blinghe H

Keywords:Solid lipid nanoparticlesNanostructured lipid carriers

tudvonicat

bination of solid and liquid) in solid lipid nanoparticle (SLN) and nanostructured lipid carrier (NLC) sys-

282 nm, with a zeta potential value of 36.57 2.67 mV, indicating the formation of a stable system.

enviroo oxid

ical antioxidants has been evaluated as a promising strategy forprevention or alleviation of the biological effects of photo-oxida-tive stress produced in the skin by ROS.

Flavonoids are natural antioxidants derived from the plantkingdom and are widely present in the human diet in the form ofnumerous edible fruits and vegetables such as onions, apples, ber-

anti-radical/anti-oxidant activity shown by quercetin has beenattributed to the presence of three active functional groups in itsstructure (Fig. 1): the ortho-dihydroxy (catechol) moiety in the Bring, the C2C3 double bond in conjunction with a 4-oxo functionand the hydroxyl substitution at positions 3, 5 and 7 (Bors et al.,1990; Saija et al., 1995).

The extremely low hydrophilicity of quercetin combined withits extensive metabolism by the gut microorganisms result in min-imal absorption from the gastrointestinal tract following its oraladministration, with no measurable plasma concentration re-ported in human volunteers from a 4 g oral dose (Gugler et al.,

Corresponding author. Current address: Department of Pharmaceutics, 160Frelinghuysen Road, Piscataway, NJ 08854, United States. Tel.: +1 732 445 3589;fax: +1 732 445 5006.

European Journal of Pharmaceutical Sciences 48 (2013) 442452

Contents lists available at

European Journal of Pha

.eE-mail address: michniak@biology.rutgers.edu (B. Michniak-Kohn).skin. Under these conditions, reactive oxygen species (ROS) suchas singlet oxygen, hydroxyl radicals, superoxide radical-anionsand hydrogen peroxide can be generated by the photodynamicreactions produced by endogenous cell photosensitizers. Thesereactive oxygen species are capable of causing damage to cutane-ous tissues (Pourzand and Tyrrell, 1999). They have also been re-ported to be involved in phototoxic reactions induced by theinteraction of UVA/visible light with drugs or chemical entities(environmental or industrial) accumulated in the skin followingtopical or systemic administration (Epstein, 1983). The use of top-

tureactivity relationships of several avonoids (myricetin, quer-cetin, kaempferol, luteolin, apigenin and chrysin) and their anti-oxidant activity in a human dermal broblast model, correlationswere obtained between the anti-oxidative efcacy and the numberof OH-groups on the avonoid structure, with quercetin(3,30,40,5,7-pentahydroxyavone) showing one of the highest activ-ities. In the same study, quercetin was also observed to reduce theexpression of matrix metalloproteinase-1 (responsible for skinwrinkling and loss of elasticity in both healthy and photoaged skin)at both the mRNA and protein levels (Sim et al., 2007). This highSonicationPhysical stabilitySolid lipidOleic acid

1. Introduction

Excessive exposure of the skin toing sun and air pollution can lead t0928-0987/$ - see front matter 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ejps.2012.12.005Release studies showed a biphasic release prole, characterized by an initial burst release followed bya more controlled release pattern from both SLN and NLC systems. The NLC system showed the highestimprovement in topical delivery of quercetin manifested by the amount of quercetin retained in fullthickness human skin, compared to a control formulation with similar composition and particle size inthe micrometer range. This study demonstrated the feasibility of nanostructured lipid carrier systemsfor improved topical delivery of quercetin.

2012 Elsevier B.V. All rights reserved.

nmental insults includ-ative stress (OS) to the

ries and red grapes. The mechanism of the anti-oxidant action ofavonoids involve their reactivity with the ROS and their chelationof transition metal ions responsible for oxygen activation via redoxreactions (Filipe et al., 2005). In a study investigating the struc-Available online 13 December 2012tems and drug loading were evaluated to produce an optimum formulation with adequate physicalstability for up to 14 weeks at 28 C. The mean particle size of the optimized formulation was aroundPreparation and characterization of lipidof quercetin

Sonali Bose a,b, Bozena Michniak-Kohn a,a Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Freb Pharmaceutical and Analytical Development, Novartis Pharmaceuticals Corporation, On

a r t i c l e i n f o

Article history:Received 19 August 2012Received in revised form 2 December 2012Accepted 4 December 2012

a b s t r a c t

The main objective of this sthe naturally occurring asolvent free probe ultrason

journal homepage: wwwll rights reserved.ased nanosystems for topical delivery

uysen Road, Piscataway, NJ 08854, United Statesealth Plaza, East Hanover, NJ 07936, United States

y was to evaluate the potential of lipid nanosystems for topical delivery ofoid quercetin. These lipid based nanosystems were manufactured using aion process. Formulation factors such as the nature of the lipid (solid/com-

SciVerse ScienceDirect

rmaceutical Sciences

lsevier .com/ locate /e jps

l of P1975). This limits the extension of the benecial effects of querce-tin observed in in vitro studies to the in vivo or clinical level with anoral delivery approach. Hence topical application of quercetin canbe a very attractive formulation approach, considering the fact thattopical application of quercetin has previously been shown to re-sult in potent inhibition of UVB-induced oxidative skin damage(Casagrande et al., 2006; Gonzalez et al., 2008).

Various formulation approaches including permeation enhanc-ers (Olivella et al., 2007), ester based prodrugs (Montenegroet al., 2007), microemulsion based approaches (Vicentini et al.,2008; Kitagawa et al., 2009) and lecithinchitosan nanoparticles(Tan et al., 2011) have been attempted to improve the penetrationof quercetin through the skin to facilitate topical/transdermaldelivery. Most of these studies have shown some degree of querce-tin penetration into the skin, but no transdermal delivery has beenreported. However, since the efcacy of quercetin in delaying ul-tra-violet radiation mediated cell damage and eventual necrosismainly occurs in the epidermal layers of the skin, topical deliveryof quercetin without a sufcient degree of skin penetration shouldbe adequate to achieve the desired pharmacological action.

Lipid based nanosystems such as solid lipid nanoparticles(SLNs) and nanostructured lipid carriers (NLCs) have been previ-ously shown to improve the delivery of various actives such as glu-cocorticoids (Maia et al., 2000; Jensen et al., 2011), Vitamin A(Jenning et al., 2000c) and betamethasone 17-valerate (Zhangand Smith, 2011) to specic skin layers, with reported localizationin the upper layers of the skin. Such a formulation approach hasalso been utilized by our group to develop solid lipid based nano-systems of quercetin and evaluate their feasibility for topical deliv-ery using full thickness human skin (Bose et al., submitted forpublication). The optimized SLN formulation showed superior top-ical delivery of quercetin compared to the control formulation withparticle size in the micrometer range, with the differences beingstatistically signicant (p < 0.05). However, an increase in the par-ticle size was observed for samples placed on stability at 28 C

Fig. 1. Chemical structure of quercetin.

S. Bose, B. Michniak-Kohn / European Journaafter 8 weeks. This increase in particle size could be attributed tosome degree of lipid transformation of the solid lipid (glyceryldibehenate) used in these nanoparticles over time, leading to theformation of a highly ordered lipid structure resulting in drugexpulsion from the SLN system (Muller et al., 2002). The lipidtransformation was conrmed from the morphology of the nano-particles visualized using TEM and also from X-ray diffractionpatterns.

Nanostructured lipid carriers (NLCs) are a second generation oflipid based nanoparticles that are obtained by substituting a part ofthe solid lipid used in the SLN formulation with a liquid lipid, in or-der to reduce the rigidity and ordered structure of the lipid matrixand introduce imperfections in the matrix to minimize drug expul-sion upon storage (Muller et al., 2002). Recently, the developmentof NLCs of quercetin for topical delivery have been reported using asolvent (chloroform/acetone) based emulsication technique(Chen-yu et al., 2012). One of the major difculties of an organic

surfactant solution heated to the same temperature. No ultrasoni-

cation step was performed on these non-homogenized controlformulations.

2.3. Freeze drying of nanoparticles

Samples of quercetin nanoparticles were freeze dried using aUsifroid Freeze Dryer (Elancourt, France). A cooling rate of 1 C/min was used to pre-cool the sample from room temperature to50 C. The sample was then maintained at 50 C for 1 h, fol-solvent based manufacturing method could be the failure to com-pletely eliminate residual solvent (Grabnar and Kristl, 2011). Theobjective of our study was to develop a solvent-free NLC formula-tion of quercetin using probe ultrasonication and evaluate its fea-sibility for topical delivery. The NLC system was fullycharacterized for particle size, zeta potential, morphology andcrystallinity. Key properties such as physical/solid state stabilityand in vitro drug release, that could affect formulation performancein vivo, were compared between the two systems.

2. Materials and methods

2.1. Materials

Highly pure (>99%) quercetin was obtained fromMerck (Darms-tadt, Germany). Compritol 888 (glyceryl dibehenate) was kindlydonated by Gattefoss (Paramus, NJ, USA). Tween 20 (polyoxyeth-ylene derivative of sorbitan monolaurate), dioctyl sodium sulfosuc-cinate (DOSS) and oleic acid were purchased from SigmaAldrichCorporation (St. Louis, MO, USA). HPLC or analytical grade of allother solvents and reagents were used.

2.2. Preparation of nanosystems of quercetin

Nanostructured lipid carrier (NLC) systems of quercetin wereprepared using probe ultrasonication by melting 0.45 g of the solidlipid (Compritol 888, glyceryl dibehenate) with 0.05 g of the li-quid lipid (oleic acid) and either 0.025 g or 0.0125 g of quercetin(corresponding to 0.05% or 0.025% drug loading in the systemrespectively) at 85 C using a water bath. The heated mixture oflipids and quercetin was then mixed with 20 mL of surfactant solu-tion (composed of 2.5% Tween 20 and 0.1% DOSS) pre-heated to thesame temperature of 85 C. The mixture was then ultra-sonicatedat 85 C at a specic speed (power setting of four) for a pre-deter-mined time interval (either 5 min or 30 min) using a Sonic Dis-membrator Model 550 (Fisher Scientic, Pittsburgh, PA). Theprimary product at the end of the sonication step was a nanoemul-sion, since the processing temperature of 85 C was at least 10 Chigher than the melting point of the solid lipid and adequate tomaintain the system in the liquid state. At the end of the sonicationprocess, the nanoemulsion was dispersed into 30 mL of an ice-coldsurfactant solution maintained in an ice bath. The nal mixturewas then ultra-sonicated at a specic speed (power setting of 1)for 10 min immersed in the ice-bath to promote the formation ofthe solid lipid nanoparticles. The corresponding solid lipid nano-particle (SLN) formulation was prepared in exactly the same way,using 0.5 g of only the solid lipid (Compritol 888, glyceryl dibeh-enate) in the composition. All formulations were stored in therefrigerator at 28 C till further analysis.

The non-homogenized control (for both SLN and NLC) formula-tion used in the skin penetration experiments were prepared bymixing the lipid and quercetin solution heated to 85 C with the

harmaceutical Sciences 48 (2013) 442452 443lowed by primary and secondary drying steps as specied inTable 1. Since the primary purpose of drying the nanoparticleswas to obtain a powder for further solid state characterization to

of Pevaluate crystallinity, no matrix formers were added to the solu-tion prior to freeze drying.

2.4. Characterization of nanoparticles

2.4.1. Particle size measurementThe particle size of NLC systems was measured using photon

correlation spectroscopy (PCS) on a Delsa Nano C particle sizeanalyzer (Beckman Coulter, Brea, CA, USA) at 25 C and at a lightscattering angle of 90. The sample (undiluted) was poured into adisposable plastic cuvette, the cuvette manually shaken for about10 s and then placed inside the sample holder of the instrument.Once the intensity of the sample was within the range recom-mended by the instrument, analysis was performed to obtain theparticle size and the polydispersity index (PI). All measurementswere performed in triplicate. All reported particle size data referto intensity weighted distributions.

2.4.2. Zeta potential measurementThe surface charge on the nanoparticles was quantied by mea-

suring the zeta potential using a Delsa Nano C (Beckman Coulter,Brea, CA, USA). An appropriately diluted nanoparticle solution wasused for the measurement, under an applied electric eld of 16 V/cm. Dilutions were performed with distilled water adjusted to aconductivity of 50 lS/cm by addition of 0.9% (m/v) sodium chlo-ride. All reported values are the mean of three separatemeasurements.

2.4.3. MorphologyTransmission electron microscopy (TEM) was used to conrm

the morphology of the nanoparticles using a FEI Tecnai G2 BioTwin

Table 1Parameters used for freeze-drying experiments.

Temperature range Rate of cooling (C/min) Hold time (min)

Primary drying cycle50 C to 45 C 1 545 C to 35 C 1 48035 C to 30 C 1 48030 C to 25 C 1 36025 C to 20 C 1 360Secondary drying cycle20 C to 10 C 1 60010 C to 0 C 1 6000 C to 10 C 1 48010 C to 25 C 1 72025 C to 30 C 1 960

444 S. Bose, B. Michniak-Kohn / European Journaltransmission electron microscope tted with a SIS Morada digitalcamera system (Fei Corporation, Hillsboro, OR, USA). Formvarcoated copper grids (200 mesh) were oated on top of 510 lL ofliquid sample for approximately 30 min, then rinsed with lteredHPLC grade water and allowed to dry at room temperature. Imageswere captured at magnications ranging from 4800 to 150,000.

2.4.4. X-ray powder diffraction (XRPD)XRPD analysis was performed using a Bruker D8 Advance (Bru-

ker-AXS, Karlsruhe, Germany) controlled by Diffrac plus XRD com-mander software. Samples (analyzed within 2 weeks aftermanufacturing) were prepared by spreading freeze dried powdersamples on PMMA specimen holder rings from Bruker. All sampleswere scanned from 2 to 40 2h using the following parameters:scanning rate of 2/min with 0.02 step size and 0.6 s/step at40 KV and 40 mA, divergence and anti-scattering slits set to 1and a stage rotation speed of 30 rpm. EVA Part 11 version14.0.0.0 software was used for data analysis.2.4.5. Modulated differential scanning calorimetry (MDSC)Modulated differential scanning calorimetry (MDSC) was per-

formed using a differential scanning calorimeter Q1000DSC (TAInstruments, New Castle, DE, USA). Instrument calibration was car-ried out using Indium supplied by TA instruments. The sample(10 mg) was placed in an aluminum DSC pan, covered with alid containing pinholes. An empty pan with pinholes in the lidwas used as the reference. The weights of the reference and samplepans were accurately recorded. The sample cell was equilibrated at0 C for 8 min, then heated to 320 C under an atmosphere of nitro-gen using modulation conditions of 0.5 C for every 60 s with anunderlying heating rate of 2 C/min.

2.4.6. Release studyThe release of quercetin from NLC systems was determined by a

dialysis based method using Slide-A-Lyzer MINI Dialysis devices,2 mL volume, 10 K MWCO (Thermo Scientic, Rockford, IL, USA). Amixture of doubly distilled water and absolute alcohol in the ratioof 65:35% v/v (Li et al., 2009) was used as the release medium. Thereceptor medium had adequate solubility of quercetin (0.358 mg/mL) to ensure sufcient sink conditions throughout the study. 2 mLof the quercetin formulation and 44.5 mL of the release mediumwere added to the donor and receptor compartments of the dialy-sis device respectively (n = 3). The experiment was performed at37 C and at a stirring speed of 100 RPM. At each sampling timepoint (2, 4, 6, 8, 24 and 30 h), 1 mL of receptor medium was with-drawn and replaced with fresh medium. The withdrawn sampleswere analyzed using the HPLC based method described inSection 2.4.7.

2.4.7. Detection of quercetin by HPLCQuercetin concentrations were determined using an Agilent

HPLC 1100 system (Agilent Technologies Inc., Santa Clara, CA,USA) consisting of a standard quarternary pump, diode arraydetector, an autosampler and vacuum degasser (Model G1311A)run by Chemstation software version B.03.01. A mixture of 80%methanol and 20% water (pH adjusted to 3.72 with glacial aceticacid) was used as the mobile phase. Chromatographic separationwas achieved using a Phalanx C18, 250 mm 4.6 mm, 5 lm col-umn (Higgins Analytical, Mountain View, CA, USA) and an isocraticmethod with the following parameters: Injection volume of 20 lL,ow rate of 1.0 mL/min, column temperature of 30 C, detectionwavelength of 370 nm and a run time of 8 min. External standardsin the mobile phase were prepared at concentrations of 0.1, 0.5, 1,5, 10, 25, 50 and 100 lg/mL and used to determine the linearity(R2 = 0.999) and the limit of detection (0.5 lg/mL).

2.4.8. Stability studyThe physical stability of selected NLC systems was monitored

for up to 8 weeks at 28 C. Physical stability testing of the mostpromising variant was continued till 14 weeks at 28 C, at whichtime the particle size and zeta potential was measured. All re-ported particle size and zeta potential data are the mean of threeseparate measurements.

2.4.9. In vitro permeation study using human skinIn vitro permeation studies (n = 56 for each formulation) were

carried out using full thickness human skin obtained from NewYork Presbyterian Hospital (New York, NY, USA). Vertical Franz dif-fusion cells (PermeGear, Inc., Hellertown, PA, USA) with a diffusionarea of 0.64 cm2 and a receptor compartment volume of 5.1 mLwere used. The skin was thawed for 30 min, hydrated by immers-ing in PBS solution for 60 min at 37 C, cut into appropriate sized

harmaceutical Sciences 48 (2013) 442452sections and mounted on the Franz diffusion cell, with the stratumcorneum facing the donor compartment (where the formulationwas applied) and the dermis facing the receptor compartment.

l of P3. Results and discussion

3.1. Rationale for selection of liquid lipid in NLC system

Systematic screening of formulation and process variables wascarried out to develop a lipid based nanosystem comprising of 5%quercetin (based on the total amount of lipid, corresponding to0.05% drug loading in the overall system), 2.5% Tween 20 and0.1% dioctyl sodium sulfosuccinate (DOSS) in a lipid matrix com-posed of Compritol 888 (glyceryl dibehenate) (Bose et al., submit-ted for publication). This system was used as the starting point forthe evaluation of NLC systems of quercetin which will be discussedin this section. 10% of the solid lipid (Compritol 888) was substi-tuted with a liquid lipid (oleic acid) in the NLC systems. Oleic acidis a well-known skin permeation enhancer and has been reportedto enhance the skin delivery of a number of compounds such asvoriconazole (Song et al., 2012), estradiol (El Maghraby et al.,2004), 5-uorouracil (Yamane et al., 1995) and tranilast (Muraka-mi et al., 1998). Its permeation enhancing effect is attributed tothe reduction of the phase transition temperature of the lipidspresent in the skin, thereby increasing the uidity of these struc-tures (Golden et al., 1987; Francoeur et al., 1990). It has also beensuggested that oleic acid might exist as a separate phase (pool)within the lipids of the stratum corneum (Ongpipattanakul et al.,1991). The selection of oleic acid as the liquid lipid was based onliterature references citing its extensive use in NLC systems (Tiwari2.4.10. Statistical analysisAll particle size, zeta potential and in vitro release rate measure-

ments were performed in triplicate. Means and standard devia-tions were calculated using Microsoft Excel 2010. Mean valueswere compared using the Students t-test using Statgraphics Plus5.1 (Statpoint Technologies Inc., Warrenton, VA), with differencesbeing considered as signicant at a level of p < 0.05.PBS solution (pH 7.4) containing 1% Tween 20 maintained at a tem-perature of 37 0.1 C and a stirring speed of 600 RPM was used asthe receptor medium. 0.5 mL of formulation (NLC or control) wasadded to the donor compartment and occluded with Paralm toprevent evaporation. For the preliminary experiments, 300 lL ofsample was withdrawn at pre-determined time intervals (2, 4, 6,8 and 24 h) from the receptor compartment and replaced by anequal volume of fresh receptor media maintained at 37 C. Thesamples were stored in the refrigerator prior to HPLC analysis.

At the end of the experiment (24 h), a glass transfer pipette wasused to remove the formulation from the donor compartment. Thesurface of the skin was thoroughly washed with distilled water toremove any excess formulation (Tan et al., 2011) and allowed todry at ambient temperature. The area of the skin in contact withthe formulation (corresponding to the diffusion area of the Franzcell) was then punched out, weighed accurately and cut into nepieces. 1 mL of methanol was added to the skin pieces and homog-enized using a Polytron PT 1035 homogenizer (Kinematica, Inc.,Bohemia, NY, USA). The homogenized residue was sonicated for60 min at 37 C using a VWR Ultrasonic B5500A-DTH sonicator(VWR International, LLC, Radnor, PA, USA), followed by centrifuga-tion at 4000 RPM for 30 min using an AllegraTM 6R centrifuge(Beckman Coulter, Brea, CA, USA). The supernatant obtained aftercentrifugation was collected and analyzed using the HPLC methoddescribed in Section 2.4.7, with an increased injection volume of50 lL.

S. Bose, B. Michniak-Kohn / European Journaand Pathak, 2011; Pardeike et al., 2011; Kuo and Chung, 2011) withspecic application to topical delivery (Silva et al., 2009; LombardiBorgia et al., 2005).3.2. Effect of process parameters on physical stability of quercetin NLCsystems

The duration of the rst sonication step in the manufacturingprocess is considered to be a critical processing parameter sinceit involves the input of energy into the system, resulting in moreefcient breakage of coarse emulsion droplets into the nanometerrange and subsequent particle size reduction. To conrm the appli-cability of the same for the production of NLC systems, two differ-ent sonication times (5 min and 30 min) at a power setting of fourwere evaluated for the rst sonication step at 85 C. The particlesize data for these two batches at the initial time point and after1 week at 28 C are shown in Fig. 2.

The polydispersity index (PI) values obtained for these batcheswere as follows: 0.285 and 0.250 for the initial and 1 week samplefor the NLC batch manufactured using a sonication time of 30 minand 0.122 and 0.293 for the initial and 1 week sample for the NLCbatch manufactured using a sonication time of 5 min. For the batchsonicated for 30 min, the difference in the particle size (both D50and D90) between the initial sample and the sample kept for1 week at 28 C was not considered to be statistically signicant(p > 0.05). However statistically signicant differences (p < 0.05)in both D50 and D90 values between the initial sample and the1 week sample were observed for the batch sonicated for 5 min.These results conrmed the criticality of using a longer sonicationtime of 30 min for the rst sonication step (homogenization step)in order to generate a physically stable nanosystem with a narrowparticle size distribution. The improved short term physical stabil-ity of the NLC batch manufactured using a sonication time of30 min can be explained by the higher input of energy into the sys-tem during the longer sonication time resulting in more efcientbreakage of coarse emulsion droplets into the nanometer rangeand subsequent particle size reduction. This observation is consis-tent with other reports in the literature, where higher sonicationtimes were found to be more efcient in generating nanoparticlesusing a probe sonication method (Siekmann and Westesen, 1994;Das et al., 2011). The zeta potential values for the initial samplesof batches sonicated for 30 min and 5 min were35.64 1.13 mV and 34.52 2.50 mV respectively, which werenot statistically signicant (p > 0.05). Based on the short termphysical stability data, a sonication time of 30 min for the rst son-ication/homogenization step was selected for the manufacturing offuture quercetin lipid based nanosystems using the probe ultrason-ication process.

3.3. Effect of oleic acid on physical stability of quercetin lipid basednanosystems

In order to evaluate the effect of oleic acid on the physical sta-bility of quercetin lipid based nanosystems, the particle size data ofbatches with and without oleic acid (NLC and SLN systems respec-tively) was compared. The data is shown in Fig. 3. No statisticallysignicant differences (p > 0.05) were observed between the SLNand NLC formulations with 0.05% quercetin loading for D50 andD90 values at the initial time point and after 8 weeks at 28 C.The zeta potential values for the SLN and NLC batches were35.83 2.11 mV and 35.64 1.13 mV respectively, which werenot found to be statistically signicant (p > 0.05). However, an in-crease in particle size for both the SLN and NLC formulations wasobserved between the initial and 8 week samples. This is similarto observations reported by Mitri et al. (2011), where increase inparticle size for both SLN and NLC formulations was observed forup to 7 days after production, beyond which no signicant change

harmaceutical Sciences 48 (2013) 442452 445in particle size was observed from day 7 till day 30. The lack of anyobserved differences between the particle size and zeta potentialvalues of SLN and NLC batches may be due to the low amount of

of P446 S. Bose, B. Michniak-Kohn / European Journaloleic acid (10%) used in the formulation. This is consistent with re-ports in the literature where no signicant difference in initial par-ticle size (volume average diameter) was observed between Nile-Red loaded SLN and NLC formulations containing 515% of oleicacid. Signicant reduction in particle size was only observed whenthe amount of oleic acid in the formulation was increased to 30% orhigher, which was attributed to the reduced viscosity of the NLCsystem at higher concentrations of oleic acid which facilitatedthe reduction of surface tension to generate smaller particles (Huet al., 2005). Teeranachaideekul et al. (2007) reported increase inoil content to have no effect on the initial particle size of Q10-loaded NLC systems. Other researchers (Ying et al., 2008; Youet al., 2007) have also reported no signicant effect of oleic acidon NLC particle size, at low oil concentrations (up to 10% w/w).

Fig. 2. Effect of sonication time on physical stability of quercetin NLC s

Fig. 3. Effect of oleic acid on physical stability of quercetin lipid based nanharmaceutical Sciences 48 (2013) 442452In our study, higher concentrations (greater than 10%) of the li-quid lipid (oleic acid) were not investigated since an increase in theconcentration of the liquid lipid has been associated with a reduc-tion in the occlusion factor (Teeranachaideekul et al., 2008), whichis one of the major aspects of topical application of lipid basednanosystems. Also, higher concentrations of oleic acid in NLC sys-tems were not evaluated since the acidic nature of fatty acids havebeen associated with dermal side effects including extraction ofthe stratum corneum lipids and damage to viable epidermal cells(Sintov et al., 1999; Touitou et al., 2002). However, the use of oleicacid in concentrations of up to 10% in topical formulations havebeen reported to have no irritating effects on the skin of animalspecies such as guinea pigs (Yu et al., 2010) or rabbits (Moreiraet al., 2010).

ystems (n = 3 measurements); PI refers to the polydispersity index.

osystems (n = 3 measurements); PI refers to the polydispersity index.

indicated that the manufacturing temperature did not induce anydegradation of quercetin and conrmed the validity of the produc-

ility

l of P3.4. Effect of drug loading on physical stability of quercetin NLCsystems

Most NLC systems described in the literature mention a rela-tively low drug loading, with loading values of 0.025% reportedfor triamcinolone acetonide (Araujo et al., 2010), 0.03% reportedfor urbiprofen (Luo et al., 2011) and 0.04% reported for ketoprofen(Cirri et al., 2012). Although the batch with 5% quercetin (based onthe total amount of lipid, corresponding to 0.05% drug loading inthe NLC system) loading demonstrated good short term physical

Fig. 4. Effect of drug loading on the physical stab

S. Bose, B. Michniak-Kohn / European Journastability with no statistically signicant differences in particle sizebetween the initial and 1 week samples, a lower drug loading of2.5% quercetin (based on the total amount of lipid, correspondingto 0.025% drug loading in the NLC system) was evaluated to deter-mine if further improvement in physical stability of the nanosys-tem could be achieved by reducing the drug loading. All othercomponents of the formulations were kept constant, so that anydifference in particle size could be attributed solely to the drugloading. The effect of drug loading (0.025% and 0.05%) on the phys-ical stability of quercetin NLC systems is shown in Fig. 4.

No statistically signicant differences (p > 0.05) in D50 and D90values were observed for initial samples of NLC batches with thetwo different drug loading. However, for samples kept at 28 Cfor 8 weeks, the differences in the D50 and D90 values for batcheswith 0.025% and 0.05% drug loading were statistically signicant(p < 0.05). The increase in particle size for the batch with 0.05%drug loading can probably be attributed to insufcient quantityof stabilizer in the formulation. When the drug loading is reducedto 0.025%, the quantity of stabilizer incorporated in the formula-tion becomes more adequate to impart long term stabilization asevidenced from the particle size data. The zeta potential valuesfor the batches with 0.025% and 0.05% drug loading were36.57 2.67 mV and 35.64 1.13 mV respectively, which werenot statistically signicant (p > 0.05). The particle size data showedbetter long term physical stability (up to 8 weeks at 28 C) for theNLC batch with the lower (0.025%) quercetin loading, which wasselected for further evaluation. In order to ensure that the hightemperature (85 C) used during the manufacturing process didnot compromise the chemical stability of quercetin, the NLC batchtion process.

3.5. Long term physical stability of selected NLC formulationwith 0.025% drug loading was also analyzed for quercetin content.98% of quercetin could be recovered from the formulation. This

of quercetin NLC systems (n = 3 measurements).

harmaceutical Sciences 48 (2013) 442452 447The NLC formulation with 0.025% quercetin loading was placedon stability at 28 C. At pre-determined time points, the particlesize of the NLC system was measured to assess the physical stabil-ity of the system. At the end of the stability study (after 14 weeks),the zeta potential of the batch was measured and compared to theinitial sample. The mean particle size and zeta potential results areshown in Table 2. Very slight increase in the mean particle size( 0.05) between initial and 14 week or between the8 week and 14 week samples.

Table 2Mean particle size and zeta potential of selected NLC formulation upon stability.

Time point Particle size, mean SD (n = 3) PIa Zeta potential (mV), mean SD (n = 3)

Initial 281.9 2.9 0.306 36.57 2.678 weeks, 28 C 309.8 3.5 0.310 Not measured14 weeks, 28 C 294.6 6.7 0.315 36.88 1.61

a PI refers to polydispersity index.

448 S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 4424523.6. Morphology of selected NLC formulation

NLC morphology was observed using transmission electronmicroscopy (TEM). The TEM image of quercetin NLC with 0.025%drug loading showed spherical particles in the nanometer range.It is worthwhile to mention here that TEM images project three-dimensional particles in a two-dimensional manner. It is quite pos-sible that the NLC particles are not truly spherical in nature but

Fig. 5. Long term physical stability of selected

Fig. 6. X-ray diffraction patterns: (A): Compritol 888 bulk material, (B): freeze dried NLCfreeze dried SLN batch, 0.05% drug loading.rather platelet like particles adhering with their at side on thesurface resulting in an overall round appearance when viewedfrom the top in the TEM image (Jores et al., 2004; Esposito et al.,2008). The mean particle size obtained from the TEM measure-ments was around 240 nmwhich was slightly lower than the meanparticle size obtained from the PCS measurements (282 nm). Thisdifference can be explained by the difference in the sample prepa-ration step between the two techniques.

NLC formulation (n = 3 measurements).

batch, 0.025% drug loading, (C) freeze dried NLC batch, 0.05% drug loading, and (D):

compared to a control formulation of quercetin in propylene glycolsolution are shown in Fig. 8. The formulations with 0.05% quercetinloading were used to obtain a direct comparison of the release ofquercetin from formulations with and without oleic acid. Querce-tin from the NLC formulation (with oleic acid) was released at afaster rate compared to the SLN formulation (without oleic acid).This is consistent with literature references comparing the releaserates from SLN and NLC formulations (Hu et al., 2005; Souto et al.,2004). The release prole from both SLN and NLC formulationswere biphasic, with an initial burst release (55% from the NLC for-mulation compared to 45% from the SLN formulation after 2 h)followed by controlled release for up to 30 h. Equilibrium was at-tained within 24 h, which was conrmed by the plateau observedin the release prole between 24 and 30 h. The incorporation of theliquid lipid (oleic acid) in the NLC formulation reduced the viscos-ity of the lipid matrix, leading to faster diffusion and release of thedrug from the NLC system compared to the SLN system that wascomposed solely of solid lipids.

Statistical analysis showed signicant differences in the rate ofrelease of quercetin from the control formulation compared to boththe SLN and NLC formulations (p < 0.05). Also, statistically signi-cant differences in quercetin release rates were observed betweenthe SLN and NLC formulations at the earlier time points up to 6 h(p < 0.05). Beyond 6 h, the differences in release rates between theSLN and NLC systems were not statistically signicant (p > 0.05).

3.10. Topical delivery of quercetin from lipid nanosystems

l of P3.7. Solid state characterization of quercetin NLC systems: X-raydiffraction (XRD) data

The X-ray diffraction patterns are shown in Fig. 6. The diffracto-gram labelled A shows the diffraction pattern of neat Compritol

888, with peaks characteristic of the orthorhombic b0 form of tri-glycerides (Chapman, 1962). In the freeze dried NLC formulationswith 0.025% and 0.05% drug loading (labelled as B and C respec-tively), an additional peak is observed at 2h values between 18and 20. This peak has been associated with the bi polymorph ofthe lipid (Jenning et al., 2000a). The appearance of this peak be-tween 2h values of 18 and 20 indicates the onset of a partial poly-morphic transformation of glyceryl behenate from the metastableb0 to the more stable bi form, as had been previously observed withthe freeze dried SLN formulation (diffractogram labelled D).

Aggregation of glyceryl behenate based nanoparticles are usu-ally accompanied by a transformation from the b0 (metastable)polymorph to the more stable bi polymorph (Jenning et al.,2000a). However, for dispersions with 10% glyceryl behenate con-tent (comparable to concentrations used for our experiments), re-ports of nanoparticle aggregation have been associated with asignicant increase in the D90 (from 0.77 0.01 lm to23.34 0.19 lm) accompanied by gelling of all samples within atime frame of 5 days (Freitas and Mller, 1999). Upon reductionof the lipid concentration from 10% to 2%, the authors reported im-proved stability of the dispersion with only a slight change(0.1 lm) in the D90 values. An increase in particle size of around0.1 lm (based on the D90 values) was observed for our experiments(Fig. 5) which is similar to the observations of Freitas et al. for dis-persions with 2% lipid content. Hence, our solid lipid nanosystemscan be concluded to be relatively stable systems, even at higher li-pid concentrations (10%) compared to what has been previouslyreported in the literature. Additionally, polymorphic transforma-tion of the lipid was found to be complete only for samples whichhad undergone complete conversion to a solid gel (Freitas and Ml-ler, 1999). Based on the fact that the solid lipid nanosystems devel-oped in our studies still retained their liquid state with no visiblegelling observed in the system, it is believed that only a partialpolymorphic transformation of the solid lipid (glyceryl behenate)occurred in these samples during the time frame studied.

3.8. Solid state characterization of quercetin NLC systems: Differentialscanning calorimetry (DSC) data

Fig. 7 shows the modulated DSC scans of the quercetin SLN andNLC formulations, with focus on the melting endotherm of Compr-itol 888. Bulk Compritol 888 melts between 63 C and 77 C,with a melting point at 72.2 C. A slight depression in the meltingpoint of Compritol 888 to around 68 C was observed for all thelipid based nanoparticle systems which is similar to values previ-ously reported in the literature (Hamdani et al., 2003; Freitas andMller, 1999). This depression in melting point can be attributedto the particle size in the nanometer range, the high specic sur-face area of the particles, the presence of surfactants in the formu-lation and the lipid polymorphism occurring from crystallization(Jenning et al., 2000b). In all the SLN and NLC formulations, themelting event of quercetin (starting around 315 C) could not bedetected (data not shown). This could be due to the presence ofquercetin in a dissolved state in the lipid matrix. During the pro-duction procedure of both SLN and NLCs, quercetin was dissolvedin the molten lipid matrix. Cooling the dispersion in an ice bathled to the formation of SLN or NLCs depending on the composition.The absence of any melting event of quercetin in the SLN and NLC

S. Bose, B. Michniak-Kohn / European Journaformulations could be due to the existence of quercetin as a super-cooled melt, in the amorphous state or in a molecularly dispersedstate within the lipid matrix. Even if a small fraction of quercetin ispresent in the nanosystem as undissolved material/in the crystal-line state, the relatively low drug loading of quercetin in the nano-systems would render it improbable to detect the melting event ofany such fraction using a DSC based technique.

3.9. Release of quercetin from lipid nanosystems

The release of quercetin from the SLN and NLC formulations

Fig. 7. Modulated DSC scans; (A): Freeze dried SLN, 0.05% drug loading, (B): freezedried NLC, 0.05% drug loading, and (C): freeze dried NLC, 0.025% drug loading.

harmaceutical Sciences 48 (2013) 442452 449In most in vitro permeation studies, the feasibility of topicaldelivery of quercetin from the optimized formulation has been

evaluated using skin from animal species such as pig (Olivella the SLN and NLC formulations), with particle size in themicrometer

Fig. 8. Release of quercetin from lipid based nanosystems (n = 3).

450 S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452et al., 2007; Vicentini et al., 2008), guinea-pig (Kitagawa et al.,2009) and mouse (Tan et al., 2011; Chen-yu et al., 2012). In onestudy, human skin was used to evaluate the in vitro permeabilityof quercetin from ester prodrugs; however only epidermal mem-branes (obtained by physical separation of the epidermis and der-mis) were used for the experiments (Montenegro et al., 2007).Since in many instances the results from skin permeation experi-ments conducted with animal skin cannot be extrapolated to hu-man skin, full thickness dermatomed human skin was used inour experiments to evaluate the feasibility of topical delivery ofquercetin from the developed lipid based nanosystems.

In vitro skin permeation studies were carried out with the SLNand NLC formulations of quercetin, the main difference betweenthe formulations being the presence of oleic acid in the NLC formu-lation. The corresponding non-homogenized formulations (withand without oleic acid, containing the same amount of lipid as inFig. 9. Topical delivery of quercetin fromrange, were used as the control formulations. Sampling from thereceptor media was carried out at 2, 4, 6, 8 and 24 h. However, noquercetin could be detected in the receptor media, indicating thelack of any transdermal delivery. This observation is consistentwithreports from other studies with quercetin (Vicentini et al., 2008;Kitagawa et al., 2009), including one study from a chitosanlecithinbased nanoparticle formulation (Tan et al., 2011). The NLC formula-tion (with oleic acid) showed the highest amount of quercetin re-tained in the skin at the end of the 24 h experimental period(Fig. 9). The difference in the amount of quercetin retained in theskin was not found to be statistically signicant (p > 0.05) betweenthe NLC and the SLN formulation. However, tighter standard devia-tion values were observed from the NLC formulation which couldpossibly be attributed to the improved physical stability of this for-mulation compared to the SLN formulation. Statistically signicantdifferences (p < 0.05) were observed between the lipid basedlipid based nanosystems (n = 45).

tern from both SLN and NLC systems. The reduction in the viscosity

fractionation and transmission electron microscopy. J. Control. Release 95,

l of Pof the NLC system due to the incorporation of the liquid lipid re-sulted in faster diffusion and release of quercetin from the NLC sys-tem. Superior topical delivery of quercetin was observed from boththe lipid based nanosystems (SLN and NLC) compared to the con-trol formulation (particles in the micrometer range), although thedifferences in the efcacy of topical delivery between the SLNand NLC systems were not found to be statistically signicant.However, the results of the in vitro skin permeation experimentsshowed a tighter standard deviation for the NLC formulation whichcould be related to the improved physical stability of this formula-tion compared to the SLN formulation. Hence, the NLC formulationis considered to be the most promising for further development asa topical delivery system of quercetin. Future studies will focus onthe feasibility of high pressure homogenization, a widely usedmanufacturing process for lipid nanosystems, to manufacturethese quercetin NLC systems.

Acknowledgements

The authors would like to acknowledge Yuechao Du and ReenaNadpara for providing assistance during batch manufacturing andDr. Paul Takishtov for providing lab access to the instrumentation.The authors would also like to thank Danielle Lazarra for help withthe skin permeation experiments, Karen Killary for the TEM imagesnanosystems with particle size in the nanometer range and theircorresponding control formulations with particle size in themicrometer range (with and without oleic acid).

The superior topical delivery of quercetin observed from boththe lipid based nanosystems compared to the control formulation(particles in the micrometer range) can be possibly explained bythe increased surface area/contact surface of the active compoundwith the skin corneocytes, higher occlusive effect and increasedhydration of the stratum corneum that has been associated in gen-eral with lipid nanoparticles (Muller et al., 2007). The absence ofany transdermal delivery of quercetin from the lipid nanoparticlesis also consistent with literature reports of lipid nanoparticles notconsidered to penetrate the horny layer (Schafer-Korting et al.,2007). Also, in this study, the presence of oleic acid in the NLC for-mulation was not observed to have any signicant effect on theskin accumulation of quercetin. Although oleic acid has been re-ported to increase the absorption of many active compounds suchas salicylic acid (28-fold increase) and 5-uorouracil (50-fold in-crease) (Goodman and Barry, 1989), there are also some literaturereferences where it has been shown to have no enhancement effecton the absorption of actives such as urbiprofen (Fang et al., 2003)and hexyl nicotinate (Tanojo et al., 1999) which is consistent withour observation.

4. Conclusions

In this study, a part of the solid lipid from the SLN formulationwas substituted with a liquid lipid (oleic acid) to produce NLC sys-tems of quercetin using the probe ultrasonication method. Longterm stability study carried out with the NLC system with 0.025%drug loading showed excellent physical stability of the systembased on particle size and zeta potential measurement values.TEM measurements showed spherical particles in the nanometerrange for the quercetin NLC system. The onset of a partial transfor-mation of glyceryl dibehenate from the metastable b0 to the morestable bi form was conrmed via XRD and DSC experiments. In vitrorelease studies showed a biphasic release prole, characterized byan initial burst release followed by a more controlled release pat-

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Preparation and characterization of lipid based nanosystems for topical delivery of quercetin1 Introduction2 Materials and methods2.1 Materials2.2 Preparation of nanosystems of quercetin2.3 Freeze drying of nanoparticles2.4 Characterization of nanoparticles2.4.1 Particle size measurement2.4.2 Zeta potential measurement2.4.3 Morphology2.4.4 X-ray powder diffraction (XRPD)2.4.5 Modulated differential scanning calorimetry (MDSC)2.4.6 Release study2.4.7 Detection of quercetin by HPLC2.4.8 Stability study2.4.9 In vitro permeation study using human skin2.4.10 Statistical analysis

3 Results and discussion3.1 Rationale for selection of liquid lipid in NLC system3.2 Effect of process parameters on physical stability of quercetin NLC systems3.3 Effect of oleic acid on physical stability of quercetin lipid based nanosystems3.4 Effect of drug loading on physical stability of quercetin NLC systems3.5 Long term physical stability of selected NLC formulation3.6 Morphology of selected NLC formulation3.7 Solid state characterization of quercetin NLC systems: X-ray diffraction (XRD) data3.8 Solid state characterization of quercetin NLC systems: Differential scanning calorimetry (DSC) data3.9 Release of quercetin from lipid nanosystems3.10 Topical delivery of quercetin from lipid nanosystems

4 ConclusionsAcknowledgementsReferences