rid of organicsolvents, and then oxybenzone-loaded NLCs were finallyobtained.

Oxybenzone suspension (without adding lipids) and theplacebo NLC dispersions (without adding the drug) wereprepared exactly in the same manner. An overview of thecomposition of the NLCs is given in Table II.

Characterization of Oxybenzone Nanostructured LipidCarriers

Transmission Electron Microscope

The morphology of the oxybenzone NLCs (selectedsamples were NLC4 and NLC8) was examined by thetransmission electron microscope (JEM-100S, Jeol Ltd.,Japan). Samples were prepared by the negative stainingtechnique. The NLCs were dispersed directly into bidistilledwater, and then copper grid coated with collodion film wasput into the solution for several times. After being stained by2% (w/v) phosphotungestic acid solution and dried at roomtemperature, the sample was ready for the transmissionelectron microscope (TEM) investigation at 70 kV (10).

Particle Size Analysis

Particle size and polydispersity index (PI) which is ameasure of the distribution of nanoparticle populationwere determined by using laser scattering particle sizedistribution analyzer (LA-920, Horiba, Japan; detectionlimit 0.2–2,000 μm). One day after production, NLCdispersions were diluted with filtered bidistilled water,sonicated for 30 s, and subsequently analyzed. Threeanalyses were performed for each sample and the averagevalues were taken. The obtained data were evaluated usingthe volume distribution (d10%, d50%, d90%) which meansthat if the diameter 90% (d90%) is registered as 1 μm, thisindicates that 90% of particles have a diameter of 1 μm orlower. The PI was measured by the span which can becalculated from the following equation (11):

Span ¼ d90% � d10%d50%

Where d90% is the particle diameter at 90% cumulative size,d10% is the particle diameter at 10% cumulative size, andd50% is the particle diameter at 50% cumulative size.

Table I. 23 Factorial Design for Preparation of Oxybenzone NLCs

Variables Levels

Liquid lipid type Miglyol 812 Oleic acidLiquid lipid conc. (% w/w)a 15 30Oxybenzone conc. (% w/w)b 5 10

a Percent with respect to lipid mixtureb Percent with respect to lipid matrix

1685Formulation of a Novel Oxybenzone-Loaded Nanostructured Lipid Carriers

Zeta Potential (ζ)

The zeta potential was measured using the Zetasizer2000 (Malvern Instruments Ltd., Malvern, UK). The solidlipid nanoparticle dispersions were diluted with 1 mM NaCland placed in the electrophoretic cell where an electric fieldof 80 mV was established. Measurements were carried out intriplicate at 25°C. The electrophoretic mobility and hencezeta potential was calculated directly from the apparatususing the Smolochowski equation (12)

Determination of Oxybenzone Entrapment Efficiency Percent

The drug-loaded NLC dispersion was uniformly mixedby gentle shaking; 1.0 ml of this dispersion was diluted with9.0 ml methanol, centrifuged by using High-Speed Refriger-ated Centrifuge (3K30, SIGMA, Germany) for 45 min at16,000 rpm and then filtered using Millipore® membrane(0.2 μm). The filtrate was collected and appropriately dilutedwith methanol and measured spectrophotometrically (Shi-madzu, model UV-2450, Kyoto, Japan) at lmax of 286 nm(13). The entrapment efficiency percent (EE%) was calcu-lated using the following equation (9):

Entrapment efficiency EE%ð Þ ¼ Wa�WsWa

� 100

Where Wa and Ws were the weight of drug added in systemand the analyzed weight of drug in supernatant, respectively.

In vitro Release Studies of Oxybenzone from NLCs: FranzDiffusion Cells

For this investigation, static Franz glass diffusion cells(Microette plus, Hanson Research, USA) were used (14).These cells consist of donor and receptor chambers betweenwhich a cellulose membrane (MEMBRA-CEL dialysis tubingwith molecular weight cutoff 3,500–7,000 Da, Serva Electro-phoresis GmbH, Germany) was positioned; area of diffusionwas 1.7 cm2. The dialysis membrane was hydrated in receptormedium [methanolic buffer solution (phosphate-bufferedsaline pH 7.4/methanol (3:2) (v/v))] for 12 h beforemounting into the Franz diffusion cell. Oxybenzone NLCdispersion (2 mg/cm2) was placed in the donor chamber andthe receptor chamber was filled with 7.5 ml receptor mediumand stirred continuously at 100 rpm at 37°C in order to ensure

the surface skin temperature of 32°C on the surface of themembrane (14). After 1, 2, 3, 4, 5, 6, 7, and 8 h, 2 ml werewithdrawn from the receptor chamber through a side-armtube. After each withdrawal of sample, an equal volume ofreceptor medium was added in the receptor chamber so as tomaintain a constant volume throughout the study. Sampleswere analyzed for oxybenzone concentration using ultravioletspectrophotometry at 289.4 nm.

Effect of Storage Temperature on Particle Sizeand Entrapment Efficiency of the Prepared NLCs

Oxybenzone NLC dispersions were divided into twosample sets of capped vials protected from light, one storedin a refrigerator at 2–8°C and the other stored at roomtemperature (25°C). Samples were withdrawn after 3 monthsand subjected to particle size and entrapment efficiencymeasurements.

Formulation of NLC-Based Hydrogel

Based on the previously mentioned characterization, andthe results of the main effects of the adopted factorial designa candidate formula NLC4 (containing 1% oxybenzone) withoptimum physicochemical properties was selected. Theselected NLC4 dispersion was formulated into hydrogel(NLC4G) by adding 1% (w/w) Carbopol 934 under magneticstirring at 800 rpm. Stirring was continued until carbopol isdispersed. The dispersions were neutralized using triethanol-amine solution (15). Hydrogel formulations containing either1% oxybenzone suspension (1% oxy G) and placebo NLC4(pNLC4G) were prepared for comparison.

Characterization of the Prepared Gels

Rheological Studies

The viscosity and rheological behavior of the gelformulations were determined using a cone and plateviscometer (Brookfield Engineering Laboratories, modelHADV-II, Middleboro, MA, USA). All measurements werecarried out at a temperature of 25°C±1, using spindle CP52.The shear rate in s−1, the shear stress in dyne/cm2 and theviscosity in centipoise were determined. The rheologicalparameters of different gels were studied (16).

Table II. Composition of the Prepared Oxybenzone NLC Dispersions

Formulation code Liquid lipid type Liquid lipid conc. (% w/w)a GMSb conc. (% w/w)a Oxybenzone conc. (% w/w)a

NLC1 Miglyol 812 14.25 80.75 5NLC2 75.75 10NLC3 28.5 66.5 5NLC4 61.5 10NLC5 Oleic acid 14.25 80.75 5NLC6 75.75 10NLC7 28.5 66.5 5NLC8 61.5 10

GMS glyceryl monostearate (the solid lipid used)a Percent with respect to total lipid concentration which is 10% w/w of the formulationbConcentration of PVA used is 1%

1686 Sanad, AbdelMalak, elBayoomy and Badawi

Skin Irritation Test

The study protocol and subject informed consent wereapproved by the institutional review board of Faculty ofPharmacy, Cairo University (IRB00007140) and the studywas conducted according to the Declaration of Helsinki (17)and the International Conference on Harmonization ofTechnical requirements for Registration of Pharmaceuticsfor Human Use Guidance for good clinical practice (18).

Ten healthy subjects (aged from 23 to 40 years) wereparticipated in this study. The participants were briefed onthe study procedures and a written informed consent wasobtained from all subjects prior to conducting procedure.

Each formulation (pNLC4, NLC4, and 1%oxybenzonesuspension) and their corresponding hydrogels (pNLC4G,NLC4G and 1% oxy G) was applied once to each volunteer,at a dose of 0.3 g on a surface area of 5 cm2 on forearm. After6 h, the test specimen was thereafter washed off by tap waterand observed for any visible change such as erythema(redness). The mean erythemal scores were recorded(ranging from 0 to 4) according to Draize scale (19). Where,0 means no erythema, 1 slight erythema, 2 moderateerythema, 3 moderate to severe erythema, and 4 severeerythema.

In vitro UV Blocking Ability

The Transpore® assay (20) is an in vitro method toinvestigate the UV blocking ability of the investigateddispersions (pNLC4, NLC4, and 1%oxybenzone suspension).A concentration of 2 mg/cm2 of the formulation was spreadevenly on top of the Transpore tape® (3M Australia Pty Ltd.,Australia) which was placed on a quartz cuvette. After adrying period of 15 min, the samples were scannedspectrophotometrically from 250 to 400 nm and theabsorption was measured.

In vitro SPF and Erythemal UVA Protection FactorMeasurement

The SPF mainly represents the protection against UVB(21). For this reason, the new developments in sunscreenshave to provide a description of the protection against notonly the UVB radiation, but also the UVA one (22). Thedetermination of the SPF of the formulations was conductedaccording to the method described by Diffey and Robson(23). Vitro-Skin™, a registered trademark of IMS, is anadvanced testing substrate used for in vitro measurement ofSPF. It contains both optimized protein and lipid componentsand is designed to have topography, pH, critical surfacetension, and ionic strength similar to human skin. It is thesubstrate that gives the most consistent correlation withpublished in vivo SPF measurements (24). It was used forsample application. It was hydrated by placing it on the shelfof a closed, controlled-humidity chamber (containing 85%water/15%glycerin in its bottom) for 16–24 h prior to its use(25) and then placed on a quartz cuvette. The intensity ofradiation transmitted through the substrate was determinedautomatically by recording the photocurrent in 5-nm stepsfrom 290 to 400 nm. An appropriate weight (2 mg/cm2) ofeach formulation (pNLC4, NLC4, and 1% oxybenzone

suspension) and their corresponding hydogels (pNLC4G,NLC4G and 1% oxyG) was applied to the substrate surface byspotting it at several sites over the application area (4.5 cm2).After a drying period of 15 min, transmission measurementswere done. These experiments were run in triplicates

The in vitro SPF can be calculated according to thefollowing equation (23):

SPF ¼X400

290

ElBl=X400

290

ElBl=MPFl� �

;

Where El, spectral irradiance of terrestrial sun light underdefined conditions; Bl, erythemal effectiveness; MPFl, themonochromatic protection factor at each wavelength incre-ment which is measured as the ratio of the detector signalintensity without sunscreen applied to the substrate, to thatwith sunscreen applied to the substrate.

Considering the UVA wavelength range (320–400 nm)and using the terms of SPF equation, the in vitro erythemalUVA protection factor could be calculated according to thefollowing equation (26).

ErythemalUV�Aprotection factor ¼X400

320

ElBl=X400

320

ElBl=MPFl� �

:

RESULTS AND DISCUSSION

In this study, 10% GMS (as solid lipid), Miglyol and oleicacid (as liquid lipids), and 1% PVA (as stabilizer) were usedto produce NLCs in order to enhance the sun screeningefficacy and safety of the chemical sunscreen oxybenzone.

NLCs were produced using the solvent diffusion method.This technique offers advantages over existing methods suchas the use of pharmaceutically acceptable organic solvents,easy handling, and a fast production process (27).

Characterization of the Prepared NLCs

Transmission Electron Microscope

Figure 1 shows the transmission electron micrographs ofoxybenzone NLCs prepared using Miglyol 812 (NLC4) oroleic acid (NLC8) as liquid lipids. Both micrographs showedspherical but not perfectly round-shaped particles that did notstick to each other.

Regarding the size, both Miglyol 812 and oleic acid NLCshad small particle size ranged from 10 to 40 nm and from 40 to60 nm, respectively. However, in order to obtain more preciseinformation on particle size, laser scattering was used.

Particle Size Analysis

Mean particle size, particle size distribution (d10%, d50%,and d90%) and polydispersity index (P.I.) of different NLCsare depicted in Table III. All oxybenzone NLCs showed aconsiderable small particle size with d90% less than 1 μm andwith P.I. ranging from 0.454 to 0.597 and from 0.528 to 0.641for oleic acid and Miglyol 812 NLCs, respectively.

The diameters determined by laser scattering wereconsiderably larger when compared to the results obtained


from TEM. This might be due to that the two methods arebased on totally different mechanisms and employing differ-ent sample preparation processes, which might lead to thediscrepancy of the outcomes. The size detection of NLCs bylaser scattering was carried out in aqueous state and in thiscase, lipid nanoparticles were well hydrated and the diame-ters were ‘hydrated diameters’, which were usually biggerthan their real diameters. In the case of TEM sample

preparation, all the free water and even some of hydratedwater was stained. This implies that the sizes of NLCs derivedfrom TEM might be considerably smaller than their realdiameters (28).

Concerning the liquid lipid type, oleic acid significantly(p<0.0001) increased the particle size when compared toMiglyol 812.This may be due to the higher viscosity of oleicacid at 20°C (36.56 cps) (29) when compared to that ofMiglyol 812 (28–32 cps) (30) at the same temperature.Consequently, this could be considered as the most importantfactor for the larger size of oleic acid NLCs. Hence, the meanparticle size of lipid nanoparticles usually increased withincreasing viscosity of the oil phase (31).

Concerning the two concentrations of both liquid lipids(Miglyol 812 and oleic acid) used, 30% gave significantly (p<0.0001) smaller particle size when compared to that of 15%.This could be attributed to that the higher liquid lipid contentwhich reduced the viscosity inside NLCs, and consequently,reduced the surface tension to form smaller and smoothersurface particles (32).

Increasing the oxybenzone concentration led to signifi-cant (p<0.0001) increase in particle size. This could be due tothe higher melting point of oxybenzone (66–68°C) (33) whencompared to that of GMS (the solid lipid used; 54–66°C) (30)which resulted in a more viscous dispersed phase, makingdifficult the mutual dispersion of the phases and originatinglarger particles (34). Similarly, Deli et al. (35) observed thatthe volume average diameter of NLCs increased as theamounts of drug increased.

Entrapment Efficiency

The entrapment efficiency of oxybenzone within thedifferent prepared nanostructured lipid carrier formulations isshown in Table III. High entrapment efficiency of oxy-benzone in all the prepared NLCs was observed, and wasfound to vary from 74.9%±3.0 for NLC6 using 15% oleic acidand 10% oxybenzone to 93.7%±3.2 for NLC4 using 30%Miglyol 812 and 10% oxybenzone (with respect to total lipidconcentration).

Concerning liquid lipid type, Miglyol 812 significantly (p<0.0001) increased the entrapment efficiency when compared tooleic acid. Miglyol 812, being mixture of triglycerides of differ-ent chain length (C8, C10) (36) form less perfect crystals withmany imperfections offering space to accommodate the drug

Table III. Particle Size Distribution, Mean Particle Size, Polydispersity Index (P.I) Entrapment Efficiency (E.E %), and Zeta Potential Valuesof Different Nanostructured Lipid Carriers

Formulation code

Mean volume distribution (μm±SD)

P.I. EE%±SD Zeta potential±SDd10% d50% d90% Mean particle size (μm ± SD)

NLC1 0.201 0.304 0.402 0.327±0.03 0.594 78.8±2.6 −14.00±0.5NLC2 0.235 0.407 0.743 0.461±0.03 0.528 79.2±1.9 −13.90±1.0NLC3 0.154 0.208 0.282 0.213±0.05 0.641 85.2±1.3 −21.03±1.2NLC4 0.195 0.313 0.593 0.366±0.09 0.558 93.7±3.2 −19.30±0.6NLC5 0.411 0.670 1.103 0.725±0.08 0.583 77.9±2.4 −29.70±1.8NLC6 0.360 0.708 1.339 0.797±0.10 0.479 74.9±3.0 −18.80±0.8NLC7 0.155 0.225 0.652 0.413±0.06 0.597 82.6±1.4 −16.70±0.5NLC8 0.204 0.405 0.941 0.501±0.02 0.454 80.2±1.5 −10.60±0.3

Fig. 1. Transmission electron microscope photograph of oxybenzonenanostructured lipid carriers using: a Miglyol (NLC4), b oleic acid(NLC8)


(37) when compared to oleic acid which is a monounsaturatedfatty acid form of stearic acid (38).

For liquid lipid concentration, 30% was found to signifi-cantly (p<0.0001) increase the entrapment efficiency whencompared to 15%. This could be attributed to the fact that drugsolubility is greater in liquid lipids than in solid lipids. Increasingliquid lipid content into the solid lipid matrix (GMS) couldincrease the solubility of the drug in the lipid matrix, whichincreased its entrapment efficiency (8). Furthermore, it wasreported (39) that the incorporation of liquid lipid to solid lipidcould led to great imperfections in the crystal lattice and leftenough space to accommodate drug molecules, thus leading toimproved drug entrapment efficiency.

Regarding oxybenzone concentration, 10% w/w (basedon total lipid concentration) was found to have a non-significant effect (p>0.05) on entrapment efficiency whencompared to 5% w/w.

Effect of Storage Time and Temperature on Particle Sizeand Entrapment Efficiency of the Prepared NLCs

The physical stability of Miglyol 812 and oleic acid NLCsformulations was suggested by the absence of visible phaseseparation and all dispersions remained in a homogeneousstate upon storage at 2–8°C and room temperature (25°C) for90 days. The mean particle size of the prepared NLCs afterstorage at both temperatures is shown in Fig. 2. Non-significant increase was observed in the particle size for thesamples stored at 2–8°C over the monitored period whilethose stored at room temperature showed slight increase inthe mean particle size but they remained less than 1.0 μm.This was in agreement with Sharma et al. (38) and Junyap-rasert et al. (40) who observed that there was no significantincrease in particle size of NLCs when stored for 3 months atdifferent temperatures.

Figure 3 shows the relationship between entrapmentefficiency and storage temperatures of NLCs. It was observedthat NLCs exhibited a good ability to reduce the drugexpulsion during storage at refrigerated (2–8°C) and roomtemperature. This could be attributed to the incorporation of

liquid lipid to solid lipid matrix, which increased theimperfection in crystal order of matrix and reduced thecrystallization process on storage, and thus prevent drugexpulsion (6).

In vitro Release Study: Franz Diffusion Cells

The in vitro release profile of oxybenzone from thenanostructured lipid carriers was investigated over 8 h. Theresults are shown in Fig. 4a and b.

Concerning liquid lipid type, Miglyol 812 NLCs pro-duced significantly (p<0.05) slower in vitro release after 8 hwhen compared to oleic acid NLCs. This might be due thehomogeneous entrapment of oxybenzone throughout thesystems when using Miglyol 812. It was previously stated(10) that the slow release of the drug from lipid nanoparticlessuggests homogeneous entrapment of the drug throughoutthe systems. Consequently, Miglyol 812NLCs with higherentrapment efficiency when compared to oleic acid NLCsshowed slower release profiles.

Concerning liquid lipid concentration, the extent of drugreleased was significantly higher (p<0.0001) upon using 30%when compared to 15%. This could be attributed to the smallerparticle size of the prepared NLCs with higher liquid lipidcontent. Similar increase in release was observed by Jenning etal. (7) and Teeranachaideekul et al. (31). It was previously stated(41) that for small particles, particularly in the nanometer size,the saturation solubility would significantly increase. Both theincrease in the saturation solubility and the enlargement of thesurface area contribute to the enhancement of dissolutionvelocity and consequently faster release rate would thereforebe expected. It was also observed from Fig. 4a and b that NLCsloaded with higher oxybenzone concentration showed signifi-cantly slower (p<0.0001) release profile when compared tolower concentrations. This could be attributed to that higherdrug concentration decreases the diffusion rate through thecarrier. This is due to the incorporation model. Higher drugconcentration led to the formation of drug-enriched core model(the drug occupies the core of the particles) which promoteslower release (14,42).

Fig. 2. Effect of storage temperature on the mean particle size of oxybenzone NLCformulations


Fig. 3. Effect of storage temperature on the entrapment efficiency of oxybenzone NLC formulations

Fig. 4. Release profile of oxybenzone from NLC formulations prepared using different liquid lipids andoxybenzone concentrations a miglyol, b oleic acid


Zeta Potential (ζ)

Zeta potential was measured to evaluate the stability ofNLC dispersions (Table III). All NLCs were negativelycharged. The negative charge was likely caused by the slightlyionized fatty acids from glycerides including the negativelycharged miglyol 812 and oleic acid at their carboxylic groups(43). Values of zeta potential ranged from −10.6±0.3 mV forNLC8 using 30% oleic acid and 10% oxybenzone to −29.7±1.8 mV for NLC5 using 15% oleic acid and 5% oxybenzone.Generally, zeta potential values of all NLCs in this study wereabove |8–9|mV, which was a prerequisite for the stability ofthe nanoparticles, prepared using a steric stabilizer (PVA)(44).

Concerning the effect of drug concentration on zetapotential, it was noticed that the absolute values of zetapotential decreased as the amounts of drug added increased.The larger particle size obtained upon increasing the drugconcentration led to the lower charge density of particle andabsolute values of zeta potential (45). No linear correlationbetween the zeta potential and liquid lipid concentration ofNLCs on the zeta potential was observed. This was inagreement with Chen et al. (46) who observed that differentsqualene (liquid lipid) ratios did not affect zeta potential ofthe prepared NLCs.

Based on the previous characterization and analysis ofthe main effects of the independent variables of the adoptedfactorial design NLC4 dispersion (see Table II for composi-tion) showed the highest EE% (93.7%±3.2), adequate zetapotential (−19.30 mV±0.6), slow drug release (46.05% after8 h), and adequate small particle size (0.366 μm±0.09), waschosen as candidate NLC dispersion which was then formu-lated into gel and its rheological properties, skin irritation,UV blocking ability, SPF, and its UVA-PF were studied andcompared with that of placebo NLC4 and 1% oxybenzonesuspension.

Characterization of the Prepared Gels

Rheological Studies

Due to the low viscosity of NLC dispersions, it could beassumed that they were not suitable as topical or dermaldelivery systems. To cope with this problem, one possibilitywould be incorporation into hydrogels or creams (40).

Carbopol 934 gel and the liquid lipid Miglyol 812 wereselected for the preparation of NLCs because of theirexcellent skin properties. Carbopol, is used due its compat-ibility with nanoparticulate dispersions, ease of preparation,esthetic appeal, thermal stability and optimum rheologicalproperties while Miglyol 812 (a mixture of triglycerides), ischosen because the lipid composition of the epidermis ismainly based on triglycerides (25%) (47).The freshly pre-pared gel bases were subjected to determination for theirrheological characteristics at 25°±1C. Rheograms are shownin Fig. 5. The results revealed that all gel formulationsexhibited pseudoplastic flow characteristics with thixotropy.NLC4 gel formulations either placebo or oxybenzone loadedhad higher thixotropy than that of 1% oxybenzone gel.Additionally, it was observed that placebo and oxybenzoneloaded NLC4 possessed higher viscosity when compared to

that of 1% oxybenzone gel (data not shown). This was due tothe presence of solid lipid (GMS) constituting the NLC4which acted as consistency imparting agent (16).

Skin Irritation Test

Results of skin irritation test are shown in Table IV. Noirritation or erythema was observed for placebo NLC4 eitherin dispersion or in gel forms, mean score±SD=0 and onlyvery slight erythema of (0.2±0.4) was observed for oxy-benzone-loaded NLC4 dispersion and the corresponding gel.Conversely, 1% oxybenzone suspension and its correspond-ing gel showed a well-defined erythema of 1.5±0.5. This couldbe attributed to the high entrapment efficiency of oxybenzonein NLC4 (93.7%±3.2) which might be beneficial to reduce theskin irritation of drug due to avoidance of the direct contactbetween drug and skin surface (48). This was in agreementwith Joshi et al. (49) who observed that NLC-based gel ofValdecoxib showed no skin irritation compared with the

Fig. 5. Rheograms of a placebo NLC4 gel, b NLC4 gel, and c 1%oxybenzone gel measured at a temperature of 25°±1C


marketed formulation containing the drug in the free form,which showed irritation upon application.

In vitro UV Blocking Ability

The wavelength scans of the placebo NLC4 (p NLC4),oxybenzone-loaded NLC4 (NLC4) and 1% oxybenzonesuspension (1% oxy) are given in Fig. 6 It was observed thatthe absorbance caused by placebo NLC4 was slightly higherthan that caused by 1% oxybenzone suspension. This wasattributed to that nanoparticles were considered as scatterers/reflectors of UV radiation (5).

It was also observed that the absorption spectrum ofoxybenzone-loaded NLC4 was shifted to higher values byabout threefold when compared to 1% oxybenzone suspen-sion. This was in accordance with Xia et al. (50) who observedthat the sunscreen-loaded NLC gave higher absorbance whencompared to that of reference emulsion with the samesunscreen content. This might be attributed to the synergisticphotoprotection caused by the incorporation of sunscreensinto a nanoparticle formulation (51).

It must be mentioned that NLC4 was the candidate NLCalthough it did not possess the smallest particle size; this wasbecause its mean particle size (0.366 μm±0.09) was adequatefor UV blocking effect. It was stated that there was anoptimal size for the maximum UV absorption (about 375 nmmean particle size). This could be explained as follows, whenthe particles became smaller the surface/volume ratio becomehigher, i.e., there are more surfaces where the light isreflected, absorbed, or refracted. But when the particles weresmaller than a definite particular size (smaller that 370 nm)they become transparent, and hence the absorption of UVradiation decreases. Moreover, for particles of smaller size,the destructive interference of the particles with the radiationbeam becomes very small, i.e., these very small particles donot absorb, refract, or reflect the light as the relatively biggerones (52).

In vitro SPF and EUVA-PF

The results of SPF and EUVA-PF of the investigatedformulations are depicted in Figs. 7 and 8. The resultsrevealed that there was no pronounced difference betweenSPF values obtained by placebo NLC4 (5.10±0.36) and 1%

Table IV. Skin Irritation Test of Placebo NLC4, Oxybenzone-LoadedNLC4 and 1% Oxybenzone Dispersions and Their Corresponding

Gels

Formulation code

Reaction in volunteers (mean±SD)

N=10

pNLC4 0±0.00NLC4 0.2±0.421%Oxy. 1.5±0.537pNLC4G 0±0.00NLC4G 0.2±0.421%Oxy.G 1.4±0.52

Fig. 6. Wavelength scans of 1% oxybenzone suspension (1%oxy), aplacebo NLC 4 dispersion (PNLC4), b and 1% oxybenzone-loadedNLC dispersion (NLC4), and (c) obtained by the Transpore® tapeassay

Fig. 7. SPF values of different oxybenzone-loaded NLCs formulationsand oxybenzone suspension using vitro skin as substrate

Fig. 8. Erythemal UVA protection factor of different oxybenzoneloaded NLCs formulations and oxybenzone suspension using vitroskin as substrate


oxybenzone suspension (4.07±0.13). However, oxybenzone-loaded NLC4 dispersion gave higher SPFs 22.20±1.00 whichis about sixfold higher than that of 1% oxybenzone suspen-sion. In case of 1% oxybenzone suspension, it had lowEUVA-PF, of about 1.90±0.20. Conversely, in the case ofoxybenzone-loaded NLC4, high EUVA-PFs of 17.10±0.85which corresponded to about eightfold increase in EUVA-PFs was observed.

The obtained SPFs and EUVA-PFs demonstrated theadvantages of the use of mixed particles composed of liquidand solid lipids (NLCs) as carrier system for UV filters.Although oxybenzone was directly responsible for the UVabsorption in those carriers, the use of Miglyol 812 wasjustified in virtue of its low viscosity enabling a gooddistribution of oxybenzone inside the GMS matrix. Similarly,Hernandez et al. (53) used the oil ‘decyl oleate’ to ensure agood distribution of sunscreen into the solid matrix of NLC toenhance its sun protection efficacy.

The SPF and EUVA-PF results also revealed that all gelformulations showed better sun protection factor comparedto their corresponding dispersions where the SPF values ofNLC4 gel reached 29.6±1.40 and the EUVA-PF reached 18.3±1.20 compared to 22.20±1.00 and 17.10±0.85 in case of thedispersions, respectively . Anderson et al. (54) demonstratedthat the increase in the viscosity of the cosmetic preparationswas directly related to an increased SPF. This could bepartially attributed to an increase in viscosity providing thepigments with a better fixation on the plates during the SPFmeasurements.

CONCLUSION

The present work has shown that NLCs containinglipophilic sunscreen oxybenzone can be produced by thesolvent diffusion method. The advantage of this method isthe instantaneous and reproducible formation of NLCsexhibiting a high degree of entrapment efficiency. Formu-lation of oxybenzone into NLCs enhanced the efficiency ofsunscreens formulation. The encapsulation of sunscreenagent in NLCs also provided the advantage of overcomingsolubility and skin irritancy problems. Oxybenzone isinsoluble in water and is difficult to incorporate in a gelbase. After entrapment of oxybenzone in NLCs, it could beeasily incorporated into gel base without any crystallizationproblem common with oxybenzone. The topical applicationof gel formulation containing NLCs of oxybenzone may bemore efficient in protecting against UVA and UVBradiation; probably due to the film formation over theskin, which itself acts as a physical barrier to UVradiations. In conclusion, the results of this study empha-size the potential of NLCs using Miglyol 812 and glycerylmonostearate as a new topical drug delivery system forenhancing the sunscreening efficacy of oxybenzone byabout sixfold while reducing its side effects

ACKNOWLEDGMENTS

I would like to express my deep thanks and sinceregratitude to Dr. Samia A. Nour, professor of Pharmaceutics,Faculty of Pharmacy, Cairo University for her support andher effort in revising the paper.

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Formulation of a Novel Oxybenzone-Loaded Nanostructured Lipid Carriers (NLCs)

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