1. Introduction

2. Materials and methods

3. In vivo study

4. Biochemical estimation

5. Statistical analysis

6. Results

8. Discussion

9. Conclusion

Original Research

Optimization of nanostructuredlipid carriers of lamotrigine forbrain delivery: in vitrocharacterization and in vivoefficacy in epilepsyTausif Alam, Jayamanti Pandit, Divya Vohora, Mohd Aqil, Asgar Ali &Yasmin Sultana†

†Jamia Hamdard (Hamdard University), Department of Pharmaceutics, Faculty of Pharmacy,

New Delhi, India

Objective: The aim of the present work was to investigate the efficacy of

nanostructured lipid carriers (NLCs) to enhance the brain targeting of lamotri-

gine (LMT) following intranasal (IN) administration.

Methods: Formulation was optimized using four-factor three levels Box--

Behnken design to establish the functional relationships between variables

on responses, that is, particle size, entrapment efficiency (EE) and percentage

cumulative drug release of LMT-loaded NLCs. NLCs were evaluated for particle

size, surface morphology, %EE and in vitro release and ex vivo permeation.

The developed formulation was subjected to stability study, in vivo efficacy

and scintigraphic study in Wistar rat model.

Results: The NLCs had a mean particle size of 151.6 ± 7.6 nm, polydispersity

index of 0.249 ± 0.035, zeta potential of 11.75 ± 2.96 mV and EE of 96.64 ±

4.27%. The drug release from NLCs followed Fickian diffusion with a flux

value of 11.73 µgcm-2h-1. Sustained drug concentration was obtained in

NLCs carrying LMT after IN administration after 24 h. g scintigraphy studies

further proved high accumulation of drug in brain.

Conclusion: Hence we can conclude that IN administration of LMT NLCs in rats

is able to maintain higher brain concentration of LMT compared to IN and

oral drug solution.

Keywords: brain delivery, epilepsy, intranasal delivery, lamotrigine, nanostructured lipid carriers

Expert Opin. Drug Deliv. [Early Online]

1. Introduction

Lamotrigine (LMT) exhibits broad spectrum efficacy and tolerability and is a goodcandidate for monotherapy in patients inadequately controlled or experiencingunacceptable side effects with other antiepileptic drugs. Compared to phenytoinand carbamazepine, LMT is associated with lesser incidences of adverse effectssuch as asthenia, dizziness and somnolence and does not negatively impact cognitivefunction [1].

Currently, LMT is available in tablet form for oral delivery. The intravenousdelivery of anticonvulsants is the most rapid way of suppressing epileptic convul-sions. It is not a desirable route due to its invasive nature and requires skilledprofessional for administration. Hence, drug delivery strategies that wouldovercome the obstacles presented by barriers such as blood--brain barrier (BBB)and the blood--cerebrospinal fluid (CSF) barrier are to be adopted.

10.1517/17425247.2014.945416 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1All rights reserved: reproduction in whole or in part not permitted

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Overexpression of P-glycoprotein in BBB limits brain accessof LMT [2]. So, it require high drug dose to attain therapeuticlevel in the brain but this attempt increases the blood concen-tration of LMT and thus its adverse effect. Due to this, intrana-sal (IN) route can be the most promising way of deliveringdrugs to the brain. Nasal delivery has many advantages in termsof its noninvasiveness, accessibility, ease of administration andpatient compliance [3]. Hence, a drug administrated into thenasal cavity and deposited on the olfactory mucosa shouldhave a good chance to reach the CSF, on diffusion across themucosa. Recent studies confirm the better bioavailability ofCNS therapeutics via nasal route [4-9].Lipid nanoparticles are promising strategy for drug delivery

to the brain, due to its rapid uptake by the brain, bioacceptabil-ity, biodegradability and less toxicity compared to the poly-meric nanoparticles. Indeed, feasibility in scale-up andabsence of burst effect make them promising carriers for drugdelivery [6,10]. Nanostructured lipid carrier (NLC) is animproved generation of lipid nanoparticles and is preferredover solid lipid nanoparticles due to its potential limitationslike low loading capacity and drug expulsion during storage [11].Objectives of this study were to use response surface meth-

odology (RSM) in conjunction with Box--Behnken design toestablish the functional relationships between four operatingvariables that is, solid lipid concentration (X1), ratio of liquidlipid to total lipid (X2), surfactant concentration (X3) andsonication time (X4) on responses of particle size (Y1), entrap-ment efficiency (EE) (Y2) and percentage cumulative drugrelease (Y3) of LMT-loaded NLC, respectively. Optimizedformulation was administered through IN route to deliverdrug in the brain to improve its clinical utility and therapeuticefficacy.

2. Materials and methods

2.1 MaterialsLMTwas kindly gifted by Kopalle Pharma Chemicals Pvt. Ltd,Hyderabad, India. Glyceryl dibehenate (Compritol 888 ATO),Cutina CP (Cetyl Palmitate), Glycerol distearate (type I) (Pre-cirol ATO 5), PEG-8 beeswax (Apifil) and Caprylic/Capric tri-glyceride (Labrafac CC) were kindly gifted by Gattefosse,France. Almond oil, castor oil, olive oil, oleic acid (OA) andTween 80 were procured from central drug house, Mumbai,India. Poloxamer was procured from BASF, Mumbai,India. Dialysis membrane-70 (molecular weight cut-off[MWCO] = 12,000 -- 14,000 Da), LA 393-1 MT was pur-chased from Hi Media, Mumbai, India. HPLC grade acetoni-trile, methanol and water were procured fromMerck India Ltd,India. All other reagents were of analytical reagents grade andwere used without any further purification.

2.2 Excipients screeningLipids showing maximum solubility of drug were selected forNLC preparation. Solubility was checked in glyceryl mono-stearate (GMS), Compritol 888 ATO, Cutina CP, Precirol

ATO 5 and Apifil. In 10 ml beaker, 1 g of lipid was takenand kept on magnetic stirrer maintained at 80

�C. Drug with

small increment was added and stirred for 30 min after eachaddition. Loss of transparency gave the saturation solubilityof drug in lipid.

Solubility of drug in various oils (olive oil, OA, castor oil,Labrafac CC, almond oil) was determined by taking 1 mloil in 5 ml glass vial. Excess drug was added to the oil andkept on biological shaker for 48 h. It was centrifuged at5000 rpm for 15 min. Supernatant was taken and dissolvedin methanol and quantified UV-spectrophotometrically (UV1700, Shimadzu, Japan) at lmax of 307.5 nm.

Surfactants were selected by assessing the particle size andstability of the placebo NLC formulation prepared with dif-ferent surfactants. Stability of prepared NLC was assessedbased on the phase separation in placebo NLCs.

2.3 Preparation of NLCsLMT-entrapped NLCs were formulated by solvent evapora-tion method as described earlier with slight modification [6,12].Briefly, GMS as solid lipid, OA as liquid lipid and LMT weredissolved in a mixture of ethanol and acetone (1:1) main-tained in water bath at 70

�C to form lipid phase. Ratio of

total lipid and mixture of organic solvent was kept at 1:20.The aqueous phase was formulated by dispersing 2% Tween80 and 0.5% Poloxamer 188 in distilled water maintainedat same temperature as that of lipid phase. Lipid phase wasadded dropwise to aqueous phase and stirred continuouslyat 70

�C until all the organic solvent was evaporated. The

hot lipid emulsion so obtained was probe sonicated at 40%amplitude for 2 min and stirred for 1 h to bring it to roomtemperature.

Final pH was adjusted to pH 1.2 by using 0.1N hydro-chloric acid and centrifuged at 30,000 rpm for 30 min at20

�C, to get NLC pellet. Pellet was then resuspended in

10 ml distilled water containing 2% mannitol and was trans-ferred to freeze-dryer (Heto Drywinner, Denmark) at -70

�C

to lyophilize.

2.4 Experimental designIn order to get an optimized formulation with best size,EE and percentage drug release, a four-factor three levelBox--Behnken design (version 8.0.7.1, Stat Ease, Inc., USA)was used. Independent and dependent variables selected forthe experimental design are shown in Table 1. This RSMrequired 29 experimental runs as show in the Table 2.A nonlinear computer-generated quadratic model definingfour-factor three-level design is given as follows:

(1)Y bo b1X1 b2X2 b3X3 b4X4 b12X1X2

b13X1X3 b14X1X4 b

= + + + + + ++ + 223X2X3 b24X2X4

b34X3X4 b11X12 b22X22 b33X32 b44X42

+ +

+ + + +

where Y is the measured response associated with each factorlevel, b0 is intercept, b1 to b4 are linear coefficient, b12 to

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b34 are linear coefficients, b11 to b44 are quadratic coefficients,X1 to X4 are coded levels of independent variables, X1X2 andXi

2 (i = 1, 2, 3 or 4) represent interaction and quadratic terms,respectively.

2.5 Particle size, polydispersity index and zeta

potential determinationParticle size, polydispersity index (PDI) and zeta potential ofNLC was measured by using a Zetasizer (Nano-ZS, MalvernInstruments, UK) after suitable dilution with distilled waterpreviously filtered with 0.45 µ membrane filter.

2.6 Transmission electron microscopyTransmission electron microscopy (TEM) of NLC was donein All India Institute of Medical Sciences, New Delhi, India.A drop of diluted NLC was negatively stained by uranylacetate, deposited over copper grid coated with carbon film,dried and then observed by TEM (CM 200, Philips BriarcliffManor, NY, USA).

2.7 Differential scanning calorimetryDifferential scanning calorimetry (DSC) (Perkin Elmer, Pyris6 DSC, USA) was done to study the melting behavior of drug,solid lipid, physical mixture of solid lipid and drug and lyoph-ilized NLC. Scanning was done at nitrogen gas flow rate of20 ml/min with scanning speed of 10

�C/min for drug and

5�C/min for physical mixture and lyophilized NLC of

LMT [13].

2.8 EE and drug loadingNLC dispersion was centrifuged at 30,000 rpm for 30 min at20�C and supernatant was estimated for the amount of drugpresent by UV spectrophotometer (UV 1700, Shimadzu,Japan) at lmax of 307.4 nm after suitable dilution [14]. EEcan be calculated by the following formula:

(2)

%EEW1 W2

W1100= ×−

Loading capacity (percentage drug loading [%DL]) wascalculated by using the following formula:

(3)

%( )

DLW W

W1 W Wlipid100= − ×1 2

2− +

where, W1, W2 and Wlipid are the weight of drug added in theformulation, analyzed weight of drug in supernatant andweight of lipid added in formulation, respectively [15].

2.9 In vitro release studyIn vitro release of drug from dialysis membrane (MWCO =12,000 -- 14,000 Da, Hi Media, Mumbai, India) was donein simulated CSF (pH 7.4, 37 ± 2

�C) [16]. About 1 ml of

the formulation and drug solution in 50% v/v dimethyl sulf-oxide (DMSO), containing equivalent to 4.832 mg of drugwere placed in separate dialysis bag and dipped in 100 mlsimulated CSF (containing 25% w/v methanol to maintainsink condition) maintained over magnetic stirrer at50 rpm. About 5 ml of sample was withdrawn at selectedtime interval and was replaced with fresh dissolutionmedium at the same time. Samples were then analyzed spec-trophotometrically after suitable dilution at 307.5 nm. Dataso obtained for the optimized formulation were treated tokinetic models such as zero order, first order, Higuchimodel, Peppas model, Hixson--Crowell model, and Weibullmodel [17].

2.10 Ex vivo permeation studyGoat nasal mucosa was obtained from local slaughter house.It was kept in phosphate buffer (pH 6.4) and then cartilagewas removed carefully. Tissue was stabilized in Franz diffu-sion cell (Logan Instrument Corporation, NJ, USA) contain-ing phosphate buffer of pH 6.4 in both donor and acceptorcompartment of capacity 5 and 11 ml, respectively, with per-meation area of 0.785 cm2. Phosphate buffer from both sideswas removed and separately 1 ml of formulation and 1 ml ofdrug suspension in phosphate buffer of pH 6.4 (as control)containing drug equivalent to 4.832 mg were placed in donorcompartment of Franz diffusion cell. Acceptor compartmentwas filled with phosphate buffer (pH 6.4) containing25% w/v methanol to maintain sink condition. About 2 mlof sample was withdrawn from acceptor compartment atappropriate time interval and was replaced with equal volumeof acceptor media to maintain sink condition. Samples wereanalyzed at 305.8 nm by UV-visible spectrophotometer.

3. In vivo study

3.1 g scintigraphy studyFor scintigraphic assessment, formulation was labeled with99mTc prepared by adding aqueous stannous chloride(SnCl2) as reducing agent as per the equation given below.About 1 ml of LMT-NLC formulation was mixed with0.02 ml of SnCl2 solution (1 mg/ml). The pH was adjustedto 6.5 with 0.5 M sodium bicarbonate buffer solution. To

Table 1. Variables in Box--Behnken design.

Factor Level used, actual (coded)

Low

(-1)

Medium

(0)

High

(+1)

Independent variablesX1 = Solid lipid (%) 3 5 7X2 = Liquid lipid: total lipid 0.1 0.2 0.3X3 = Surfactant (%) 1 4 8X4 = Sonication time (min) 2 5 8Dependent variablesY1 = Size (nm)Y2 = Entrapment efficiency (%)Y3 = % drug release

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this, aqueous 99mTcO4- (2 mci/ml) was added and the whole

suspension was incubated for 15 min. The amount of SnCl2,pH of the solution and encapsulation period are the factorswhich effect maximum labeling, so all these were previouslyoptimized to get maximum labeling efficiency out of it.

The labeling efficiency of the radiolabeled formulation

was determined by paper chromatography using acetone as

mobile phase. Paper (stationary phase) was cut well above

the spot of formulation and the region below the solvent

front. The piece of paper was then put in g counter to check

the radioactivity which ensures that the formulation has

been radiolabeled.About 200 µl of radiolabeled formulation was administered

through IN route to Wistar rat (100 µl in each nostril). The

rat was anesthetized using intramuscular injection of 0.4 mlketamine (50 mg/ml) and placed on the imaging board.Imaging was performed using single-photon-emission com-puterized tomography (LC 75-005, Diacam, Siemens AG,Erlanger, Germany) g camera.

3.2 AnimalsThe study protocol was approved by the Institutional AnimalEthics Committee (949/IAEC/13).

Sixteen male Wistar rats (body weight: 150 -- 200 g) fromcentral animal house facility of Jamia Hamdard were usedfor the in vivo studies. The animals were housed in colonycages with free access to food and water, under uniform hous-ing in the environmentally controlled conditions (22 -- 30�C,12-h light--dark cycle).

Table 2. Observed responses in Box--Behnken design for NLC of lamotrigine.

Formulation

code

Independent variables Dependent variables

X1 (%) X2 X3 (%) X4 (min) Y1(nm) Y2(%) Y3(%)

T1 0 -1 0 -1 181.6 74.52 66.72T2 0 -1 0 +1 177.5 71.2 68.11T3 0 +1 0 -1 151.6 96.64 76.97T4 0 0 +1 +1 154.3 87.25 73.16T5 +1 0 0 -1 202.2 97.32 58.13T6 +1 0 0 +1 194.8 91.64 65.64T7 +1 +1 0 0 186.4 97.88 75.41T8 0 0 0 0 172.4 86.12 73.64T9 0 0 +1 -1 164.2 84.42 72.78T10 0 0 0 0 173.3 85.59 73.25T11 0 0 0 0 179.6 86.72 69.18T12 -1 +1 0 0 137.4 71.46 88.35T13 0 -1 +1 0 183.2 72.9 62.68T14 -1 0 +1 0 141.4 65.45 86.24T15 0 0 -1 -1 212.5 72.51 58.22T16 -1 0 0 +1 148.4 63.65 84.82T17 0 0 0 0 171.1 85.47 74.32T18 0 -1 -1 0 198.2 69.17 59.49T19 -1 -1 0 0 154.6 61.62 82.24T20 0 0 0 0 174.6 84.35 71.61T21 +1 -1 0 0 204.2 91.45 47.38T22 0 0 -1 +1 203.7 67.42 57.79T23 0 +1 +1 0 146.3 91.32 77.49T24 -1 0 0 -1 153.8 67.8 83.31T25 -1 0 -1 0 168.4 60.32 77.40T26 +1 0 -1 0 227.4 71.38 51.22T27 +1 0 +1 0 209.4 89.56 56.64T28 0 +1 -1 0 188.4 73.79 61.43T29 0 +1 0 +1 144.6 89.84 78.21

Note: X1: Solid lipid (%); X2: Ratio of liquid lipid to total lipid; X3: Surfactant concentration (%); X4: Sonication time (min); Y1: Size (nm); Y2: Entrapment

efficiency (%); Y3: Percentage cumulative drug release.

NLC: Nanostructured lipid carrier.

(4)

LMT NLCs mTcOReducation in presence of Sn2

− + 99 4− ⎯ →⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯+( )⎯⎯⎯ 99mTc labeled LMT NLCs( )−

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3.2.1 Induction of maximal electroshock seizureAll rats were given electric shock (when unmedicated)through ear electrode (current 150 mA, duration 0.2 s, fre-quency 60 Hz) 48 h before study. Animals showing tonichind limb extension (THLE) were selected for the study.Animals were divided into four groups each containing fouranimals. Group 1 was control (without any treatment), group2 was given drug solution (50% v/v DMSO) orally, group3 was given drug solution (50% v/v DMSO) via nasal routeand group 4 was given NLC formulation via nasal route.The rats were held from the back in slanted position duringadministration of the preparations through nasal route. Allthe animals were under light ether anesthesia before dosing.Latency and duration of THLE were recorded for each groupafter 1 h and 24 h post-dose.

3.2.2 Collection of plasma and brain homogenateAfter 24 h, animals were anesthetized by ether, and blood wascollected by retro-orbital route in heparinized tube and thenwas centrifuged at 4000 rpm for 15 min. Supernatant soobtained were stored in deep freezer until analysis. Anesthe-tized rats were sacrificed by cervical dislocation; head wasseparated out and skull of the rats was opened. Whole brainwas collected and washed with isotonic phosphate buffer(pH 7.4), weighed and then homogenized in PBS, pH7.4 to make 10% of brain homogenate.

3.2.3 Extraction of drug from plasma and brain

homogenateMethod used for extracting LMT from brain and plasma wasbased on earlier experiment done by Castel-Branco et al. [18].Briefly, to 1 ml plasma, 1 ml of 2 M sodium hydroxide and5 ml ethyl acetate was added and vortexed for 1 min. It wasthen kept on horizontal shaker at 200 oscillations for 20 minand centrifuged at 4000 rpm for 20 min at 15

�C. Upper

organic layer was transferred in 10 ml glass test tube and wasevaporated under stream of nitrogen at 45

�C. Pellet so formed

was reconstituted with 200 µl mobile phase (0.05M potassiumphosphate: acetonitrile: methanol [50:35:15% v/v/v]) filteredthrough 0.25 µ membrane filter and kept for analysis.

To extract drug from brain homogenate, 100 µl of 20%trichloroacetic acid (TCA) was added to 1 ml of brain homog-enate and then centrifuged at 4000 rpm for 20 min at 15

�C.

Supernatant so obtained was treated similarly as for the plasma.

3.2.4 Analysis of LMT in plasma and brain extractThe drug concentration in brain and plasma was analyzed bythe HPLC method reported by Matar et al. with slight modifi-cation [19]. Mobile phase eluted at a flow rate of 1 ml/minthrough C18 column. Retention time was 3.875 min. Detec-tion was monitored at 214 nm by UV detector. The assayshowed good linearity within the concentration range of0.05 to 10 µg/ml with R2 = 0.996. Limit of detection and limit

of quantitation of this method were 22.9 and 69.4 ng/ml,respectively.

4. Biochemical estimation

Maximal electroshock seizure (MES)-induced seizures causeda significant increase in malondialdehyde (MDA) levels anda decrease in glutathione (GSH) levels [20]. So the levels ofGSH and MDA were estimated in the brain homogenate aftertreatments. One more animal group (group 5) was includedfor this study which acted as vehicle control and was treatedwith placebo. No seizure was induced in this particular group.

4.1 GSH estimationGSH was estimated by the method described by Sedlak andLindsay [21]. To 2 ml of 10% brain homogenate, 2.5 ml of0.02 M EDTA was added and shaken vigorously. To 2 mlof this mixture, 4 ml of cold distilled water and 1 ml of50% TCA were added and shaken for 10 min. Thereafter,the content was centrifuged at 3000 rpm for 15 min. About2 ml of the supernatant was mixed with 4 ml of 0.4 M trisbuffer (pH 8.9) and 0.1 ml of 0.01 M 5,5’-dithiobis-(2-nitro-benzoic acid) (DTNB). The absorbance was read within5 min of addition of DTNB at 412 nm against reagent blankwith no homogenate [21,22]. For blank, the homogenate wassubstituted by 2 ml of distilled water.

Total GSH (tissue) was calculated using the followingformula:

(5)Co = /A D×( ) e

where A is absorbance at 412 nm, D is dilution factor, " is themolar extinction coefficient (" = 13600 M-1cm-1) and Co isthe concentration of GSH expressed as micromole/gram ofwet tissue.

4.2 MDA estimationDetermination of brain MDA content was performed by athiobarbituric acid (TBA) method given by Ohkawa et al.[23]. To 1 mL of 10% brain homogenate, 1 ml of 30% TCAand 1 ml of 0.8% TBA reagent were added. The tubes werecovered with aluminum foil and kept in a shaking waterbath for 30 min at 80�C. After 30 min, the tubes were takenout and kept in ice-cold water for 30 min. The content wasthen centrifuged at 3000 rpm for 15 min. The absorbanceof the supernatant was read at 535 nm at room temperatureagainst appropriate blank. Blank consisted of 1 ml distilledwater, 1 ml of 30% TCA and 1 ml of 0.8% TBA. The contentof MDA, expressed as n moles formed per gram of the wettissue, was calculated using the following formula:

(6)C = A D× /( ) e

where A is absorbance, D is dilution factor and " is extinctioncoefficient (1.56 � 105 M-1cm-1).

Optimization of NLCs of LMT for brain delivery

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5. Statistical analysis

All the results are reported as mean ± standard deviation.Comparison between the two groups were made usingStudent’s t-test, and comparison for more than two groupswere done by using one-way ANOVA, followed byBonferroni’s post hoc test. In all the cases p < 0.05 was consid-ered statistically significant.

6. Results

6.1 Excipients screeningBased on the maximum solubility of drug in solid and liquidlipid, GMS (60 mg/g) and OA (33.9 mg/ml) was selected assolid and liquid lipid, respectively. About 2% Tween 80 and0.5% Poloxamer 188 were selected as surfactant and as co-surfactant, respectively, based on the stability of formulationprepared with different surfactants.

6.2 Experimental design

6.2.1 Fitting of data to the modelWhen the observed responses were fit to 29 runs, it wasobserved that best-fitted model for all the three dependentvariables was quadratic model with coefficient of correlationnearly equal to 1.As shown in Table 3, value of the correlation coefficient

(R2) of Equation 1 was found to be 0.9564, indicating goodfit. The ‘predicted R-squared’ of 0.7602 is in reasonableagreement with the ‘adjusted R-squared’ of 0.9129. ‘Adequateprecision’ measures the signal-to-noise ratio. A ratio > 4 isdesirable; the ratio of 19.165 indicates an adequate signal.The value of the correlation coefficient (R2) of Equation 2was found to be 0.9461, indicating good fit (Table 3). The‘predicted R- squared’ of 0.6930 is in reasonable agreementwith the ‘adjusted R-squared’ of 0.8922. The signal-to-noiseratio of 14.944 indicates an adequate signal. R2 for Equation3 was 0.9614, indicating good fit (Table 3). The ‘predictedR-squared’ of 0.7996 is in reasonable agreement with the

‘adjusted R-squared’ of 0.9227. The signal-to-noise ratio of17.069 indicates an adequate signal.

6.2.2 Response analysis for optimization6.2.2.1 Particle sizeSmallest and largest mean particle sizes observed for LMT-loaded NLCs were 137.4 for T12 and 227.4 for T26, respec-tively (Table 2). As the concentration of total lipid increases,the size also increases, but with increase in the amount of liq-uid lipid, the total lipid size decreases. Increase in the amountof liquid lipid reduces particle size. Surfactant causes a signif-icant decrease in size as its concentration increases to 2.5%,but as it rises to 4%, size reduces but not to a significant level.Increase in sonication time was found to have nonsignificantdecrease in particle size (Figure 1A).

6.2.2.2 Entrapment efficiencyHighest and lowest EEs were 97.88 and 61.62% for T7 andT19, respectively (Table 1). It was found that with the increasein concentration of total lipid, EE increases significantly. Incre-ment of the lipid content also reduces the escaping of drug intothe external phase [15,24]. An increased EE was observed withthe increase in the amount of liquid lipid (Figure 1B). It wasobserved that increase in surfactant concentration first increasesand then decreases the %EE of drug in NLC. Increase in son-ication time leads to nonsignificant decrease in EE.

6.2.2.3 In vitro drug releaseMaximum and minimum drug releases were 88.35 and47.38% for T12 and T21, respectively (Table 1). As shownin Figure 1C, increase in the amount of total lipid causes sig-nificant decrease in percentage drug release. Increase in surfac-tant concentration up to 2.5% increases drug release; a furtherincrease in surfactant concentration decreases the drug release.Increase in sonication time was found to have nonsignificanteffect on percentage cumulative drug release.

Based on the response of various dependent variables inrespect to composition of independent variable, formulationT3 was selected as optimized formulation for the study.

Table 3. Summary of results of regression analysis for responses Y1, Y2 and Y3.

Quadratic model R2 Adjusted R2 Predicted R2 Adequate

precision

SD CV (%)

Response (Y1) 0.9564 0.9129 0.7602 19.165 7.08 4.02Response (Y2) 0.9461 0.8922 0.6930 14.944 3.79 4.77Response (Y3) 0.9614 0.9227 0.7996 17.069 2.98 4.25Regression equations of the fitted model*

Y1 = +174.20 + 26.70x1 -- 12.05x2 -- 16.65x3 -- 3.55x4 -- 0.15x1x2 + 2.25x1x3 -- 0.50x1x4 -- 6.77x2x3 -- 0.73x2x4 -- 0.27x3x4 +2.51x1

2 -- 6.79x22 + 11.14x3

2 -- 2.39x42 ……………… (1)

Y2 = +85.65 + 12.41x1 + 6.67x2 + 6.36x3 -- 1.85x4 -- 0.85x1x2 + 3.26x1x3 -- 0.38x1x4 + 3.45x2x3 -- 0.87x2x4 + 1.98x3x4 -- 4.99x12 --

0.96x22 -- 7.99x3

2 -- 0.65x42 ………………………… (2)

Y3 = +72.40 -- 12.33x1 + 5.94x2 + 5.29x3 + 0.97x4 + 5.48x1x2 -- 0.85x1x3 + 1.50x1x4 + 3.22x2x3 -- 0.038x2x4 + 0.20x3x4 +1.32x1

2 -- 0.22x22 -- 6.46x3

2 -- 0.29x42 ………………………... (3)

*Only the terms with statistical significance are included.

SD: Standard deviation.

T. Alam et al.

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Siz

eE

ED

R

100

90

80

70

60

50

80

70

60

50

40

0.30

0.250.20

0.150.10 3.00

4.00

4.00

1.001.60

2.202.80

3.405.00

6.007.00

4.003.00

2.00

5.006.00

7.008.00

40

50

60

70

80

90

0.300.25

0.200.15

0.10 1.001.60

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6.007.00

8.00

3.004.00

5.00

6.007.00

0.30

0.25

0.20

0.15

0.10A:TL D:ST

D:ST

D:ST

A:TL

A:TL

B:LL:TL

B:LL:TL

B:LL:TL

3.00

4.00

5.00

6.00

7.00

2.00

100

90

80

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1.001.60

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7.008.00

6.00

120

140

160

180

200

220

220

180

160

140

120

200

A.

B.

C.

C: Surfactant

C: Surfactant

C: Surfactant

Figure 1. Three-dimensional plots showing relative effects of different process parameters on (A) particle size, (B) EE and

(C) drug release.A:TL: Total lipid; B:LL:TL: Liquid lipid to total lipid ratio; D:ST: Sonication time; EE: Entrapment efficiency.

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6.3 Particle size, PDI, zeta potential and EE

determinationMean particle size, PDI and EE of optimized formulation(T3) were 151.6 ± 7.6 nm, 0.249 ± 0.035, 96.64 ± 4.27%,respectively. Loading capacity was calculated to be 8.81 ±1.2% and zeta potential was 11.75 ± 2.96 mV.

6.4 Transmission electron microscopyTEM image (Figure 2A) of NLC shows particle of uniformsize distribution and roughly spherical in shape. Thisuniform particle size distribution was in accordance withthe small PDI (0.249) as obtained by photon correlationspectroscopy.

Peak = 219.467 °C

Peak = 64.005 °C

Peak = 62.498 °C

Peak = 158.939 ˚CPeak = 59.387 °C

Area = 277.586 mJDelta H = 277.586 J/g

Area = 1690.962 mJDelta H = 1690.962 J/g

Area = 802.822 mJDelta H = 160.564 J/g

Area = 229.073 mJDelta H = 229.073 J/g

Area = 122.286 mJDelta H = 122.286 J/g

Hea

t fl

ow

en

do

UP (

mW

)

A.

A

B

B.

D

C

Temprature (°C)

Figure 2. A. TEM photomicrograph of lamotrigine-NLC and B. Differential scanning calorimetric profiles of (A) lamotrigine, (B)

GMS, (C) lamotrigine--GMS physical mixture and (D) formulation are shown.GMS: Glyceryl monostearate; NLC: Nanostructured lipid carriers; TEM: Transmission electron microscopy.

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6.5 Differential scanning calorimetryDSC thermogram is shown in the Figure 2B. The thermal anal-ysis of DSC curve C, revealed that the drug and GMS mixture(1:10) shows no extra peak when compared to DSC curve ofdrug (Figure 2B(A)) and GMS alone (Figure 2B(B)). So, thedrug and GMS combination is compatible. Drug peak in theformulation (NLC) disappeared showing that drug is fullypresent inside the lyophilized drug-loaded NLC (DSC curveD). Recrystallization index of lyophilized NLC was found tobe 13.54%, showing reduction in crystalline state of GMS.

6.6 In vitro release studiesIn vitro drug release study showed 66.97 ± 4.76% drug releasein 24 h, whereas almost all drug was released from drug solu-tion within 8 h. Drug release from NLC was biphasic showingburst release (20.42% in 1 h) followed by sustained release,

showing 21.12% drug release in next 11 h (Figure 3A). Dataobtained by in vitro release experiment were fitted to variousrelease models and value of coefficient of correlation wasfound nearer to 1 (R2 = 0.973) for zero-order release, so thismodel was selected as best fit model. Drug solution was justtaken to show that enhancement of solubility of drug by anymeans does not have any effect on the achievement of sus-tained release profile of drug. The drug release from NLCwas found to be Fickian diffusion [17,25].

6.7 Ex vivo permeation studiesThe steady-state flux and permeability coefficient through drugsolution and formulation after 24 h of study was found to be5.815 µgcm-2h-1, 1.163 � 10-3 cm-2h-1 and 11.73 µgcm-2h-1,2.346 � 10-3 cm-2h-1, respectively. The flux and permeabilitycoefficient of NLC were almost double than those obtained

Cu

mu

lati

ve d

rug

rel

ease

(%

)

100

90

80

70

60

50

40

30

20

10

00 2 4 6 8 10 12 14 16 18 20 22 24

A.

NLC Drug solution

Time (h)

B.

i ii iii

Figure 3. A. In vitro release study of drug-loaded NLC and drug solution, B. g scintigraphy images of rat following intranasal

administration at (i) 30 min, (ii) 2 h and (iii) 6 h.NLC: Nanostructured lipid carriers.

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for drug solution. Student’s t-test reveals that difference amongthe results were statistically significant at p < 0.05.

7 In vivo study

7.1 g scintigraphy studyRadioactivity accumulation of NLC of LMT in brain isclearly seen in the Figure 3B (i), (ii) and (iii) after 0.5, 2 and6 h of study, respectively. After 0.5h, radioactivity can beseen only in the nostrils. After 2 and 6h, radioactivity accu-mulation in the brain is clearly visible. A good amount ofactivity was also noticed in esophagus and in the abdominalregion which could be due to absorption of a part of the for-mulation from gastrointestinal tract [26,27].

7.2 In vivo efficacy study in animalsAs shown in Table 4, rats were fully protected after 1 h ofdosing by IN formulation (NLCs). Oral and IN solutionshad significant effect on latency and duration of THLE ascompared to control and each other as well. One-wayANOVA showed significant difference among groups inlatency (F[2,9)] = 44.63, p < 0.0001) and duration (F[2,9]= 299.4, p < 0.001). On post hoc analysis, differences betweencontrol and each treated group were also found significant.After 24 h, when MES seizure was induced, 75% of ratswere protected against THLE; however, unprotected ratsshowed highly significant difference in latency and durationof THLE. One-way ANOVA showed significant differenceamong groups in latency (F[3,12] = 19.13, p < 0.0001) andduration (F[3,12] = 510.8, p < 0.001). Post hoc analysisshowed significant difference between each group and con-trol. IN solution had shown significant improvement inlatency and duration of THLE.

7.3 Analysis of LMT in brain and plasmaThe LMT concentration in brain and plasma is given inthe Figure 4A. Estimated concentrations of LMT in brain withdifferent treatment were 1746.443, 1261.756, 342.365 ng forIN formulation, IN Solution, oral solution, respectively.Thus, concentration of LMT in brain was in the following

order, IN formulation > IN solution > oral solution. Theseresults revealed the effectiveness of the formulation in control-ling THLE even after 24 h.

7.4 Biochemical estimationThe GSH and MDA concentrations in different LMTformulation-treated rats (either intranasally or orally) was stud-ied and compared to the vehicle control as shown in Figure 4B

and C.The vehicle control rat showed GSH concentration of

780.36 ± 47.79 nmol/g of wet tissue. The GSH concentrationwas significantly lower (312.78 ± 11.27 nmol/g of wet tissue)in MES control as compared to vehicle control rat (p < 0.01).The GSH concentration in rat treated with LMT solutionadministered either orally or IN was 397.05 ± 38.97 and412.01 ± 14.93 nmol/g of wet tissue, respectively, whichshowed statistically nonsignificant result among each other(p > 0.05) but relatively significant result as compared tovehicle control and MES-induced seizure in rats (p < 0.05).The GSH concentration in rats treated with formulationadministered intranasally was 529.44 ± 41.82 nmol/g of wettissue. These results illustrated that rats treated with formula-tion showed significantly higher GSH level as compared tothe MES rats (p < 0.01), whereas it shows nonsignificantresults in vehicle control rat (p > 0.05).

MDA level as shown in Figure 4Cwas 115.82 ± 16.37 nmol/gof wet tissue in vehicle control. MES group showed highlysignificant increase (413.23 ± 33.57 nmol/g of wet tissue) inMDA content as compared to vehicle control (p < 0.01). Smalldecrease in MDA content was found in rat treated with LMTsolution administered either orally or intranasally, having360.55 ± 15.39 and 355.17 ± 21.31 nmol/g of wet tissue,respectively, which shows nonsignificant result among eachother (p > 0.05) but relatively significant result as comparedto vehicle control (p < 0.05). However, LMT-NLC-treatedrat showed significantly lower MDA content (139.76 ±128.96 nmol/g of wet tissue) as compared to MES-treated rats(p < 0.01). The study showed that MDA content was signifi-cantly lower in LMT-NLC-treated rats [27,28].

Table 4. Effects of lamotrigine on MES-induced seizure in mice.

Groups MES induction post-1 h of dosing MES induction post-24 h of dosing

THLE THLE

Latency

(s)

Duration

(s)

% Protection

(s)

Latency

(s)

Duration

(s)

% Protection

(s)

Control 2.55 ± 0.22 13.78 ± 0.57 0 2.715 ± 0.53 12.44 ± 0.25 0Oral solution (24.3 mg/kg) 3.90 ± 0.21* 2.46 ± 0.25z 0 3.727 ± 0.46§ 11.07 ± 0.37§ 0IN solution (4.83 mg/kg) 5.13 ± 0.13{ 2.03 ± 0.24z 0 4.585 ± 0.43* 0 9.5 ± 0.48* 0IN formulation (4.83 mg/kg) Nil Nil 100 5.46{ 1.45z 75

Data represent mean ± SEM (n = 4), *p < 0.01, zp < 0.0001, §p <0.05, {p < 0.001, as compared to control group.

IN: Intranasal; MES: Maximal electroshock seizure; THLE: Tonic hind limb extension.

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500

450

400

350

300

250

200

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100

50

0

900

800

700

600

500

400

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0

3000

2500

2000

1500

1000

500

0

Brain Brain Brain Plasma

Vehiclecontrol

MEScontrol

Plasma Plasma

IN formulation

IN formulation

IN solution

IN solution

Oral solution

Oralsolution

Vehiclecontrol

MEScontrol

IN formulationIN solutionOralsolution

Co

nce

ntr

atio

n (

ng

/ml)

A.

B.

C.

GS

H (

nm

ol/g

of

wet

tis

sue)

MD

A (

nm

ol/g

of

wet

tis

sue)

Figure 4. (A) Concentration of LMT in brain and plasma after MES test, effects of different LMT formulations via different

routes on GSH (B) and MDA (C) levels in different group of rats are shown.GSH: Glutathione; IN: Intranasal; LMT: Lamotrigine; MDA: Malondialdehyde; MES: Maximal electroshock seizure.

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8. Discussion

8.1 OptimizationThe LMT-loaded NLCs were developed and optimized byfour-factor three-level Box--Behnken statistical design. We

have tried different parameters such as lipids concentration,lipid ratio, surfactant concentration and sonication time to

obtain small particle size, maximum loading and encapsula-tion efficiency with optimum drug release. Fitting of thedata for observed responses to various models, it was observed

that the best-fitted model for all the four dependent variableswas the quadratic model (Table 3).With the increase in total lipid concentration an increase in

particle size can be due to decrease in emulsifying efficiency of

surfactant and increase in particle agglomeration [15]. As there

is increase in the amount of liquid lipid, a reduction in parti-

cle size was observed because of decrease in the viscosity of

formulation and surface tension inside NLC [29].An increase in total lipid concentration increases EE sig-

nificantly by providing more space for the accommodation

of drug particle, as increment of the lipid content also

reduces the escaping of drug into the external phase [15,24].

Also, as concentration of solid lipid (GMS) increases with

increase in total lipid concentration it solubilizes drug due

to presence of monoglycerides, diglycerides and triglycerides

of fatty acid [30]. A significant increase in EE was obtained

with the increased liquid lipid concentration as more of

the drug particles were entrapped inside the oil-enriched

lipid core. This can be explained due to more imperfection

in highly ordered solid lipid crystal due to incorporation of

spatially incompatible liquid lipid [11]. Increase in the

amount of total lipid causes significant decrease in cumula-

tive percentage drug release. This is due to the fact that

increase in lipid concentration increases the size of the nano-

particle, thereby decreasing the effective surface area available

to interact with the releasing medium and hence decrease in

drug release.Surfactant concentration significantly affects the EE and

drug release. It first increases and a decrease in the EE wasobserved after further rise in surfactant concentration. Thisdecrease may be attributed to entrapment of surfactant mole-cule itself into the NLC at higher concentration of surfactant.An increase in the drug release characteristics till particularconcentration of surfactant is due to its ability to decreasethe particle size, a further increase in surfactant concentrationcauses decrease in EE which decreases the amount of drugrelease. Nanodispersion can be stabilized by two differentmechanisms: steric stabilization and electrostatic repulsion.Tween 80 and Poloxamer 188 being non-ionic stabilizersstabilize the system by steric stabilization due to the presenceof dense hydrophobic tail which does not allow particles tocome closer to each other and thus particle agglomeration isprevented [31]. These surfactants also impart negative zetapotential of 11.75 ± 2.96 mV to the nanoparticulate system

which causes electrostatic repulsion of nanoparticles andthey are prevented to form aggregate.

8.2 Differential scanning calorimetryLyophilized NLCs did not show peak for the drug as the drugis fully present inside the lyophilized drug-loaded NLC. Thisresult can be explained by Kelvin effect which states that amaterial (LMT) will melt at a temperature lower than thematerial (GMS) isolating that particle (here that particle isLMT) whose melting point has depressed from 219.46

�C

to < 59.387�C, which is the melting point of GMS. Decrease

in melting point of NLC (Gibbs--Thomson effect, the equa-tion derived from Kelvin equation) as compared to pureGMS was due to reduced crystallinity of GMS. This resultis due to the blend of oil with solid lipid and is in accordancewith that obtained by Kovacevic et al. and Tiwari and Pathak[13,29].

8.3 In vitro release studyInitial burst release was due to presence of drug-enrichedliquid lipid content in the outer shell of NLCs. Sustainedrelease pattern was due to drug solubilized liquid lipid coreof NLCs [32]. Formulation was showing zero-order releaseprofile by following Fickian diffusion.

8.4 Ex vivo permeation studiesThe high drug permeation effect of formulation compared todrug suspension was due to the nanosized particle of formula-tion and presence of surfactants on the surface of NLC, whichare well known as penetration enhancer. Poloxamer 188 hasbeen reported to form adhesion with mucous through second-ary forces followed by their diffusion into the mucous wherethey undergo molecular interaction [33].

8.5 g scintigraphy studyg scintigraphy studies showed accumulation of formulation inrat brain. Radioactivity of formulation after 6 h showedlonger retention time of drug in brain. This long retentiontime was further confirmed by MES seizure test and estima-tion of LMT in brain. A significant activity was also presentin gastrointestinal tract because of intragastric ingestion of apart of formulation by way of throat [26].

8.6 In vivo efficacy studyIN solution had shown significant improvement in latencyand duration of THLE. This effect is due to direct nose-to-brain drug delivery as compared to oral solution which showshepatic first-pass metabolism and has to cross gastrointestinalbarrier and BBB before finally reaching the brain. Brain alsoexpresses P-glycoprotein which further limits the brain accessof LMT [2]. These results demonstrate the efficacy of INformulation after 1 and 24 h of seizure induction. This maybe due to the fact that nanoformulations increase nose-to-brain drug delivery as compared to drug solution of equiva-lent dose as shown earlier by in vitro permeation study.

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Protection of the drug from efflux back into the IN cavitymay also be the reason for this effect of nanoparticles as solu-tions are rapidly cleared from the nasal cavity. The effect ofnanoformulation maintained till 24 h was due to its sustaineddrug release profile. Tween 80 and Poloxamer 188 alsoenhance the penetration of drug through nasal mucosa.P-glycoprotein expressed in olfactory epithelium causes drugefflux which is prevented by Poloxamer 188, resulting inhigher drug concentration in brain after IN administrationof formulation as compared to solution [6]. Hence it showsthe effectiveness of the developed formulation in controllingTHLE even after 24 h.

8.7 Biochemical estimationThe increased GSH content and decrease in TBA contentcould be due to antioxidant property of LMT which hasbeen already established in earlier reports [34,35].

9. Conclusion

Optimized LMT-loaded NLC (T3) was developed usingBox--Behnken statistical experiment design which gives theoptimum concentration of lipids and surfactants in the for-mulation to get the minimum size and maximum EE. Drugrelease was found to follow zero-order kinetics with Fick’slaw of diffusion according to Korsmeyer--Peppas model. Thepresent study clearly demonstrated that IN administration of

optimized nanoformulation in rats is efficient in maintainingthe effect of LMT with much higher brain concentration dueto direct brain targeting and increased residence time of drugin brain as compared to IN and oral administration of drugsolution. The higher protective effect of IN administrationcompared to oral administration was seen with lower doses(one-fifth of oral dose) indicating a remarkable delivery routefor targeting brain with an appropriate dosage form design.

Acknowledgment

The authors are also thankful to Kopalle Pharma ChemicalsPvt. Ltd (Hyderabad, India) for providing drug sample andto Gattefosse (St-Priest, France) for providing gift samplesof solid lipids.

Declaration of interest

The authors are grateful to University Grant Commission,Government of India, New Delhi for financial assistance.The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financialinterest in or financial conflict with the subject matter ormaterials discussed in the manuscript. This includes employ-ment, consultancies, honoraria, stock ownership or options,expert testimony, grants or patents, received or pending, orroyalties.

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2002;54:557-66

AffiliationTausif Alam1, Jayamanti Pandit1, Divya Vohora2,

Mohd Aqil1, Asgar Ali1 & Yasmin Sultana†1

†Author for correspondence1Jamia Hamdard (Hamdard University),

Department of Pharmaceutics, Faculty of

Pharmacy, M. B. Road, New Delhi 110062,

India

Tel: +91 9811539489;

E-mail: yas2312003@yahoo.com2Jamia Hamdard (Hamdard University),

Department of Pharmacology, Faculty of

Pharmacy, M. B. Road, New Delhi 110062,

India

T. Alam et al.

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