Formulation and evaluation of Nimodipine-loaded solid lipid nanoparticles delivered via lymphatic transport system

Colloids and Surfaces B: Biointerfaces 97 (2012) 109–116

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Formulation and evaluation of Nimodipine-loaded solid lipid nanoparticles delivered via lymphatic transport system Shailesh S. Chalikwar ∗ , Veena S. Belgamwar, Vivek R. Talele, Sanjay J. Surana, Mrunal U. Patil Department of Pharmaceutics and Quality Assurance, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur 425405, Maharashtra, India

a r t i c l e

i n f o

Article history: Received 29 December 2011 Received in revised form 12 April 2012 Accepted 18 April 2012 Available online 25 April 2012 Keywords: Palmitic acid Intestinal lymphatic targeting High pressure homogenisation Oral bioavailability Pharmacokinetic evaluation

a b s t r a c t In an attempt to increase oral bioavailability and to target intestinal lymphatic transport system, Nimodipine loaded solid lipid nanoparticles (NMD-SLNs) were prepared. Nimodipine (NMD) is highly lipophilic antihypertensive drug having (log P 3.41) and 13% oral bioavailability. NMD-SLNs were prepared with palmitic acid (PA), poloxamer 188 and soya lecithin as a lipid, surfactant and co-surfactant respectively using high pressure homogeniser. A (23 ) factorial design was employed; three factors such as lipid, surfactant and co-surfactant concentration were used. Parameters investigated includes particle size, polydispersity index (PDI), zeta potential, drug entrapment efficiency (EE %), drug loading efficiency (LE %), in vitro drug release of the SLNs. Optimised SLNs (F8) had particle size of 116 ± 21 nm, zeta potential of −10 ± (−4.8) mV, EE of 93.66 ± 9.72% and cumulative drug release of 87.52 ± 2.54% in 10 h. The pharmacokinetic study of optimised SLNs conducted in male Albino Wistar rats showed 2.08-fold increase in relative bioavailability than that of NMD solution, when administered orally. Differential scanning calorimetry study revealed absence of any chemical interaction between NMD and PA while SEM study confirmed the non spherical shape of optimised SLNs. Accelerated stability studies showed that there was no significant change in the mean particle size and PDI after storage at 25 ± 2 ◦ C/60 ± 5% RH for the period of three months. Due to enhanced bioavailability, these NMD-SLNs are considered to be promising vehicles for oral delivery. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Solid lipid nanoparticles (SLNs) are worthy carriers for oral delivery of lipophilic and to some extent for hydrophilic drug candidates [1]. SLNs, important colloidal carriers for lipophilic drugs are composed of a solid lipid (i.e. those lipids which are solid at room temperature and at the body temperature too) core in which drug is entrapped to target it to a specific organ or system of the human body [2–4]. Recently, SLNs have been used orally aiming at targeted delivery and enhancing oral bioavailability. Previously reported research work showed enhanced bioavailability of poorly water soluble drugs when encapsulated in lipid-base vehicles [2,5,6]. Why is there such increasing interest in SLNs? What are the possible reasons? The main reasons for the interest in these carriers can be summed up in few words: components, size and related narrow distribution, biocompatibility, and different possible administration routes [7].

∗ Corresponding author at: Department of Pharmaceutics & Quality Assurance, R. C. Patel Institute of Pharmaceutical Education & Research, Karwand Naka, Shirpur 425405, Dhule, Maharashtra, India. Tel.: +91 2563 255189; fax: +91 2563 251808; mobile: +91 9850104541. E-mail address: [email protected] (S.S. Chalikwar). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.04.027

SLN not only has combine advantages of colloidal drug carrier systems such as liposomes, polymeric nanoparticles and emulsions but also avoid the drawbacks associated with respective carrier system [5,8]. Proposed advantages include compatible degradation in in vivo, possibility of controlled drug release and drug targeting, and avoidance of organic solvents. Polymeric nanoparticles have disadvantages like residual contamination, possible toxicity, lack of suitable sterilisation method and stability, which can be avoided by SLNs. The mechanism of bioavailability enhancement by SLNs is attributed to adhesive properties that make them adhere to gut wall and release the drug exactly where it should be absorbed [9]. Lymphatic uptake was found to be the major absorption mechanism for drugs encapsulated in SLN [10]. One of the prominent advantages of SLNs prepared by high pressure homogenisation is its ability for production at industrial scale up [11]. Nimodipine (NMD), a calcium channel blocker, is used in the treatment of senile dementia for prophylaxis of the vascular hemicranias, stroke, and hypertension [12,13]. NMD belongs to Biopharmaceutical Classification System (BCS) “Class II” drugs having plasma half life of 7–8 h. In clinical studies, NMD has shown low oral bioavailability of 4–13% in healthy human subjects mainly due to its low solubility and extensive first pass metabolism [14]. Therefore, 60 mg of its oral dose has to be administered every 4 h, such high dosing frequency further results into patient inconvenience

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and incompliance. An alternative intravenous route of administration could have provided greater bioavailability, however, it causes the patient incompliance such that patient needs to be hospitalised and its safety problems [15]. SLNs administered through oral route have advantages with lymphatic drug transport which bypasses first pass metabolism and higher amount of drug concentration is subjected for lymphatic exposure than that attained in systemic blood [16]. Lipid nanoparticles passes through digestive and absorption phase followed by circulatory uptake for enhancing the oral bioavailability of the lipid encapsulated drug [17]. Literature survey revealed that NMD loaded SLNs (NMD-SLNs) have not been reported so far. Low solubility, extensive first pass metabolism, low bioavailability, high dosing frequency, highly lipophilic nature (log P 3.41) and half life makes NMD promising drug for formulation of SLN’s to enhance its oral bioavailability. Delivering the existing drug molecules, by advanced technology will be the more preferred strategy so as to enhance its therapeutic efficiency. A 23 factorial design was employed for preparing different batches of NMD-SLNs. Oral bioavailability of NMD for optimised formulation was investigated. Sterilisation is of utmost importance and stringent requirement for development of parenteral and ophthalmic drug delivery systems [18,19]. However, in the present investigation authors have attempted to develop Nimodipine loaded SLNs for oral administration targeting to intestinal lymphatics, for which sterilisation is not required [2,5,6]. 2. Experimental 2.1. Materials NMD was a generous gift from Micro Labs Ltd., Hosur, Tamilnadu, India. Palmitic acid (PA) and Poloxamer 188 were purchased from HiMedia Lab. Pvt. Ltd., Mumbai, India. Soya lecithin was received as a gift sample from Phospholipid GmbH, Germany. All other chemicals used for the study were of HPLC grade. Water used in all the studies was distilled and filtered through 0.22 ␮m nylon filter paper before use. 2.2. Partitioning behaviour of NMD in different lipids NMD (20 mg) was dispersed in a blend of melted lipid (2 g) and hot distilled water (2 mL), after which it was shaken for 45 min over a hot water bath. Aqueous phase was then separated after cooling by centrifugation at a speed of 3000 rpm and analysed by HPLC (Agilent 1200 Series) for NMD content to study its partitioning behaviour with different lipids [20].

2.4. Experimental design To design the formulation of lipid based nanoparticles, it was essential to recognise the parameters in the formulation as these variables can affect the properties of desired formulation. Various batches of NMD loaded SLNs were planned based on the 23 factorial designs, to study the effect of different variables on its properties. Independent variables include lipid concentration (X1 ), surfactant concentration (X2 ) and concentration of co-surfactant (X3 ). All independent variables, their levels along with actual and coded values of these variables are shown in (Table 1). Respective particle size of the SLN (Y1 ) and (EE %) drug entrapment efficiency (Y2 ) were taken as response parameters as the dependent variables. It was observed that the best-fitted models were linear for both particle size and EE. 3FI model was studied because it showed the interactive effect of independent variables on responses which were not seen in case of non-linear model and 3FI had P-value < 0.05 which is considered to be statistically significant. Evaluation of predicted R2 was pursued to confirm best suitability of model [22]. Three dimensional surface plots were used to ascertain the relationship between variables and responses. For optimisation, the desirability function of particle size was in the minimum level while that of entrapment efficiency was in the maximum level [23]. 2.5. Determination of particle size, PDI and zeta potential of the NMD-SLNs Mean diameters of the SLNs and PDI as a measure of the width of particle size distribution were determined by photon correlation spectroscopy (PCS) using a Zetasizer (Nano ZS 90, Malvern Instruments, UK). SLN formulations were diluted with double distilled water to get optimum 50–200 kilo counts per second (kcps) for measurements. Based on the Smoluchowski equation, the surface charge of the SLNs was determined by measuring the zeta potential of SLNs using the same equipment. Zeta potential measurements were run at 25 ◦ C with electric field strength of 23 V/m [24,25]. 2.6. Determination of entrapment efficiency (EE %) and loading efficiency (LE %) of the NMD-SLNs Percent EE was calculated by determining the amount of nonencapsulated NMD in the aqueous surfactant solution [26]. The aqueous medium was separated by using the cooling centrifuge (Remi Instruments Ltd., Mumbai, India). Volume of 1.5 mL of the SLN dispersion of NMD was placed in the Eppendorf tubes and speed of centrifuge was kept at 15,000 rpm for 30 min at 4 ◦ C. The concentration of NMD in the aqueous phase was determined using UV–visible spectrophotometer (UV 1700, Shimadzu, Japan) at max 237 nm. Values of EE % and LE % were calculated using Eqs. (1) and (2), respectively [27].

2.3. Preparation of NMD loaded SLNs NMD-SLNs were prepared using high pressure homogenisation method. PA concentration at different levels (Table 1) and 50 mg of drug was utilised. Drug was dissolved in the melted lipid phase and this solution was then dissolved in 20 mL of ethanol. Poloxamer 188 and soya lecithin as a surfactant, co-surfactant respectively were dissolved in 80 mL of distilled water. The lipid phase, preheated to 10 ◦ C above the melting point of lipid, was transferred to the hot aqueous surfactant solution under stirring at 500 rpm for 20 min using a mechanical stirrer (Remi Instruments Ltd., Mumbai, India). This resulted into formation of a pre-emulsion which was then subsequently homogenised in a high pressure homogeniser (Panda 2K, Niro Soavi Ltd., Italy) for 5 homogenisation cycles at a pressure of 600 bar. Later the mixture was cooled to room temperature yielding NMD-SLN [21].

EE % =

LE % =

total mass of NMD − mass of NMD in supernatent × 100 total mass of NMD (1) total mass of NMD − mass of NMD in supernatent × 100 total mass of lipid (2)

2.7. Differential scanning calorimetry (DSC) studies DSC analysis was carried out using differential scanning calorimeter (DSC 1 STARe System, Mettler-Toledo, Switzerland) at a heating rate of 10 ◦ C/min in the range of 35–300 ◦ C. DSC studies

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Table 1 Independent variables along with their code, levels and respective average size, PDI, zeta potential, EE % and LE % of different batches of NMD-SLNs (n = 3). These results are mean ± standard deviation. Code

X1 (mg)

X2 (%)

X3 (%)

Particle size (nm)

F1 F2 F3 F4 F5 F6 F7 F8

15 15 15 25 25 25 25 15

1.50 0.50 0.50 0.50 0.50 1.50 1.50 1.50

0.50 0.50 1.50 1.50 0.50 0.50 1.50 1.50

367.9 162.9 199.7 394.2 525.5 429.9 452.8 116.0

± ± ± ± ± ± ± ±

12 25 18 31 34 47 38 21

PDI 0.265 0.054 0.019 0.237 0.173 0.192 0.417 0.330

Zeta potential (mV) ± ± ± ± ± ± ± ±

0.08 0.05 0.70 0.06 0.05 0.07 0.21 0.06

−16.3 −12.8 −10.3 −10.3 −17.7 −9.63 −15.7 −10.0

± ± ± ± ± ± ± ±

(−6.4) (−2.3) (−4.8) (−9.1) (−2.8) (−1.0) (−3.7) (−4.8)

% EE 89.67 84.45 90.8 92.96 91.36 93.96 90.66 93.66

% LE ± ± ± ± ± ± ± ±

2.8 9.2 7.18 11.2 8.71 1.73 17.4 9.7

15.7 14.0 15.3 10.2 8.13 9.39 9.06 9.39

± ± ± ± ± ± ± ±

3.8 2.8 5.7 8.6 8.9 2.5 5.2 2.5

X1 = lipid concentration in mg/mL (high level-25, low level-15); X2 = surfactant concentration in % (high level-1.5, low level-0.5); X3 = co-surfactant concentration in % (high level-1.5, low level-0.5).

were conducted for NMD, bulk PA, physical mixture of NMD with PA in 1:1 ratio and freeze dried NMD-SLNs of the optimised batch [28]. 2.8. Scanning electron microscopy (SEM) Morphology of SLNs was studied by using scanning electron microscope. The optimised formulation was kept onto metal plate and dried under vacuum to form a dry film which was then observed under the scanning electron microscope (LEO 440i; Leo Electron Microscopy Ltd., Cambridge, UK). 2.9. Determination of in vitro drug release from NMD-SLNs The drug release from NMD-SLNs was performed in phosphatebuffer saline (PBS) solution (pH 6.8), using the dialysis-bag method [29]. Dialysis membrane with a pore size of 2.4 nm and molecular weight cut off between 100 kDa was used. The membrane was soaked in PBS solution for 12 h prior usage. The NMD-SLN dispersion was placed in a dialysis bag and sealed at both ends. The dialysis bag was placed in a beaker containing 250 mL of dissolution medium phosphate buffer (PBS pH 6.8) at 37 ± 2 ◦ C and magnetically stirred at 100 rpm [3]. Samples were withdrawn at predetermined time intervals of 60 min for 10 h and sink condition was maintained by replacing with fresh pre-warmed PBS solution at same temperature. The content of NMD in the samples was determined UV spectrophotometer (1700, Shimadzu, Japan) at max 237 nm [30,31]. 2.10. In vivo studies 2.10.1. Oral administration of drug to rats Male Albino Wistar rats weighing 250 ± 20 g, maintained on pellet diet and water ad libitum were used for oral bioavailability studies. The protocol for all animal experiments was approved by CPCSEA and the local animal ethical committee. Animals were kept in the animal house at an ambient temperature of 25–30 ◦ C and 45–55% RH with 12 h of dark and light cycle. Animals were fasted overnight but had free access to drinking water. Two groups of male Wistar rats each with 6 animals were used. The first group (control group) received 8 mg/kg of NMD solution, and in the second group (treated group), the suspension containing the same amount of NMD-SLNs (F8) was administered. The formulations were administered orally with the aid of a syringe and infant feeding tube. Blood samples were collected by retro-orbital venous plexus puncture with the aid of glass capillary at 0, 15, 30, 45, 60, 120 and 240 min post oral dose. All samples were collected in heparinised Eppendorf tubes and centrifuged (Remi Instruments Ltd., Mumbai) at 5000 rpm for 15 min; plasma was collected and stored at −22 ◦ C until analysis [5,6].

2.10.2. Quantification of rat plasma by HPLC NMD concentration in the plasma samples was determined using HPLC method [32]. An Agilent HPLC system with UV detector was used which is composed of quaternary pump and diode array detector. Chromatography was performed on a reverse-phase C18 column (Eclipsed XDB 5 ␮m, 4.6 mm × 150 mm, Singapore) using Methanol–water (65:35) as mobile phase. Elution was performed isocratically at 40 ◦ C at a flow rate of 1.0 mL/min at 237 nm. The data was acquired and processed by means of Ezchrome Elite software. 2.10.3. Data analysis The non compartmental model was considered as a best suited model for calculation of the different pharmacokinetic parameters. The maximum plasma concentration (Cmax ) and time of its occurrence (Tmax ) were directly computed from the plasma concentration against time plot [33]. The trapezoidal method was used to calculate the concentration–time curve from time 0 min to 240 min (AUC0→240 ). The Kinetica 5 (Thermo Fisher Scientific Demo version) software was employed for study. Relative bioavailability (F) was calculated with reference to oral NMD solution. 2.11. Accelerated stability studies The lyophilised powder sample of final optimised formulation (batch F8) was utilised for carrying out accelerated stability studies according to International Conference on Harmonisation (ICH) Q1A (R2) guidelines and previously reported method by Souto et al. [2]. For the products stored in refrigerator ICH guidelines suggested long term stability at 5 ± 3 ◦ C and accelerated stability study at 25 ± 2 ◦ C/60% RH ± 5% RH (relative humidity). Accelerated stability study was performed with the prime aim to assess the stability of SLNs at 25 ± 2 ◦ C/60 ± 5% RH with respect to particle size, PDI and EE. Freshly prepared freeze dried powder was filled in 3 different amber coloured glass vials, sealed and placed in stability chamber (CHM-10S, Remi Instruments Ltd., Mumbai, India) maintained at 25 ± 2 ◦ C/60 ± 5% RH for a period of total 3 months. The dried powder samples subjected for stability test were re-dispersed in distilled water and analysed with a sampling interval of 1 month for particle size, PDI and EE of the NMD-SLNs over 3 month period [2]. 2.12. Statistical analysis Design-Expert software (version 8.0.1; Stat-Ease, Inc., Minneapolis, MN, USA) was utilised for statistical analysis and graph plotting. The results of one-way analysis of variance (ANOVA) for the dependent variables were utilised for the selection of model which could be considered significant for both the response variables.

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Table 2 Summary of results of regression analysis for responses Y1 and Y2 and analysis of variance for particle size and EE. Parameters Particle size (Y1 ) Model Residual Total EE (Y2 ) Model Residual Total

DF

SS

MS

F

P-value

R2

SD

Coefficient of variation (%) 13.26

3 4 7

24,520.05 7705.06 32,225.11

8097.26 1926.27 10,023.53

26.27 – –

0.0043 significant – –

0.951 – –

43.89

4 3 7

101.57 2.04 103.61

25.39 0.68 26.07

37.29 – –

0.003 significant – –

0.980 – –

0.83

0.910

DF, degrees of freedom; SS, sum of square; MS, mean sum of square; F, Fischer’s ratio.

3. Results and discussion 3.1. Partitioning behaviour of NMD Calibration curve of NMD in mobile phase was utilised for estimating the concentration of the NMD in the aqueous phase. Partition coefficients obtained were 26.96 ± 2.63, 57.21 ± 1.46, 68.02 ± 1.02, 112.37 ± 4.32, 167.34 ± 1.89, 358.47 ± 2.53, 450.72 ± 1.59 and 634.41 ± 2.48 for trimyristin, tristearin, tripalmitin, glycerol monostearate, tribehenin, lauric acid, stearic acid and PA, respectively. From the above results, it can be claimed that the highly lipophilic drug NMD is soluble to greater extent in PA than any other lipid used. Thus, the use of PA as the lipidic phase for the study of NMD loaded SLNs was considered to be the best. 3.2. Preparation of NMD encapsulated SLNs The high pressure homogenisation method is the only method which can be utilised for the industrial production of the SLNs. Ethanol (20%) was incorporated for homogenous distribution of NMD inside lipid phase (PA). The homogeniser pressure was optimised to 600 bar for 5 cycles. 3.3. Optimisation data analysis for the NMD-SLNs Responses observed for eight formulations prepared were fitted to various models using Design-Expert® software 8.0.1. It was observed that the linear models were best-fitted for both particle size and EE. 3FI model was studied because it showed the interactive effect of independent variables on responses that non-linear model does not show. All values of R2 , SD and % coefficient of variation are depicted in (Table 2). The regression equation generated for each response were given as, Y1 = 331.11 + 119.49X1 + 10.54X2 − 40.44X3 − 19.79X1 X2 + 13.34X1 X3 − 16.81X2 X3 + 55.36X1 X2 X3

Y2 = 90.41 + 1.82X1 + 0.27X2 + 1.86X3 − 0.19X1 X2 − 2.28X1 X3 − 0.38X2 X3 − 0.85X1 X2 X3 Results of ANOVA in (Table 2) for the dependent variables demonstrated that the model was significant for both the response variables. 3.4. Response surface plots Response surface plots are important three dimensional surface curves for studying the interaction patterns. Three dimensional

response surface plots generated at different levels by the DesignExpert® software are presented in (Fig. 1) for the studied responses, i.e. particle size and EE. In all presented figures, the third factor was kept at a constant level. From Fig. 1, it can be concluded that the particle size goes on decreasing as the lipid concentration (A) increases and surfactant concentration (B) decreases. It is summarised that lipid (A) and surfactant (B) concentration has significant effect on the EE, i.e. with the increase in lipid concentration and decrease in surfactant concentration, increases the EE. Response surface plots revealed that the lipid and surfactant concentrations were statically significant. The observation was in agreement with the results obtained from previous study on particle size and EE [18]. 3.5. Particle size, polydispersity index (PDI) and zeta potential (ZP) of NMD-SLNs Particle size, PDI and zeta potential of the NMD loaded SLNs are reported in (Table 1). The mean particle sizes of the total eight formulations were found to be in the range of 116–525.5 nm. An optimised formulation based on quantitative and sophisticated arrangements was selected and formulated. PDI values of the batches F1–F8 was in the range of 0.019–0.417. The SLNs to be preferably transported using lymphatic transport; their size should be in the range of 0.3 and 1.0 ␮m [34,35]. Particle size obtained in present study displayed the aforesaid range. This particle size range did not cause any changes in lymphatic uptake of nanoparticles. An electric charge on each particle surface forms electrical barrier which results in ‘Repulsion phenomenon’ is the zeta potential of a particular formulation. Zeta potential of all batches was found to be towards negative side in the range of (−17.7 to −9.63 mV). This can be explained by the fact that PA used for the preparation of NMD-SLNs possesses negative charge and there may be the presence of the PA residues which might have contributed for zeta potential. The pH of the resulting formulation was found to be 6.5 ± 0.8. 3.5.1. Effect of surfactant concentration on the particle size, PDI and zeta potential Different surfactant concentrations have shown very predominant effect on the particle size of the NMD-SLNs. The particle size was found to decrease with increase in the surfactant concentration at a constant amount of lipid (i.e. inversely proportional). A higher surfactant concentration reduces the surface tension and facilitates partitioning during homogenisation [36]. The decrease in the particle size is accompanied by a tremendous increase in the surface area. Thus, the process of primary coverage of the newer surfaces competes with the agglomeration of the uncovered surface. Hence, increase in the surfactant concentration in the primary dispersion resulted in rapid coverage of the newly formed particle surface (Fig. 2). As the particle size of the SLNs is towards lower side, the absorption through peyer’s patches will be higher and thus, plasma concentration achieved will be higher. There was

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Fig. 1. Response surface plots (a) for the lipid concentration and surfactant concentration on particle size and (b) for the lipid concentration and surfactant concentration on EE.

no significant effect seen on the PDI and zeta potential with the change in surfactant concentration. 3.5.2. Effect of lipid concentration on the particle size The concentration of lipid which solubilises the drug in formulation has significant effect on the particle size of the SLNs. Fig. 2 clearly represents that, with the increase in PA concentration from 300 mg to 500 mg, the mean particle size of the formula-

tion was also increased in each case (i.e. directly proportional). Batches F4, F5, F6, F7 which had the highest concentrations of lipid, comparatively showed the larger particle size (in the range of 400–500 nm) than that of batches having low lipid concentrations (in the range of 100–300 nm). This can be explained on the basis of inability of the poloxamer 188 to completely cover the surface of the NMD-SLNs which resulted in the larger particle sizes.

Fig. 2. Effect of (a) surfactant concentration on particle size, (b) lipid concentration on particle size, (c) co-surfactant concentration on particle size and (d) effect of lipid concentration on EE %.

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Fig. 3. DSC thermograms of (A) NMD, (B) physical mixture of NMD and PA, (C) bulk PA, and (D) NMD loaded lyophilised SLNs.

3.5.3. Effect of co-surfactant concentration on the particle size The co-surfactant used in the present study was soya lecithin which in combination with poloxamer 188 reduces the particle size and also stabilised the SLN dispersion. The co-surfactant soya lecithin along with the poloxamer 188 can effectively cover the SLNs and thus, prevents the particle agglomeration. Increment in the quantity of soya lecithin showed decreases in particle size. This can be well observed from the batch F8 and batch F1. Batch F8, which had the highest co-surfactant concentration, i.e. 1.5% (w/v) and had the particle size of 116 nm while in batch F1, which had the co-surfactant concentration of 0.5% (w/v) had the particle size of 367.9 nm. In both the batches, lipid and surfactant concentration was kept constant. Thus, this implies that as there is increase in the co-surfactant concentration, the particle size is significantly reduced (Fig. 2). 3.6. Entrapment efficiency (EE %) and LE (%) of NMD-SLNs The EE is mainly dependent on the nature of the drug and the lipidic phase in which the drug is encapsulated. As the NMD is a lipophilic drug and its solubility is also greater in the PA (conclusion drawn from partition coefficient study), the EE obviously was found to be higher, i.e. in the range of 84–93%. The LE was found to be in the range of 8–15% (Table 1). 3.6.1. Effect of lipid concentration on the EE (%) The effect of lipid concentration on the EE % was found to be significant (Fig. 2). Increasing concentration of the lipid showed increasing EE % of the NMD in the PA. This can be explained on the fact that as there was increase in the lipid phase, more amount of the lipid was available for the NMD to dissolve. Also, NMD has the highest partition coefficient for the PA which may be another reason for the higher EE, with increase in lipid concentration. Thus, it might ultimately decrease the dose in the formulation and attain the higher plasma concentration through the lymphatic transport system.

Fig. 4. SEM of NMD loaded SLNs optimised batch F8.

interaction between NMD and PA. The DSC thermogram of lyophilised SLNs showed two endothermic peaks. The first endotherm was observed at around 62 ◦ C due to existence of lipidic phase PA and second at around 165 ◦ C due to the presence of mannitol which was used as cryoprotectant during the process of freeze drying. NMD endothermic peak was not observed in DSC thermogram of lyophilised SLNs owing to the molecular inclusion of NMD in the lipid matrix of PA. This suggests that NMD exists in amorphous state in SLNs. Similar results have been reported earlier by [18,20].

3.7. DSC study DSC studies were performed to confirm the absence of drugexcipients interactions. The DSC thermograms of NMD, bulk PA, physical mixture of NMD and PA in 1:1 ratio and drug loaded freeze dried SLNs are depicted in Fig. 3. The DSC thermogram showed a sharp endothermic peak for NMD at 125 ◦ C and for PA at 62 ◦ C. No considerable shift in the position of endothermic peaks was observed in the DSC thermogram of physical mixture of NMD and PA. Thus, DSC study concluded the absence of any chemical

Fig. 5. In vitro drug release profile (optimised batch F8) of NMD-SLNs (n = 3, mean ± SD).

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Fig. 6. The mean concentration–time curve obtained in blood plasma after a single oral dose (8 mg/kg) of NMD solution and NMD-SLNs (n = 6, mean ± SD).

3.8. Scanning electron microscopy (SEM)

3.11. Accelerated stability studies

SEM photomicrograph of the NMD loaded SLNs is shown in Fig. 4. Optimised formulation showed non-spherical shaped particles with an average particle size of 150 nm. These SLNs can preferably be absorbed by peyer’s patches and transported to intestinal lymphatic, bypassing the liver, thereby, enhancing the oral bioavailability of the drug NMD.

Accelerated stability studies were conducted on optimised SLNs (F8) using the particle size, PDI and EE as the prime parameters. There was a slight increase in the particle size during the three months storage from the 116 ± 5.62 nm to 128.56 ± 7 .80 nm with not much change in PDI (i.e. initially which was 0.330 ± 0.06 and that after 3 months it was 0.348 ± 0.01). The EE (%) of the optimised batch initially was found to be 93.66 ± 9.72% while that after 3 months was found to be 91.67 ± 0.14% indicating that the drug can retain within the nanoparticles for the sufficient period of time. On storage of the SLNs (F8), there were no significant alterations in the size, PDI and EE % of the SLNs. Hence, they were found to be stable at 25 ± 2 ◦ C/60 ± 5% RH for a total period of 3 months.

3.9. In vitro drug release In vitro drug release study of the NMD loaded SLNs showed the sustained release behaviour in 6.8 pH phosphate buffer (Fig. 5). Almost all formulation batches have shown the burst release with the 40% of drug release within first two hours followed by the sustained release from the NMD loaded SLNs. The presence of the free NMD in the external phase and on the surface of the nanoparticles may be the reason for this burst release. The low solubility of the NMD in aqueous phase could be the reason for the slow release of the drug from the lipid matrices after initial burst release. The increase in lipid concentration had significant effect on the NMD release which prolonged the release of the NMD from SLNs. It may be due to the equal distribution of drug within the lipid matrix and good entrapment of NMD in PA. Thus, with the use of the NMD loaded SLNs, it is possible to achieve the loading dose of the drug due to initial burst release and followed by maintenance dose due to the sustained release. In fact this will prevent the fluctuations in the drug plasma level.

3.10. In vivo pharmacokinetic study The in vivo pharmacokinetic study was carried out on male Albino Wistar rats using the single dose (8 mg/kg) of the NMD solution and the optimised batch (F8) NMD-SLNs have shown significant results. The plasma concentration against time curves of NMD in the plasma medium is depicted in Fig. 6. After the single oral dose administration of the NMD-SLNs, the relative bioavailability was found to be 2.08-fold higher than that obtained after the administration of single oral dose of NMD solution. The value of Tmax obtained was found to be earlier (28 min) for the NMD-SLNs than that of the NMD solution (42 min). Thus, it can be concluded from the results that the NMD loaded SLNs were successfully transported to intestinal lymphatics.

4. Conclusions The NMD encapsulated SLNs were successfully prepared by hot high pressure homogenisation technique. In vivo pharmacokinetic study of optimised batch on the rat model revealed that SLNs are promising carriers for transporting the lipophilic drugs to the intestinal lymphatic region which resulted in increased oral bioavailability of the drug along with reduction in dosing frequency and better patient compliance. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements The authors are very much thankful to All India Council for Technical Education (AICTE), New Delhi, India, for the financial support of this project under research promotion scheme (Project no. 8023/BOR/RID/RPS–132/2009-10). The authors are also grateful to the Management of R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur for providing the necessary facilities, support and encouragement. References [1] M. Muchow, P. Maincent, R.H. Muller, Lipid nanoparticles with a solid matrix (SLN® , NLC® LDC® ) for oral drug delivery, Drug Dev. Ind. Pharm. 34 (2008) 1394–1405.

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[2] M.R.A. Alex, A.J. Chacko, S. Jose, E.B. Souto, Lopinavir loaded solid lipid nanoparticles (SLN) for intestinal lymphatic targeting, Eur. J. Pharm. Sci. 42 (2011) 11–18. [3] L. Serpe, R. Canaparo, M. Daperno, R. Sostegni, G. Martinasso, E. Muntoni, L. Ippolito, N. Vivenza, A. Pera, M. Eandi, M.R. Gasco, G.P. Zara, Solid lipid nanoparticles as anti-inflammatory drug delivery system in a human inflammatory bowel disease whole-blood model, Eur. J. Pharm. Sci. 39 (2010) 428–436. [4] S.A. Wissing, O. Kayser, R.H. Muller, Solid lipid nanoparticles for parenteral drug delivery, Adv. Drug Deliv. Rev. 56 (2004) 1257–1272. [5] J. Varshosaz, M. Minayian, E. Moazen, Enhancement of oral bioavailability of pentoxifylline by solid lipid nanoparticles, J. Liposome Res. 20 (2010) 115–123. [6] J. Varshosaz, M. Tabbakhian, M.Y. Mohammadi, Formulation and optimization of solid lipid nanoparticles of buspirone HCl for enhancement of its oral bioavailability, J. Liposome Res. 20 (2010) 286–296. [7] M.R. Gasco, Lipid nanoparticles: perspectives and challenges, Adv. Drug Deliv. Rev. 59 (2007) 377–378. [8] A.A. Date, M.D. Joshi, V.B. Patravale, Parasitic diseases: liposomes and polymeric nanoparticles versus lipid nanoparticles, Adv. Drug Deliv. Rev. 59 (2007) 505–521. [9] D. Zhao, X. Shuyu, Z. Luyan, W. Yan, W. Xiaofang, Z. Wenzhong, Preparation and in vitro, in vivo evaluations of norfloxacin loaded solid lipid nanopartices for oral delivery, Drug Deliv. 18 (2011) 441–450. [10] H. Yuan, J. Chen, Y.Z. Du, F.Q. Hu, S. Zeng, H.L. Zhao, Studies on oral absorption of stearic acid SLN by a novel fluorometric method, Colloids Surf. B: Biointerfaces 58 (2007) 157–164. [11] R.H. Muller, K. Mader, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art, Eur. J. Pharm. Biopharm. 50 (2000) 161–177. [12] Y.S.R. Krishnaiah, P. Bhaskar, V. Satyanarayana, Formulation and evaluation of limonene-based membrane-moderated transdermal therapeutic system of nimodipine, Drug Deliv. 11 (2004) 1–9. [13] S.A. Giannakou, P.P. Dallas, D.M. Rekkas, N.H. Choulis, In vitro evaluation of nimodipine permeation through human epidermis using response surface methodology, Int. J. Pharm. 241 (2002) 27–34. [14] D. Yang, J. Zhu, Y. Zheng, L. Ge, Preparation, characterization, and pharmacokinetics of sterically stabilized nimodipine-containing liposomes, Drug Dev. Ind. Pharm. 32 (2006) 219–227. [15] F.Q. Hu, Y. Zhang, Y.Z. Du, H. Yuan, Nimodipine loaded lipid nanospheres prepared by solvent diffusion method in a drug saturated aqueous system, Int. J. Pharm. 348 (2008) 146–152. [16] N.L. Trevaskis, W.N. Charman, C.J.H. Porter, Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update, Adv. Drug Deliv. Rev. 60 (2008) 702–716. [17] S. Chakraborty, D. Shukla, B. Mishra, S. Singh, Lipid – an emerging platform for oral delivery of drugs with poor bioavailability, Eur. J. Pharm. Biopharm. 73 (2009) 1–15. [18] R. Cavalli, O. Caputo, M.E. Carlotti, M. Trotta, C. Scarnecchia, M.R. Gasco, Sterilization and freeze-drying of drug-free and drug-loaded solid lipid nanoparticles, Int. J. Pharm. 148 (1997) 47–54. [19] C. Schwarz, W. Mehnert, J.S. Lucks, R.H. Muller, Solid lipid nanoparticles (SLN) for controlled drug delivery. I. Production, characterization and sterilization, J. Control. Release 30 (1994) 83–96.

[20] V. Venkateswarlu, K. Manjunath, Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles, J. Control. Release 95 (2004) 627–638. [21] M.D. Triplett II, J.F. Rathman, Optimization of ␤-carotene loaded solid lipid nanoparticles preparation using a high shear homogenization technique, J. Nanopart. Res. 11 (2009) 601–614. [22] C.F.J. Wu, M. Hamada, Experiments: Planning, Analysis and Parameter Design Optimization, Wiley Publisher, NY, USA, 2000, pp. 37–42. [23] J. Varshosaz, S. Ghaffari, M.R. Khoshayand, F. Atyabi, S. Azarmi, F. Kobarfard, Development and optimization of solid lipid nanoparticles of amikacin by central composite design, J. Liposome Res. 20 (2010) 97–104. [24] A. Helbok, C. Decristoforo, G. Dobrozemsky, C. Rangger, E. Diederen, B. Stark, R. Prassl, E.V. Guggenberg, Radiolabeling of lipid-based nanoparticles for diagnostics and therapeutic applications: a comparison using different radiometals, J. Liposome Res. 20 (2010) 219–227. [25] L.H. Reddy, K. Vivek, N. Bakshi, R.S.R. Murthy, Tamoxifen citrate loaded solid lipid nanoparticles (SLNTM ): preparation, characterization, in vitro drug release, and pharmacokinetic evaluation, Pharm. Dev. Technol. 11 (2006) 167–177. [26] M.A. Kalam, Y. Sultana, A. Ali, M. Aqil, A.K. Mishra, K. Chuttani, Preparation, characterization, and evaluation of gatifloxacin loaded solid lipid nanoparticles as colloidal ocular drug delivery system, J. Drug Target. 18 (2010) 191–204. [27] N.P. Aditya, S. Patankar, B. Madhusudhan, R.S.R. Murthy, E.B. Souto, Artemetherloaded lipid nanoparticles produced by modified thin-film hydration: pharmacokinetics, toxicological and in vivo anti-malarial activity, Eur. J. Pharm. Sci. 40 (2010) 448–455. [28] Z. Rahman, A.S. Zidan, M.A. Khan, Non-destructive methods of characterization of risperidone solid lipid nanoparticles, Eur. J. Pharm. Biopharm. 76 (2010) 127–137. [29] S. Chakraborty, D. Shukla, P.R. Vuddanda, B. Mishra, S. Singh, Utilization of adsorption technique in the development of oral delivery system of lipid based nanoparticles, Colloids Surf. B: Biointerfaces 81 (2010) 563–569. [30] U.M. Dhana lekshmi, G. Poovi, N. Kishore, P.N. Reddy, In vitro characterization and in vivo toxicity study of repaglinide loaded poly (methyl methacrylate) nanoparticles, Int. J. Pharm. 396 (2010) 194–203. [31] A.Z. Muhlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for controlled drug delivery – drug release and release mechanism, Eur. J. Pharm. Biopharm. 45 (1998) 149–155. [32] M. Qian, J.M. Gallo, High-performance liquid chromatographic determination of the calcium channel blocker nimodipine in monkey plasma, J. Chromatogr. B: Biomed. Sci. Appl. 578 (1992) 316–320. [33] R. Pandey, S. Sharma, G.K. Khuller, Oral solid lipid nanoparticle-based antitubercular chemotherapy, Tuberculosis 85 (2005) 415–420. [34] J.S. Alexander, V.C. Ganta, P.A. Jordan, Gastrointestinal lymphatics in health and disease, Pathophysiology 17 (2010) 315–335. [35] N. Hussain, V. Jaitley, A.T. Florence, Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics, Adv. Drug Deliv. Rev. 50 (2001) 107–142. [36] S. Kheradmandnia, E. Vasheghani-Farahani, M. Nosrati, F. Atyabi, Preparation and characterization of ketoprofen-loaded solid lipid nanoparticles made from beeswax and carnauba wax, Nanomedicine: NBM 6 (2010) 753–759.