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Colloids and Surfaces B: Biointerfaces 88 (2011) 483– 489

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

ormulation design, preparation and physicochemical characterizations of solidipid nanoparticles containing a hydrophobic drug: Effects of process variables

urajit Dasa,∗, Wai Kiong Nga, Parijat Kanaujiaa, Sanggu Kima, Reginald B.H. Tana,b

Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, SingaporeDepartment of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore

r t i c l e i n f o

rticle history:eceived 4 April 2011eceived in revised form 11 July 2011ccepted 15 July 2011vailable online 23 July 2011

eywords:olid lipid nanoparticleydrophobic drugretinoinrug releasencapsulation

a b s t r a c t

This study aimed to prepare solid lipid nanoparticles (SLNs) of a hydrophobic drug, tretinoin, byemulsification–ultrasonication method. Solubility of tretinoin in the solid lipids was examined. Effectsof process variables were investigated on particle size, polydispersity index (PI), zeta potential (ZP), drugencapsulation efficiency (EE), and drug loading (L) of the SLNs. Shape and surface morphology of theSLNs were investigated by cryogenic field emission scanning electron microscopy (cryo-FESEM). Com-plete encapsulation of drug in the nanoparticles was checked by cross-polarized light microscopy anddifferential scanning calorimetry (DSC). Crystallinity of the formulation was analyzed by DSC and powderX-ray diffraction (PXRD). In addition, drug release and stability studies were also performed. The resultsindicated that 10 mg tretinoin was soluble in 0.45 ± 0.07 g Precirol® ATO5 and 0.36 ± 0.06 g Compritol®

888ATO, respectively. Process variables exhibited significant influence in producing SLNs. SLNs with<120 nm size, <0.2 PI, >I30I mV ZP, >75% EE, and ∼0.8% L can be produced following the appropriate for-mulation conditions. Cryo-FESEM study showed spherical particles with smooth surface. Cross-polarized

light microscopy study revealed that drug crystals in the external aqueous phase were absent whenthe SLNs were prepared at ≤0.05% drug concentration. DSC and PXRD studies indicated complete drugencapsulation within the nanoparticle matrix as amorphous form. The drug release study demonstratedsustained/prolonged drug release from the SLNs. Furthermore, tretinoin-loaded SLNs were stable for 3months at 4 ◦C. Hence, the developed SLNs can be used as drug carrier for sustained/prolonged drugrelease and/or to improve oral absorption/bioavailability.

. Introduction

The factors like poor aqueous solubility, low gastro-intestinalGI) absorption, and rapid metabolism of drug play a major rolen disappointing in vivo results [1,2]. Although some colloidalrug carriers (e.g., micelles, nanoemulsions, nanosuspensions,

iposomes, polymeric nanoparticles) have been investigated tovercome the solubility and bioavailability related problems, theyave several drawbacks, such as limited physical stability, aggre-ation, drug leakage on storage, presence of solvent residues leftver from production, cytotoxicity of polymers etc. [3–7]. Hence,

he researchers have focused on the biocompatible lipids as carri-rs for the delivery of poorly soluble drugs to avoid/minimize the

∗ Corresponding author at: Institute of Chemical and Engineering Sciences,*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island,ingapore 627833, Singapore. Tel.: +65 6796 3853; fax: +65 6316 6183.

E-mail addresses: surajit das@ices.a-star.edu.sg, surajitdas1982@yahoo.com (S.as), reginald tan@ices.a-star.edu.sg (R.B.H. Tan).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.07.036

© 2011 Elsevier B.V. All rights reserved.

above mentioned problems [8–10]. Solid lipid nanoparticles (SLNs)have emerged as potential drug carriers for this purpose [9,11].

The matrix of SLNs is solid at room and body temperature. Thelipid matrix employed in lipid nanoparticles is usually a physiologi-cal lipid (biocompatible and biodegradable) with negligible toxicity[6]. It has been reported that lipids may promote oral absorptionof some drugs mainly due to lymphatic transport and enhance-ment of GI permeability [12–17]. It has also been reported thatnanoparticles (120–200 nm) rarely undergo blood clearance by thereticuloendothelial system [3,18,19]. In addition, sustained drugrelease from SLNs is possible due to the solid matrix of the particles[9]. Furthermore, sufficient drug loading [20,21], long-term shelfstability [1,3,22–24], and easy large-scale production [1,3,25–27]are advantageous. Hence, SLNs are very attractive carriers for oraldrug delivery to enhance oral bioavailability and/or sustained deliv-ery of drugs [8,9].

Tretinoin (all-trans retinoic acid) has shown anticancer activ-

ities against several types of cancer [28–30]. However, its utilityis strongly limited by its poor aqueous solubility and low oralbioavailability. Therefore, its solubility and oral bioavailabilityproblems should be resolved for clinical application. An oral

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ormulation with a high degree of oral absorption might solve thisroblem. In view of this, exploring the potential of SLNs loaded withretinoin seems worthwhile.

In this study, the formulation parameters, such as homoge-ization time (HT), sonication time (ST), surfactant concentrationSC), lipid concentration (LC), drug concentration (DC), lipid type,nd surfactant type were optimized for the formulation of SLNsy emulsification–ultrasonication method. Tretinoin was used asodel drug. Solubility of tretinoin in the solid lipids was deter-ined to screen the suitable lipid for formulation. The effects of

he process variables on the particle size, polydispersity index (PI),eta potential (ZP), drug encapsulation efficiency (EE), and drugoading (L) of SLNs were investigated. The SLNs were physicochem-cally characterized by cryogenic field emission scanning electron

icroscopy (cryo-FESEM), cross-polarized light microscopy, differ-ntial scanning calorimetry (DSC), and powder X-ray diffractionPXRD). Furthermore, drug release study was performed to observehe drug release profile from the developed SLNs. Finally, stabilityf the developed formulation was evaluated.

. Materials and methods

.1. Materials

Tretinoin and Chremophore® EL was generous gift from BASFGermany). Precirol® ATO 5 (glycerol distearate), and Compritol®

88 ATO (glyceryl dibehenate/behenate) were kindly donatedy Gattefossé (France). Dynasan® 114 (glycerol trimyristate) andynasan® 118 (glycerol tristearate) were gift samples from Sasol

Germany). Tween® 80 and dialysis tube were purchased fromigma (USA). HPLC grade acetonitrile and methanol were obtainedrom Fisher Scientific (USA) and J. T. Baker (USA), respectively.hosphoric acid was bought from Kanto Chemical Co. Inc. (Japan).urified water from Millipore-Q® Gradient A10TM ultra-pure waterystem (Millipore, France) was used throughout the study.

.2. Measurement of tretinoin solubility in lipids

As equilibrium solubility study was not possible due to the solidature of the lipids, an alternative method was adopted to measureolubility of drug in the solid lipids [31]. Briefly, 10 mg tretinoin waseighed accurately and placed in a screwcapped glass bottle cov-

red with aluminum foil. About 200 mg of lipid was added in theottle and heated at 80 ◦C under continuous stirring. Then addi-ional lipid was added in portions under continuous stirring andeating at 80 ◦C until a clear solution was formed. Total amount of

ipid added to get a clear solution was recorded.

.3. Formulation procedure

SLNs were prepared by emulsification–ultrasonication method32]. Briefly, solid lipid was melted at 80 ◦C and dispersed in thequeous surfactant solution at 80 ◦C using a homogenizer (IKA®

-10 basic Ultra-Turrax®, Germany) at ∼14,000–15,000 rpm. Thebtained emulsion was ultrasonicated using a probe sonicatorVibracellTM 700W; Sonics, USA) at 80 ◦C. The resulting nanoemul-ion was cooled down in an ice bath to produce nanoparticleispersion. Different formulations were prepared by varying theritical process variables (Section 2.4).

.4. Formulation design

SLNs were prepared by varying:

a) HT (1, 2.5, 5 and 10 min); while ST = 10 min, SC = 2.5% (w/v),LC = 5% (w/v), DC = 0.05% (w/v), lipid = Precirol® ATO5 and sur-factant = Tween® 80.

iointerfaces 88 (2011) 483– 489

b) ST (1, 2.5, 5 and 10 min); while HT = 5 min, SC = 2.5% (w/v),LC = 5% (w/v), DC = 0.05% (w/v), lipid = Precirol® ATO5 and sur-factant = Tween® 80.

c) SC (0.5, 1, 2, 2.5 and 3%, w/v); while HT = 5 min, ST = 10 min,LC = 5% (w/v), DC = 0.05% (w/v), lipid = Precirol® ATO5 and sur-factant = Tween® 80.

) LC (1, 2.5, 5 and 10%, w/v); while HT = 5 min, ST = 10 min,SC = 2.5% (w/v), DC = 0.05% (w/v), lipid = Precirol® ATO5 and sur-factant = Tween® 80.

e) DC (0, 0.025, 0.05, 0.075, 0.1 and 0.2%, w/v); while HT = 5 min,ST = 10 min, SC = 2.5% (w/v), LC = 5% (w/v), lipid = Precirol® ATO5and surfactant = Tween® 80.

f) Lipid (Precirol® ATO5, Compritol® 888ATO, Dynasan® 114 andDynasan® 118); while HT = 5 min, ST = 10 min, SC = 2.5% (w/v),LC = 5% (w/v), DC = 0.05% (w/v) and surfactant = Tween® 80.

g) Surfactant (Tween® 80, Chremophore® EL and Lutrol® F68);while HT = 5 min, ST = 10 min, SC = 2.5% (w/v), LC = 5% (w/v),DC = 0.05% (w/v) and lipid = Precirol® ATO5.

2.5. Determination of particle size and polydispersity index

Particle size (z-average diameter) and polydispersity index(PI) were measured by photon correlation spectroscopy usingMalvern Zetasizer Nano ZS (Malvern Instruments, UK) at 25 ◦C.The instrument contains a 4 mW He–Ne laser operating at 633 nmwavelength. The nanoparticle dispersion was appropriately diluted(particle count rate between 100 and 1000 s−1) with ultra-purifiedwater before measurement. The measurement was conducted at173◦ detection angle.

2.6. Zeta potential measurement

Zeta potential (ZP) reflects the electric charge on the particlesurface. It is a useful parameter to predict the physical stabil-ity of colloidal systems. ZP was determined by the measurementof the electrophoretic mobility using a Malvern Zetasizer NanoZS (Malvern Instruments, UK). The field strength applied was20 V cm−1. The conversion into the zeta potential was performedusing the Helmoltz–Smoluchowski equation:

� = EM × 4��

ε

where, � is zeta potential, EM is electrophoretic mobility, � is vis-cosity of the dispersion medium, and ε is dielectric constant. Priorto the measurement, all samples were diluted using ultra-purifiedwater.

2.7. Cryogenic field emission scanning electron microscopy

Shape and surface morphology of the SLNs prepared at 0.05%DC were examined by cryogenic field emission scanning electronmicroscopy (cryo-FESEM). Approximately 2–3 drops of SLN disper-sion (∼30 �L) were placed on a copper stub and frozen in nitrogenslush at −196 ◦C. Samples were then stored in liquid nitrogen andtransferred into the cryo preparation chamber (GATAN ALTO 2500,UK) attached to a FESEM (JEOL JSM-6700F, Japan). The sample wasfreeze-fractured and sublimed at −95 ◦C for 30 s. The fracturedsample was sputter-coated with platinum for 120 s in the cryopreparation chamber and then introduced onto the specimen stageof the FESEM at −140 ◦C and examined at an excitation voltage of5 kV. On the other hand, tretinoin powder was analyzed by normal

FESEM. Briefly, tretinoin powder was placed on a copper stub withthe help of double-sided adhesive tape and sputter-coated withgold at 20 mA for 120 s. Then the sample was analyzed in FESEM(JEOL JSM-6700F, Japan) at an excitation voltage of 5 kV.

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solubility studies are shown in Fig. 1. Solubility of tretinoin wastested in 4 lipids, Precirol® ATO5, Compritol® 888ATO, Dynasan®

114, and Dynasan® 118. Among them, Compritol® 888ATO showedhighest solubilization capacity followed by Precirol® ATO5. Amount

S. Das et al. / Colloids and Surfac

.8. HPLC analysis of tretinoin

Agilent HPLC (Agilent 1100 series; USA) attached with a reversehase C18 column (ZORBAX Eclipse Plus C18; 4.6 mm × 250 mm,

�m; Agilent, USA) was used for the assay. Mobile phase usedas acetonitrile and 0.1% aqueous solution of phosphoric acid

9:1) at 1 mL min−1 isocratic flow. Temperature of the columnnd detection wavelength were set at 35 ◦C and 350 nm, respec-ively. An injection volume was set at 25 �L for all standards andamples. Tretinoin was eluted at a retention time of 7.9 min. Thessay was linear (r2 = 0.9996) in the tretinoin concentration range0–1000 ng mL−1.

.9. Encapsulation efficiency and drug loading

The freshly prepared SLN dispersion was filtered through 3 �mitrocellulose membrane filter (Millipore, Ireland) to get rid ofnentrapped drug crystals (undissolved drug). The filtered formu-

ation was dissolved in methanol and vortexed to extract drugrom lipid. The mixture was centrifuged at 5000 rpm for 15 min.he supernatant was appropriately diluted with methanol andrug concentration was measured by HPLC assay. The amount ofrug dissolved in the aqueous phase was determined by ultra-ltration method using centrifugal filter tubes with a 10 kDaolecular weight cut-off (Centricert 1; Sartorius, Germany). Briefly,

anoparticle dispersion was placed into a centrifugal filter tubehich was then centrifuged at 5000 rpm for 30 min. After cen-

rifugation, the amount of soluble free drug in the aqueoushase was detected by HPLC. Drug loading (L) was presenteds percent drug to lipid ratio. Encapsulation efficiency (EE) wasalculated as:

E (%) = amount of drug in filtered formulation − soluble free druginitial amount of drug added during preparation of SLNs

× 100%

.10. Cross-polarized light microscopy

SLN formulations were examined under cross-polarized lighticroscope (Olympus, Japan) to see the presence of tretinoin crys-

als in the aqueous phase. SLN formulations prepared at 0.05 and.2% DC were examined. Furthermore, SLN formulation preparedt 0.2% DC was also examined after filtration through 3 �m nitro-ellulose filter (Millipore, Ireland). Freshly prepared SLN dispersionfiltered/unfiltered) was diluted with ultra-pure water (1:10) and 1rop was placed on a glass slide and covered with a cover slip. Thenhe slide was placed on the microscope stage and cross-polarizedight was passed through the sample. The images were capturedy a digital camera (Sony, Japan) equipped with the microscopetilizing a software (analySIS pro).

.11. Differential scanning calorimetry

Drug-free and drug-loaded SLNs were lyophilized (VirTisenchtop lyophilizer, USA). The thermograms of the individualomponents and lyophilized SLNs were recorded by a DSC (Perkinlmer, USA). Samples (∼4–5 mg) sealed in standard aluminum pans

ere kept under isothermal condition at 25 ◦C for 10 min. DSC studyas performed at 10 ◦C min−1 from 25 to 200 ◦C under a nitrogen

tmosphere with a flow rate of 20 mL min−1. An empty sealed panas used as reference.

iointerfaces 88 (2011) 483– 489 485

2.12. Powder X-ray diffraction

PXRD measurements of pure tretinoin, Precirol® ATO5,lyophilized drug-free SLNs, and lyophilized tretinoin-loaded SLNswere carried out with a powder X-ray diffractometer (D8-ADVANCE, Bruker, Germany) using Cu K ̨ radiation as X-ray source.Samples were placed in glass sample holders and scanned from 2◦

to 80◦ with a scan angular speed (2� min−1) of 2◦ min−1. Operatingvoltage was 35 kV and current was 40 mA.

2.13. Drug release study

Drug release study was performed following dialysis bagmethod [33]. The dialysis bag retains the SLNs but allows thetransfer of the dissolved/released drug molecules into the releasemedia. Briefly, dialysis tube (molecular cut off = 10 KDa) was pre-pared according to the protocol provided by Sigma and soaked inthe release media overnight prior to the study. Release media was10 mM phosphate buffer (pH 7.4) containing 2% Tween® 80. Oneend of the dialysis tube was tightly tied and 2 mL of SLN dispersionwas placed in the tube. Other end of the tube was also tightly tiedand the tube was then placed in an amber colored glass bottle con-taining 10 mL release media. The bottle was capped and placed on ahorizontal rotary shaker (Sartorius, Germany) rotating at 100 rpm.At the predetermined time points, 1 mL of release media (sample)was withdrawn from the bottle and replaced by 1 mL fresh releasemedia to maintain the sink condition. The samples were analyzedby HPLC as mentioned earlier.

2.14. Stability study

The stability of the developed SLN formulation was evaluated for3 months. Briefly, samples were stored in the sealed amber coloredglass vials at 4 ◦C. After 3 months, the samples were characterizedwith respect to particle size, ZP, PI, EE, and L.

3. Results and discussion

3.1. Solubility of tretinoin in lipids

Solubility of drug in lipid is one of the most important factorsfor determining drug loading capacity of the SLNs. Results from the

Fig. 1. Solubility profile of tretinoin in the solid lipids. Data represent mean ± SD.

486 S. Das et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 483– 489

Table 1Size, PI, ZP, EE, and L of the nanoparticles (data represent mean ± SD).

Variables Size (nm) PI ZP (−mV) EE (%) L (%)

HT (min) 1 117.3 ± 1.47 0.202 ± 0.012 30.7 ± 0.49 77.89 ± 4.70 0.77 ± 0.052.5 117.8 ± 0.36 0.177 ± 0.019 28.0 ± 0.04 80.88 ± 1.60 0.80 ± 0.025 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.0410 117.4 ± 0.85 0.193 ± 0.028 30.2 ± 0.23 75.02 ± 3.54 0.78 ± 0.04

ST (min) 1 322.9 ± 9.42 0.551 ± 0.029 31.6 ± 0.72 72.17 ± 1.61 0.74 ± 0.022.5 244.1 ± 8.70 0.417 ± 0.061 33.4 ± 0.07 76.23 ± 2.19 0.78 ± 0.025 172.4 ± 6.21 0.299 ± 0.011 30.0 ± 1.50 77.10 ± 4.16 0.77 ± 0.0410 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.04

SC (% w/v) 0.5 392.1 ± 17.25 0.428 ± 0.071 40.6 ± 0.32 64.08 ± 4.22 0.64 ± 0.041 243.6 ± 4.78 0.308 ± 0.022 40.1 ± 0.80 67.45 ± 3.85 0.68 ± 0.042 140.9 ± 2.19 0.231 ± 0.005 36.6 ± 1.48 73.74 ± 2.18 0.77 ± 0.022.5 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.043 107.6 ± 1.97 0.200 ± 0.026 29.8 ± 1.48 80.79 ± 4.35 0.81 ± 0.04

LC (% w/v) 1 37.53 ± 1.00 0.375 ± 0.020 23.7 ± 0.83 21.75 ± 1.46 1.08 ± 0.072.5 69.61 ± 1.05 0.181 ± 0.011 26.2 ± 0.35 50.19 ± 2.47 1.00 ± 0.055 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.0410 239.5 ± 3.57 0.264 ± 0.009 34.5 ± 0.12 84.16 ± 3.36 0.42 ± 0.02

DC (% w/v) 0 102.2 ± 0.76 0.169 ± 0.013 42.0 ± 1.37 - -0.025 110.7 ± 1.59 0.211 ± 0.010 36.3 ± 0.42 94.60 ± 3.66 0.58 ± 0.020.05 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.040.075 119.9 ± 0.50 0.191 ± 0.023 42.8 ± 1.33 64.80 ± 1.33 0.86 ± 0.020.1 224.2 ± 58.84 0.340 ± 0.058 37.4 ± 2.76 54.49 ± 2.03 1.11 ± 0.040.2 263.9 ± 19.30 0.476 ± 0.040 32.0 ± 1.29 27.83 ± 1.46 1.11 ± 0.06

Lipid Precirol® ATO5 113.5 ± 0.89 0.182 ± 0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.04Compritol® 888ATO 146.2 ± 2.36 0.201 ± 0.035 24.0 ± 1.04 77.63 ± 4.83 0.78 ± 0.05Dynasan® 114 165.0 ± 2.19 0.207 ± 0.009 35.2 ± 0.38 55.95 ± 2.47 0.56 ± 0.02Dynasan® 118 223.7 ± 3.86 0.345 ± 0.044 22.6 ± 0.05 42.60 ± 2.31 0.43 ± 0.02

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Surfactant Tween 80 113.5 ± 0.89 0Chremophore® EL 90.9 ± 1.34 0Lutrol® F68 176.9 ± 1.15 0

f Dynasan® 114 (0.98 ± 0.14 g) and Dynasan® 118 (1.33 ± 0.22 g)equired to solubilize 10 mg tretinoin was significantly higherhan Precirol® ATO5 (0.45 ± 0.07 g) and Compritol® 888ATO0.36 ± 0.06 g). This study indicated that tretinoin loading capac-ty of Precirol® ATO5 and Compritol® 888ATO might be more thanynasan® 114, and Dynasan® 118.

.2. Effects of process variables

The emulsification–ultrasonication method was found to be effi-ient and quick to produce SLNs. Effect of different process variablesn size, PI, ZP, EE, and L are discussed in the following sections andresented in Table 1.

.2.1. Homogenization timeHomogenization time (HT) did not show any effect on parti-

le size. This should be expected as homogenization step is onlyor emulsification of lipid in aqueous phase. This step did not pro-uce final particles; rather it is an intermediate step. However,igher PI was observed in case of 1 and 10 min homogenization.enerally, ZP value can predict physical stability of the nanodis-ersion [11,22]. The ZP indicates the degree of repulsion betweenlose and similarly charged particles in the nanodispersion. High ZPndicates highly charged particles. Generally, high ZP (negative orositive) prevents aggregation of the particles due to electric repul-ion and electrically stabilizes the nanoparticle dispersion. ZP morehan I30I mV indicates stable nanodispersion [22]. In this case, ZPas always higher than I28I mV and highest (−38.7 mV) in case of

min homogenization. There were no significant differences in EEnd L among different HTs. However, EE was slightly lower in casef 10 min homogenization.

.2.2. Sonication timeSonication time (ST) showed huge influence on particle size and

I. Size and PI significantly decreased with increasing ST. Thesebservations are reasonable as sonication was responsible for final

0.007 38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.04 0.015 37.9 ± 0.78 73.26 ± 2.97 0.73 ± 0.03 0.069 35.3 ± 1.24 59.87 ± 1.09 0.60 ± 0.01

particle size of SLNs, which broke the coarse emulsion drops tonanoemulsion droplets. Longer sonication time put more soni-cation energy to the SLN dispersions, which reduced size of thenanoemulsion droplets and decreased size distribution. ZPs weremore than I30I mV indicating stable system. ST of 1 min producedparticles with lowest EE. Otherwise, EE and L were not significantlydifferent among the groups.

3.2.3. Surfactant concentrationConcentration of surfactant demonstrated huge influence on

particle size and PI. Particle size decreased with increasing surfac-tant concentration (SC). Similar observations were also reported byother researchers [33,34]. PI also decreased with increasing SC upto 2.5% SC. These observations might be due to production and sta-bilization of smaller lipid droplets (nanoemulsion) at higher SC asenough surfactant was present to stabilize the nanodroplets. How-ever, surprisingly PI was higher at 3% SC than 2.5% SC. ZPs weremore than I36I mV which predict good physical stability of the SLNdispersions. EE and L increased with increasing SC (except L of 3%SC). This observation was supported by other researchers [34]. Thiscould be due to the presence of sufficient SC which helped the drugto remain within the lipid particles and/or on the surface of theparticles.

3.2.4. Lipid concentrationParticle size significantly increased with increasing lipid con-

centration (LC). Distribution of sonication energy in the dilute (lowLC) dispersion is better than concentrated dispersion, which wasresponsible for more efficient particle size reduction. Although par-ticle size was lowest in case of 1% LC, PI was very high. On the otherhand, both size and PI was high in case of 10% LC. In all LCs, ZP wasmore than I23I mV. As expected, EE significantly increased with

increasing LC. This is because, amount of drug encapsulation intothe lipid particles depends on the availability of the amount of lipid.Higher amount of lipid was available for drug encapsulation at highLC, which led to higher EE. Opposite trend was noticed for low LC. In

es B: Biointerfaces 88 (2011) 483– 489 487

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ontrary, L significantly decreased with increasing LC. This was dueo fixed DC used during formulation. DC remained same in spite ofncreased LC, which led to reduction of drug to lipid ratio (i.e., L)

ith increasing LC.

.2.5. Drug concentrationThere were insignificant differences in particle size among dif-

erent batches prepared with drug concentration (DC) from 0.025 to.075%. Particle size significantly increased with higher drug con-entration after that (0.075–0.2%). PI increased with higher DC from.05 to 2.0%. However, 0.025% DC showed slightly higher PI than.05 and 0.075% DC. Higher size and PI at higher DC were due tohe presence of high amount of drug. ZPs were more than I32I mVn all cases, which suggests good stability of the SLN dispersions.E significantly decreased with increasing DC. L also increased withigher DC up to 0.1% DC. EE directly depends on the amount of drugdded (i.e., DC). As lipid has certain drug loading capacity, additionf excess drug led to increase of unencapsulated drug (i.e., decreasef EE). Furthermore, after reaching maximum drug loading capac-ty, L cannot increase, which signify that L reached its maximumoint at 0.1% DC.

.2.6. Lipid typeAmong 4 types of lipids, Precirol® ATO5 produced smallest

articles with lowest PI. Particle size and PI increased in theollowing order: Precirol® ATO5 < Compritol® 888ATO < Dynasan®

14 < Dynasan® 118. ZPs were more than I22I mV for all lipids.recirol® ATO5 produced SLN with highest ZP indicating best phys-cal stability of the particles in the nanodispersion. There were noignificant differences in EE and L between SLNs produced withrecirol® ATO5 and Compritol® 888ATO. EE and L were signifi-antly lower in case of Dynasan® 114 SLNs, lowest for Dynasan®

18 SLNs. These observations can be explained by the solubilitytudy that demonstrated higher solubilization capacity of Precirol®

TO5 and Compritol® 888ATO than Dynasan® 114 and Dynasan®

18. The results indicate that Dynasan® 114 and Dynasan® 118ere unsuitable for the preparation of desired SLNs.

.2.7. Surfactant typeSmall particle size was observed when Tween® 80 or

hremophore® EL was used. However, Chremophore® EL exhib-ted lower particle size than Tween® 80 although PI was slightlyigher in case of Chremophore® EL than Tween® 80. Lutrol® F68roduced SLNs with higher particle size and PI. In all batches, ZPsere more than I35I mV suggesting physically stable formulations.

E and L were slightly higher when Tween® 80 was used thanhremophore® EL. EE and L were significantly low when Lutrol®

68 was used. The results indicate that Lutrol® F68 was unsuitableor the preparation of desired SLNs.

From the results discussed above, the optimized formula-ion conditions were decided as follows: HT = 5 min, ST = 10 min,C = 2.5% (w/v), LC = 5% (w/v), DC = 0.05% (w/v), lipid = Precirol®

TO5, and surfactant = Tween® 80. SLNs prepared according tohese formulation conditions were subjected to the following stud-es (unless specifically mentioned).

.3. Shape and morphology

Shape and surface morphology of the nanoparticles was checkedy cryo-FESEM. Cryo-FESEM helps to obtain more informationbout particle size and shape by investigating the samples in frozenondition as the samples are investigated close to their natural

tate [11]. Smooth surface morphology of the SLNs was observedFig. 2A). SLNs were almost spherical in shape within 75–300 nmange, which was in agreement with the size data determinedy PCS. Agglomeration of nanoparticles was also evident, which

Fig. 2. SEM images of tretinoin-loaded SLNs (A) and tretinoin powder (B).

could be due to the lipid nature (stickiness) of the carriers. FESEMimage of tretinoin powder exhibited regular crystalline-like struc-ture (Fig. 2B). Such block-like structures of tretinoin were absentin the SLNs (Fig. 2A), which suggest the absence of unencapsulateddrug crystals in the SLN dispersion.

3.4. Microscopic analysis

The cross-polarized light microscopy was used to investigate thepresence of unencapsulated tretinoin crystals that might be sus-pended in the external aqueous phase of SLN dispersion (Fig. 3A).This technique is also a well established method to support EEstudy [35]. The formulation prepared at 0.2% DC showed presence ofmany unencapsulated drug crystals in the external aqueous phase(Fig. 3A). Whereas, same formulation filtered through 3 �m nitro-cellulose syringe filter did not show any trace of drug crystal inthe external aqueous phase (Fig. 3B). This indicates that all unen-capsulated drug crystals observed were >3 �m in size (Fig. 3A),which retained on the filter paper during filtration. Nevertheless,formulation prepared at 0.05% DC did not show any unencapsulateddrug crystals in the external aqueous phase even before filtration

through 3 �m nitrocellulose filter (similar to Fig. 3B). This indi-cates that there were almost no unencapsulated drug crystals andalmost all drugs were encapsulated inside SLNs as EE study revealedthat amount of solubilized tretinoin in the aqueous phase was very

488 S. Das et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 483– 489

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the nature of SLNs. An explanation to this observation is that thetretinoin was entrapped in the lipid core of SLNs.

ig. 3. Cross polarized microscopic images of SLNs prepared at 0.2% DC before fil-ration (A) and after filtration (B).

ow (data not shown). SLNs prepared at 0.2% DC encapsulated cer-ain amount of drug according to their capacity and rest of therug remained as unencapsulated crystals in the aqueous phases tretinoin is poorly water soluble. Another aspect was that theormulation prepared at 0.2% DC was deep yellow (color of drug)ue to presence of drug crystals in the external aqueous phase,hereas the formulation prepared at ≤0.05% DC was almost white

r light yellow due to complete drug encapsulation. These find-ngs also confirm the EE data obtained through filtration technique

hich revealed higher EE of the formulation prepared at 0.05% DChan the formulation prepared at 0.2% DC (Table 1).

.5. Thermal analysis

DSC thermogram of Precirol® ATO5, tretinoin, drug-free SLNs,nd tretinoin-loaded SLNs are presented in Fig. 4. Crystallineretinoin demonstrated a sharp peak at 184 ◦C corresponds to

elting temperature of tretinoin. The tretinoin-loaded SLNs andrug-free SLNs were lyophilized to protect the physical state of

ipid. Tretinoin-loaded SLNs showed a broad endothermic peakt 60.9 ◦C, whereas drug-free SLNs and Precirol showed a broadndothermic peak at 61.4 ◦C. This slight shift in peak was probablyue to drug loading in lipid matrix. In case of SLN formulations,

n additional peak at ∼54 ◦C was probably due to the presencef surfactant within the SLNs. Absence of the peak at 184 ◦C inretinoin-loaded SLNs indicates either formation of amorphous dis-

Fig. 4. DSC thermogram of Precirol ATO5, tretinoin, drug-free SLNs, and tretinoin-loaded SLNs.

persion of tretinoin in lipid matrix or solubilization of tretinoin inlipid matrix upon heating.

3.6. X-ray diffractogram

PXRD data (Fig. 5) confirmed the results demonstrated by DSCstudy. The diffraction pattern of tretinoin showed 5 distinct sharppeaks at 2� = 5.2◦, 13.6◦, 14.8◦, 15.6◦, and 20.9◦; and few other peaksof lower intensity between 17.4◦ and 29.7◦ and at 10.4◦. These peakscould not be detected in diffractogram of drug-loaded SLNs whichindicate that tretinoin was solubilized within the lipid matrix ofSLNs and stabilized in amorphous form. Due to its poor aqueoussolubility, if tretinoin was located outside the lipid matrix, crys-tallization would occur which should have affected the diffractionpatterns of drug-loaded SLNs. This suggests that the drug was suc-cessfully incorporated into the lipid matrix of the developed SLNs.In addition PXRD spectra obtained between angles 2� = 18–25◦

indicated lower intensity in SLNs than Precirol® ATO5, which sup-port lower crystallinity of the lipid matrix of SLNs in comparison tobulk solid lipid reflecting less ordered structure of the lipid matrixin SLNs. Also, looking at the diffraction patterns of drug-free SLNsand tretinoin-loaded SLNs, there was not much difference in thepattern, indicating that the addition of tretinoin did not changed

Fig. 5. PXRD profile of Precirol® ATO5, tretinoin, drug-free SLNs, and tretinoin-loaded SLNs.

S. Das et al. / Colloids and Surfaces B: Biointerfaces 88 (2011) 483– 489 489

Table 2Size, PI, ZP, EE, and L of the nanoparticles after 3 months storage at 4 ◦C (data represent mean ± SD).

Storage Size (nm) PI ZP (−mV) EE (%) L (%)

Fresh 113.5 ± 0.89 0.182 ± 0.007

3 months 129.7 ± 3.62 0.191 ± 0.073

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Fig. 6. Drug release profile from the formulation. Data represent mean ± SD.

.7. Drug release

As aqueous solubility of tretinoin is very low, 2% (w/v) Tween®

0 was added to the release media to maintain sink condition. Drugeleased from the SLNs and diffused through the dialysis membranento the release media, while the dialysis bag retained the SLNs.ence, drug released from the SLNs were measured by determiningrug concentration in the release media. Percent cumulative drugelease versus time was plotted to demonstrate the drug releaseattern (Fig. 6). The result demonstrated sustained/prolonged drugelease from the formulation. The drug release after 80 h was3.39 ± 3.79%. Such sustained/prolonged drug release from theLNs was also observed by other researchers [33].

.8. Stability

The tretinoin-loaded SLNs showed minor enhancement of par-icle size and PI, but slight reduction of ZP, EE, and L after 3 monthstorage at 4 ◦C (Table 2). However, the changes were insignificant,hich indicates good physical stability of the SLNs during their

torage at 4 ◦C for 3 months.

. Conclusion

In this study, SLNs were successfully prepared bymulsification–ultrasonication method. Precirol® ATO5 andompritol® 888ATO demonstrated reasonable tretinoin solubiliza-ion capacity. Poorly aqueous soluble drug tretinoin was efficientlyncapsulated into the nanoparticles. Most of the process variableshowed significant effect on the formulation properties. Particlesrepared at proper formulation conditions were spherical with

120 nm size. Physicochemical characterization showed that SLNsrepared at 0.05% DC efficiently encapsulated almost all drug. Therug release study exhibited sustained/prolonged drug releaserom the SLNs. Furthermore, SLNs were stable for 3 months at 4 ◦C.

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38.7 ± 0.56 78.18 ± 4.26 0.82 ± 0.0434.63 ± 0.96 74.22 ± 4.97 0.75 ± 0.05

Hence, the developed SLNs can be used for sustained/prolongeddrug delivery and/or to improve oral absorption/bioavailability.However, further studies should focus on the improvement of drugloading in the SLNs.

Acknowledgements

This work was supported through the grant ICES/09-222A05 (S.Das) provided by the Science and Engineering Research Council ofA*STAR (Agency for Science, Technology and Research), Singapore.We are grateful to Miss Annie Wong for her assistance in the exper-iments. We would like to thank Mr. Ng Jun Wei and Mr. Mark Ngfor their technical assistance.

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