Engineering of a nanostructured lipid carrier for the poorly water-soluble drug, bicalutamide: Physicochemical investigations

Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

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Engineering of a nanostructured lipid carrier for the poorly water-soluble drug, bicalutamide: Physicochemical investigations Dipak D. Kumbhar, Varsha B. Pokharkar ∗ Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune 411 038, Maharashtra, India

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

 We developed a nanostructured lipid carrier (NLC) for the poorly watersoluble drug, bicalutamide (BCT).  BCT phase transition occurred during the NLC processing and was subsequently studied by DSC, PXRD and Raman analysis.  The presence of hydrophilic surfactants was significant to modulate BCT release from NLC.  Developed NLC showed potential to entrap the poorly water-soluble BCT and revealed good stability for six months.

a r t i c l e

i n f o

Article history: Received 13 July 2012 Received in revised form 14 October 2012 Accepted 18 October 2012 Available online 26 October 2012 Keywords: Nanostructured lipid carrier Bicalutamide Poorly water-soluble High-pressure homogenization Crystalline Form I Raman analysis

a b s t r a c t The purpose of this study was to develop an optimized nanostructured lipid carrier (NLC) for bicalutamide (BCT), a poorly water-soluble drug, and to investigate its phase transition behavior during the NLC processing. BCT loaded NLCs (BCT-NLCs) were prepared using a hot high-pressure homogenization (HPH) technique. Factorial design (23 ) was used to identify the key formulation variables influencing particle size, percent drug encapsulation, and zeta potential of the NLC. The optimized batch (NLC-2) revealed spherical morphology with a smooth surface under scanning electron microscopy (SEM). NLC-2 achieved a high drug encapsulation of 98.48 ± 0.70% and demonstrated good stability for six months. Drug–lipid interaction was investigated using Fourier transform infrared spectra (FT-IR) and proton nuclear magnetic resonance (1 H NMR). BCT phase transition occurred during the NLC processing and BCT crystalline Form I was identified in NLC-2. The same was confirmed by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Raman analysis. The in vitro release study of NLC-2, revealed peppas release kinetics with Fickian diffusion (n < 0.5) as drug release mechanism. The presence of hydrophilic surfactants was significant to modulate BCT release from NLC-2. Finally, NLCs made of Precirol® ATO 5 (solid lipid) and triacetin (oil) posses the potential to entrap the poorly water-soluble drug, bicalutamide and the system can be tailor-made to meet the desired drug release. This may provide better prospects for the oral delivery of bicalutamide. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +91 20 25437237; fax: +91 20 25439383. E-mail address: [email protected] (V.B. Pokharkar). 0927-7757/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2012.10.031

Prostate cancer is the second most common type of newly diagnosed cancer and the sixth leading cause of cancer deaths among men worldwide [1]. The treatment of prostate cancer generally includes surgery, radiation therapy, and the hormonal therapy [2]. Bicalutamide [BCT] is an orally active non-steroidal antiandrogen

D.D. Kumbhar, V.B. Pokharkar / Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

used to treat prostate cancer [3,4]. However, its pharmacokinetic after oral administration is highly variable. This could be due to its high lipophilicity (log P; 2.92) and the poor aqueous solubility (5 mg/L) [5]. Moreover, a high pKaa value of 12 is responsible for the poor solubility of the BCT at physiological pH [6]. This results in reduced gastro-intestinal (GI) absorption of BCT and thereby may account for its oral bioavailability of 60%. Thus, the low aqueous solubility is a major factor limiting the oral bioavailability of BCT molecule. Therefore, there is a need to develop strategies that would allow overcoming the drug solubility issue and thereby maximizing the therapeutic benefit. Thus far, a few strategies, such as solid dispersion [5], ␤-cyclodextrin inclusion complexation [6], and nanosizing [7], reported to improve dissolution rate of BCT. Lipid-based systems, such as solid lipid nanoparticles (SLNs) and the nanostructured lipid carriers (NLCs) are the promising carriers for a lipophilic molecule [8]. This could be because of their potential to increase the solubility of lipophilic drug [8,9]. SLNs loaded with a poorly water-soluble drug have been reported for the oral administration by few researchers [10–13]. NLCs are the second-generation of lipid nanoparticles developed from a blend of solid lipid and liquid lipid (oil). NLCs offer many advantages, such as good biocompatibility, controlled drug release and the possibility of production on large industrial scale [14]. Moreover, a high drug loading efficiency can be achieved with the use of NLC [15,16]. Literature search revealed no reports on the NLC system for BCT. Therefore, the primary objective of the current work was to develop an optimized nanostructured lipid carrier for the encapsulation of bicalutamide, and further to investigate the influence of process and formulation variables on its performance and characteristics, using a 23 factorial design. The NLCs were prepared by hot homogenization technique using a high-pressure homogenizer (HPH). BCT is a flexible molecule that undergoes conformational polymorphism and exists in two crystalline polymorphs, Form I and Form II [17]. Though, both (Form I and II) have the same chemical composition, they differ in physicochemical properties due to the difference in their internal crystal structures [18]. Besides, a low aqueous solubility of the drug can be related with its ability to undergo polymorphic transition [19]. Therefore, another aspect of the study was to investigate the BCT phase transition during the NLC processing. For this purpose, a detailed investigation was performed on the drug–lipid melt and the BCT loaded NLC using DSC, PXRD and Raman analysis. Moreover, the presence of both proton donors and proton acceptors makes BCT a favorable site for the hydrogen bonding. The functional groups, such as amide (N H) and hydroxyl (O H) and the functional groups, such as carbonyl (C O) and sulfonyl (O S O) can act as proton donors and proton acceptors, respectively [20]. Therefore, to understand the microenvironment and a possible hydrogen bonding interaction between the drug and the carrier lipid, proton nuclear magnetic resonance (1 H NMR) investigation was performed. The surface morphology of the NLC was investigated by scanning electron microscope (SEM). The developed NLC was further evaluated for the in vitro drug release and stability.

2. Experimental 2.1. Materials BCT was received as a gift sample from Cipla Ltd. India. Precirol® ATO 5 was the generous gift of Gattefossé GmbH, USA. Phosal® 53MCT and Pluronic® F-127 were received from BASF Corporation, New Jersey, USA. Captex® 500P (triacetin) was a kind gift from Abittec Corporation, USA. Sodium taurocholate was a kind gift from Profotti Chemici, Italy. Distilled water was used throughout the

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experiments. All other chemicals were of analytical grade and used as received. 2.2. Screening of components and preformulation study The solubilization potential of BCT in different solid lipids and oils were obtained using earlier reported methods [21,22]. The solid lipids screened for the purpose included, Compritol® ATO 888, Precirol® ATO 5, Dynasan® 116, and Dynasan® 118. The different oils screened for the purpose included, Labrafac® CC, Labrafil® M 1944 CS, Lauryglycol ® 90, Lauroglycol ® FCC and Captex® 500P. Various surfactants such as Pluronic® F-127, sodium taurocholate and lecithin (Phosal ® 53 MCT) were studied at different ratios to identify optimum stabilizer blend. The processing variables such as homogenizer pressure and the number of cycles were studied as a function of particle size of the drug loaded NLCs. 2.3. Production of BCT-NLC dispersion BCT-NLC dispersion was prepared using a hot high-pressure homogenization (HPH) technique [23,24]. Briefly, accurately weighed Precirol® ATO 5 [melting point (m.p.): 54 ◦ C], Phosal® 53 MCT and Captex® 500 P were heated to melt at 65 ◦ C (≈10 ◦ C above m.p. of solid lipid) to form an oily phase, to which the required amount of BCT was added. At the same time, aqueous surfactant solution comprising of Pluronic® F-127 and sodium taurocholate was prepared by heating at the same temperature. The hot surfactant solution was added to the oily phase under magnetic stirring. For adequate homogenization, the dispersion was processed under ultra-turrax (T 25 Basic, Ika Werke, Stanfer, Germany) at 11000 rpm for 1 min. The processed sample was then subjected to the highpressure homogenizer under optimized conditions of 1000 bar pressure (1 bar = 10 × 104 N/m2 ) and 7 cycles. Finally, the prepared hot dispersion was allowed to cool to room temperature during which lipid recrystallizes to form nanoparticles. The NLC dispersion thus obtained was subsequently stored at 4 ◦ C until further analysis. 2.4. Preparation of drug–lipid melt Drug–lipid melt was used as a control to study the BCT phase transition during the NLC processing. The melt was prepared by dispersing the pure BCT (150 mg) into the molten lipids comprising of Precirol® ATO 5 (solid lipid) and Captex® 500 P (oily lipid) in the ratio of 70:30. The molten lipid mixture was allowed to cool and solidify at room temperature. 2.5. Effect of variables A 23 factorial design was used to study the effect of formulation variables on the performance and characteristics of nanostructured lipid carrier. The amount of drug (X1 ), lipid concentration (X2 ) and the stabilizer concentration (X3 ) were selected as the three independent formulation variables. Particle size (Y1 ), zeta potential (Y2 ) and the percent drug entrapment (Y3 ) were selected as the dependent response variables. Solid to liquid lipid (oil) ratio 70:30 and surfactant (Phosal® 53 MCT, pluronic® F-127 and sodium taurocholate) ratio 3:2:1 was established through preliminary studies and kept constant throughout the study. Values of all variables and codes of the different batches are as shown in Table 1. 2.6. Characterization 2.6.1. Particle size analysis Size analysis and measurement of the polydispersity index (PDI) of the nanoparticles were performed by photon correlation

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Table 1 Experimental design with coded levels of variables and actual values. Batch code

Variable X1 amount of drug (mg)

Variable X2 lipid concentrationa (%, w/v)

Variable X3 stabilizer concentrationb (%, w/v)

NLC-1 NLC-2 NLC-3 NLC-4 NLC-5 NLC-6 NLC-7 NLC-8

150 (+1) 150 (+1) 150 (+1) 150 (+1) 10 (− 1) 10 (− 1) 10 (− 1) 10 (− 1)

3 (+1) 3 (+1) 1 (−1) 1 (−1) 3 (+1) 3 (+1) 1 (− 1) 1 (− 1)

1.5(+1) 0.5 (−1) 0.5 (−1) 1.5 (+1) 0.5 (−1) 1.5 (+1) 1.5 (+1) 0.5 (−1)

A value in parentheses indicates coded level. a Comprises solid to liquid lipid (oil) at ratio 70:30. b Comprises blend of phosal 53 MCT, pluronic F-127 and sodium taurocholate at ratio 3:2:1.

spectroscopy (PCS) using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Distilled water was used as a dispersant and the system was maintained at 25 ◦ C. 2.6.2. Zeta potential measurement (ZP) Zeta potential of the nanoparticles was measured using Malvern Zetasizer Nano ZS (Malvern Instruments). The samples were diluted appropriately with the distilled water and the zeta potential was determined. 2.6.3. Percent entrapment efficiency (% EE) The free (unentrapped drug) was separated from the drug entrapped in the lipid carrier using the reported sephadex minicolumn centrifugation technique [25] with slight modification. Briefly, sephadex ® G25M aqueous solution (10%, w/v) was prepared and kept overnight for swelling. To prepare the minicolumn, Whatman filter pad was inserted in a 1 ml syringe. The formed sephadex gel was added slowly to the prepared minicolumn with due care to avoid air entrapment. The gel incorporated minicolumns were then centrifuged (Eppendorff centrifuge 5810 R, Hamburg, Germany) at 2000 rpm for 3 min. This resulted in removal of excess water and subsequently formed a compact packing of sephadex in the column. BCT-NLC dispersion (100 ␮L) was slowly added on the prepared column and centrifuged as earlier. The resultant elute was further processed through a fresh sephadex minicolumn. The free drug remained bound to the gel, while drug loaded vesicles traveled through the gel and was collected from first and second stage of centrifugation. The eluted nanoparticles were ruptured using sufficient volume of methanol and the amount of drug present was analyzed spectrophotometrically at 272 nm using dual beam UV spectrophotometer (Jasco, Japan). The percent drug entrapment was calculated mathematically using Eq. (1). %EE =

Qe × 100 Qt

(1)

where Qe is the amount of encapsulated BCT and Qt is the total amount of BCT present in 100 ␮L of NLC dispersion. The method was validated by applying a free drug suspension instead of drug loaded NLC dispersion [26]. 2.6.4. Lyophilization Lyophilization of an optimized NLC-2 dispersion was carried out with and without cryoprotectant using Virtis-Bench Top Lyophilizer, Spinco Biotech Pvt. Ltd. The dispersion was pre-frozen (−75 ◦ C) for 12 h and subsequently lyophilized at a temperature of −25 ◦ C for 24 h followed by a secondary drying phase of 12 h at 20 ◦ C. Mannitol (3.5%, w/v) was used as the cryoprotectant. 2.6.5. Scanning electron microscopy (SEM) analysis The surface morphology of lyophilized NLC-2 and the drug–lipid melt was visualized using scanning electron microscope (VEGA

MV2300T/40, TS 5130 MM, TESCAN). Before observation, the lyophilized nanoparticles were fixed on a double-sided sticky tape that was previously mounted on aluminum stubs and then coated with gold in an argon atmosphere. The scanning was performed at an accelerating voltage of 10 kV. 2.6.6. Fourier transform infrared spectroscopy (FT-IR) analysis FT-IR spectra were recorded for BCT, Precirol® ATO 5, lyophilized NLC-2, and the drug–lipid melt using IR spectrophotometer (Jasco4100, Japan). The samples were prepared in a KBr disc (2 mg sample in 200 mg KBr) with a hydrostatic press at a force of 5.2 N/m2 for 3 min. The scanning range was 1500–3900 cm−1 . 2.6.7. Thermal analysis The DSC thermograms of BCT, Precirol® ATO 5, lyophilized NLC2, and the drug–lipid melt were recorded in nitrogen environment using a METTLER TOLEDO thermal analysis system (USA). The heating rate employed was 10 ◦ C per min. The crystallinity index (CI) of the lipid nanoparticles (NLC-2) was calculated using Eq. (2). CI (%) =

enthalpy (lyophilized NLC) enthalpy (bulk material) × concentration of lipid phase (2)

2.6.8. Powder X-ray diffraction (PXRD) analysis PXRD analysis of BCT, Precirol® ATO 5, lyophilized NLC-2, and the drug–lipid melt was performed using Brucker D8 advanced diffractometer coupled with the Diffrac plus V1.01 software. The diffraction pattern was measured at 2 value of 2–60◦ , a voltage of 40 kV and a current of 40 mA. 2.6.9. Raman spectroscopy Raman spectra were recorded for BCT, lyophilized NLC-2 and the drug–lipid melt using a Raman spectrometer (Jobin Yvon Ramanor HG 2S). The scanning was performed at three different ranges, 100–200, 1400–1800 and 2100–2300 cm−1 . 2.6.10. Proton nuclear magnetic resonance (1 H NMR) study 1 H NMR spectrum was recorded for the lyophilized NLC-2 at 300 MHz with a Varian Mercury YH-300 spectrometer using DMSOd6 as a solvent. The 1 H NMR chemical shift values were reported on the ı scale in ppm, relative to TMS (ı = 0.00) as an internal standard. 2.6.11. In vitro release studies The release profile of NLC-2 dispersion and the pure drug suspension was obtained by dialysis bag method using cellophane membrane (molecular weight cut off 12 000–14 000 Da). Fivemilliliters of suspension was placed inside the dialysis bag, tied at both ends, and immersed in the dissolution medium (1%, w/v, sodium lauryl sulfate, SLS). The medium was stirred on a magnetic stirrer at 100 rpm and the temperature maintained at 37 ± 0.2 ◦ C.

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Two-milliliter aliquots were withdrawn at pre-set time intervals and replaced by an equal volume of fresh dissolution medium. After suitable dilution with methanol, the samples were analyzed spectrophotometrically at 272 nm. The drug release kinetics was studied by using PCP disso V3 software (Poona College of Pharmacy, Pune), based on the following equations: Zero order :

%R = k0 t

First order :

log % unreleased =

Higuchi-matrix :

%R = km t

Korsmeyer–Peppas : Hixson–Crowell :

(3) k1 t 2.303

0.5

%R = kp t n

(% unreleased)1/3 = kh t

(4) (5)

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Table 2 Responses obtained for studied parameters from experimental batches. Batch code

Sizea (nm)

PDIa

Zeta potential (mV)

%Entrapment

NLC-1 NLC-2 NLC-3 NLC-4 NLC-5 NLC-6 NLC-7 NLC-8

230 160 240 204 158 212 180 130

± ± ± ± ± ± ± ±

0.3 0.1 0.2 0.2 0.3 0.2 0.3 0.2

−18.4 −25.1 −21.3 −18.2 −21.7 −21.3 −19.7 −22.8

95.48 98.48 82.23 84.43 93.46 90.68 92.11 92.25

5.2 1.2 4.1 4.5 5.5 3.7 5.5 4.8

± ± ± ± ± ± ± ±

4.3 3.2 3.2 4.7 5.4 3.2 4.2 4.1

± ± ± ± ± ± ± ±

0.51 0.70 0.89 0.51 0.37 0.40 0.46 0.50

Values are presented as the mean ± S.D. (n = 3). a Values were determined by dynamic light scattering.

(6)

3.2. Statistical data

(7)

Responses of different batches obtained using factorial design are shown in Table 2. Obtained data were subjected to multiple regression analysis using StatSoft, Inc. STATISTICA version 8, USA. The data were fitted to the second order polynomial (Eq. (8)) and the generated response surfaces are depicted in Fig. 1. The adequacy of a fitted model was checked by analysis of variance (ANOVA).

where, %R is percent drug release; t is time; k0 , k1 , km , kp , and kh are the rate constants for zero order, first order, Higuchi-matrix, Korsmeyer–Peppas and Hixson–Crowell respectively, “n” indicates release exponent.

Y = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ12 X1 X2 + ˇ13 X1 X3 + ˇ23 X2 X3 (8) 2.6.12. Stability study Stability study of the NLC-2 dispersion was carried out for a six months at the three different conditions of temperature and relative humidity (%RH) as 40 ◦ C/75%RH, 25 ◦ C/60%RH and 4 ◦ C. The particle size and the percent drug entrapment were evaluated as stability parameters. 3. Result and discussion 3.1. Screening of components and preformulation study The objectives of the preliminary studies included selection of the solid and liquid lipid, selection of the appropriate surfactant blend and its ratio, and to establish the optimum processing parameters such as the homogenization pressure and number of cycles. Among the different solid lipids and oils screened (data not shown), Precirol® ATO 5 (solid lipid) and Captex® 500 P (oil) gave the highest drug solubility of 3% w/w and 20 ± 0.8 mg/ml respectively, hence, were selected for the preparation of BCT-NLC. For a colloidal system such as NLC, appropriate stabilizers are required to obtain the desired size and to impart stability to the nanoparticles on storage. It has been established that a blend of stabilizers having suitable HLB values helps in achieving above objective [27]. In the present study, we have chosen three stabilizers namely, Phosal® 53 MCT (HLB ≈ 4), Pluronic® F-127 (HLB 18-23) and sodium taurocholate (HLB 20-25). Upon preliminary screening, Phosal® 53 MCT, Pluronic® F-127 and sodium taurocholate in 3:2:1 ratio was found to be optimum as a stabilizer blend. Further studies on the processing by high-pressure homogenization resulted in optimized conditions of 1000 bar pressure and 7 cycles to achieve the desired size and polydispersity index (PDI) of the BCT loaded NLCs. Here, the reduction in particle size was mainly due to the cavitational forces in the homogenization gap and subsequent diminution of the lipid droplets to the nanosize [28]. It was found that at high lipid concentration of about 4% (w/v) and more, large particles (>1 ␮m) were produced while lipid concentration between 1 and 3% (w/v) produced smaller NLCs with good PDI values. The amount of drug, the concentration of lipid, and the concentration of stabilizer, being crucial in the preparation and stabilization of NLC, were selected as formulation variables in the 23 factorial design (Table 1). The formulation variables were studied as a function of size, zeta potential and the percent drug entrapment.

3.3. Effect of variables 3.3.1. Effect of formulation variables on particle size The average particle diameters of different batches of BCT-NLC ranged from 130 ± 4.8 to 240 ± 4.1 nm as summarized in Table 2. The influence of the formulation variables on the particle size of the NLC is presented in Eq. (9) and found to be statistically significant (p value 0.042, r2 = 0.874). Y1 (size) = 189.25 + 19.25X1 + 0.75X2 + 17.25X3 − 14.25X1 X2 − 8.75X1 X3 + 13.75X2 X3

(9)

The particle size analysis revealed positive relationship with all the three formulation variables namely, amount of drug (X1 ), lipid concentration (X2 ) and the stabilizer concentration (X3 ). However, as evidenced by the strong positive coefficients for X1 and X3 in Eq. (9), the amount of drug and the stabilizer concentration are the major factors influencing size of NLC. A concentration dependent increase in particle size with the stabilizer could be the result of deposition of the pluronic F-127 onto a lipid vesicle [29]. On the other hand, an increase in the lipid concentration also led to a concentration dependent increase in the particle size. This was in agreement with the report of Muller–Goymann [30]. Besides, the size distribution analysis of NLC-2 dispersion (Fig. 2) revealed a narrow and unimodal distribution with the PDI value of 0.1. 3.3.2. Effect of formulation variables on zeta potential (ZP) The average zeta potential values of the NLC containing BCT was in the range of −18.2 ± 4.7 to −25.1 ± 3.2 mV as summarized in Table 2. The influence of formulation variables on zeta potential of NLC is presented in Eq. (10) and found to be statistically significant (p value 0.023, r2 = 0.866). Y2 (ZP) = −21.06 + 0.312X1 − 0.562X2 + 1.66X3 − 0.437X1 X2 + 0.787X1 X3 + 0.112X2 X3

(10)

ZP is the electric charge on particle surface that creates an electrical barrier and acts as a ‘repulsive factor’ in the process of emulsion stabilization [31]. NLCs found to possess negative surface charges due to the adsorption of hydroxyl ions of lipid, Precirol®

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D.D. Kumbhar, V.B. Pokharkar / Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

Fig. 1. Estimated response surfaces for particle size (A), zeta potential (B) and percent drug entrapment (C and D) of NLC as a function of formulation variables.

ATO 5, at the o/w interface. This was indicated by the negative coefficient for X2 in Eq. (10). The strong positive coefficient observed for the stabilizer concentration (X3 ) could be the result of shielding of surface charges and the steric stabilization provided by Pluronic® F-127 [32]. 3.3.3. Effect of formulation variables on percent drug entrapment The percent drug entrapment of the NLC containing BCT varied from 82.23 ± 0.89 to 98.48 ± 0.70% as summarized in Table 2. NLC2 achieved the maximum drug entrapment of 98.48 ± 0.70%. The influence of the formulation variables on the percent drug entrapment of the NLC is presented in Eq. (11) and found to be statistically significant (p value 0.002, r2 = 0.996). Y3 (%EE) = 91.14 − 0.985X1 + 3.385X2 − 0.46X3 + 3.44X1 X2 + 0.26X1 X3 − 0.98X2 X3

(11)

encapsulation [15]. Thus, the achieved BCT encapsulation could be the combined result of hydrophobic nature of Precirol® ATO 5 and the influence of the oily component. The negative regression coefficients for X1 and X3 indicated that the amount of drug and the stabilizer concentration had an inverse relationship with the percent drug entrapment. The increase in amount of drug, without increase in the lipid concentration could lead to decrease in the percent drug entrapment. This can be attributed to the insufficient amount of lipid available for the drug to encapsulate. However, as evidenced by the strong positive coefficient for X1 X2 , simultaneous increase in both, the amount of drug and the lipid concentration could result in improved drug encapsulation. On the other hand, Pluronic® F-127 is a surface modifying agent with the ability to influence porosity of a material [34]. This could render diffusion of encapsulated drug to the external aqueous phase and thus may account for the reduced drug encapsulation. 3.4. Lyophilization

As evidenced by the strong positive regression coefficient for X2 in Eq. (11), concentration of the carrier lipid is the major factor governing percent drug entrapment. This could be related to esterification of glycerol by the long chain fatty acids and the absence of PEG esters in Precirol® ATO 5 [33]. This renders, Precirol, a pronounced hydrophobic character (HLB 2) that may account for the encapsulation of BCT. Moreover, the presence of oil (triacetin) may enhance imperfections in the lipid matrix and thereby assist drug

The lyophilization of NLC-2 dispersion without cryoprotectant resulted in a sticky mass. The crystallization of ice may exercise a mechanical stress on the nanoparticles and thereby lead to their destabilization [35]. Hence, the use of cryoprotectant before freezing is desirable to protect these fragile systems. For lyophilization of the NLC-2 dispersion, mannitol (3.5%, w/v) was found to be optimum as a cryoprotectant. This resulted in a free flowing dry powder

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Fig. 4. FT-IR spectra of pure BCT (A), lyophilized NLC-2 (B), Precirol® ATO 5 (C) and drug–lipid melt (D).

3.6. Fourier transform infrared spectroscopy (FT-IR) analysis Fig. 2. Particle size distribution of NLC-2 dispersion.

that was further subjected to the various solid-state characterizations. 3.5. Scanning electron microscopy (SEM) analysis Fig. 3 shows SEM photomicrographs of drug–lipid melt (Fig. 3A) and the BCT-loaded lyophilized NLC-2 (Fig. 3B). Lipid crystallization was clearly observed in the photomicrograph of the drug–lipid melt (Fig. 3A). On the other hand, nonporous, spherical particles with a smooth surface were observed for the lyophilized NLC-2 (Fig. 3B). The observed particle diameters were found to be consistent with the PCS diameters.

The drug–lipid interaction was studied by FT-IR spectroscopy and depicted in Fig. 4. The IR spectrum of the pure drug (Fig. 4A) revealed the absorption bands at 3339 cm−1 [ NH stretch], 1688 cm−1 [C O stretch], 2228 cm−1 [C N stretch], and 3587 cm−1 [H bonded OH stretch]. These are the characteristic peaks of BCT Form I [19]. For Precirol® ATO 5, IR absorption peaks were recorded at 1737 cm−1 (C O stretch), 1625 cm−1 (C C stretching) and 2916 cm−1 (C H stretch) (Fig. 4C). The IR spectrum of lyophilized NLC-2 (Fig. 4B) revealed the absorption peaks at 3339 cm−1 , 1688 cm−1 and 2228 cm−1 . This was clearly suggestive of the presence of BCT crystalline Form I in NLC-2. The same was further confirmed using DSC, PXRD and the Raman analysis. The IR spectrum of drug–lipid melt (Fig. 4D) showed that Precirol® ATO 5 does not affect BCT signature bands. However, the intensity of the

Fig. 3. SEM photomicrographs of drug–lipid melt (A) and lyophilized NLC-2 (B).

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Fig. 5. DSC thermograms of pure BCT (A), lyophilized NLC-2 (B), Precirol® ATO 5 (C) and drug–lipid melt (D).

drug peaks, especially at, 3339 cm−1 , 2228 cm−1

reduced, while the

Fig. 6. Powder X-ray diffraction patterns for pure BCT (A), Precirol® ATO 5 (B), drug–lipid melt (C) and lyophilized NLC-2 (D).

peak at 1688 cm−1 broadened. 3.7. Differential scanning calorimetry (DSC) analysis Fig. 5 depicts DSC thermograms of pure BCT, Precirol® ATO 5, drug–lipid melt, and the lyophilized NLC-2. Pure BCT and Precirol® ATO 5 revealed the melting endothermic peak at 195 ◦ C (Fig. 5A) and 54 ◦ C (Fig. 5C) respectively. Total disappearance of the characteristic drug melting endotherm was observed in the DSC thermogram (Fig. 5D) of drug–lipid melt. This indicated molecular dispersion of BCT within the lipid matrix, suggestive of its amorphous nature in the melt sample. In contrast, NLC-2 revealed a melting endotherm at 195 ◦ C (Fig. 5B) corresponding to BCT crystalline Form I. This clearly indicated that the amorphous BCT in the melt sample recrystallized to the Form I during NLC processing. As cited in literature, the amorphous form of bicalutamide spontaneously undergoes transition to the crystalline Form I upon mechanical activation [36]. This was well in the agreement with our findings. The high-pressure homogenization (1000 bar pressure, 7 cycles) and the elevated temperature (≈65 ◦ C) used in the production of NLC might have served as mechanical activators to bring about BCT phase transition. This transition was subsequently confirmed by the techniques such as, PXRD and Raman analysis. On the other hand, the melting enthalpy (H) was evaluated to study recrystallization of the lipid. There was a sharp decline in melting enthalpy from the bulk material to the nanoparticles. The melting endotherm of Precirol® ATO 5 in the lyophilized NLC-2 (Fig. 5B) has been shifted to 51 ◦ C (H 124.21 J/g) from 54 ◦ C (H 138.60 J/g). The decrease in melting enthalpy suggested lower ordered lattice arrangements with possible lipid recrystallization that might influence the loading capacity [37]. Moreover, it is important to note that, more the imperfections in the lipid matrix, better will be the drug loading. Considering the enthalpy of bulk lipid (Precirol® ATO 5) at 138.60 J/g as 100%, the crystallinity index (CI) of lipid nanoparticles (NLC-2) was calculated

using Eq. (2) and found to be 29.87% indicating partial recrystallization of lipid. 3.8. Powder X-ray diffraction (PXRD) analysis X-ray diffractograms of Pure BCT, Precirol® ATO 5, drug–lipid melt and the lyophilized NLC-2 are shown in Fig. 6. There are significant differences between the diffraction patterns of drug, lipid and the drug–lipid melt. Pure BCT showed diffraction peaks at 2 values of 12.1◦ , 16.7◦ , 18.8◦ , 19.2◦ , 23.5◦ , 24.8◦ , 29.5◦ , and 31.1◦ (Fig. 6A). These are the characteristic diffraction peaks of BCT crystalline Form I [38]. In the diffractogram of Precirol® ATO 5 (Fig. 6B), an intense signal was observed at 20.92◦ , typical of waxy materials [39]. The absence of crystalline peaks of drug in the diffractogram of drug–lipid melt (Fig. 6C) suggested amorphous nature of BCT in the melt sample. On the other hand, NLC-2 revealed the diffraction peaks at 2 values of 12.1◦ , 16.6◦ , 18.8◦ , 19.2◦ , 23.6◦ , 24.8◦ , 29.5◦ and 31.2◦ (Fig. 6D). This clearly indicated the phase transition of BCT from amorphous to crystalline Form I during the NLC processing. Mechanical activation during processing (high-pressure homogenization) could increase the stored energy of the amorphous phase, and thereby result in nuclei of the thermodynamically stable crystalline Form I [40]. 3.9. Raman analysis Raman analysis was performed to characterize the phase transition of BCT during the NLC processing. Fig. 7 depicts Raman spectra of pure BCT, drug–lipid melt, and the lyophilized BCT-NLC (NLC-2). The analysis was carried at wavenumbers 100–200 cm−1 (Fig. 7A), 1400–1800 cm−1 (Fig. 7B), and 2100–2300 cm−1 (Fig. 7C). The Raman spectra of pure BCT had distinct peaks at 1516 cm−1 [N H in plane bending], 1614 cm−1 [C C ring stretching vibration],and at

D.D. Kumbhar, V.B. Pokharkar / Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

39

Fig. 7. Raman analysis of pure BCT, drug loaded NLC (BCT-NLC) and drug–lipid melt.

2230 cm−1 (C N stretching). Moreover, peaks were also recorded at 142 cm−1 and 158 cm−1 . The observed characteristic maxima confirmed the structure of BCT crystalline Form I [17,20]. In lyophilized NLC-2, peaks were recorded at 1514 cm−1 , 1614 cm−1 , and 2230 cm−1 . The region below wavenumber 200 cm−1 was also found to be useful to distinguish between the amorphous and the crystalline nature of BCT. Unlike drug–lipid melt, the Raman spectra of NLC-2 revealed peaks at a low wavenumber region within 130–160 cm−1 . This could be related with the difference in backbone deformation, liberation, and lattice vibration that appears in this region [17]. This gave the strong confirmation on the presence of BCT crystalline Form I in NLC-2. On the other hand, in the Raman spectra of drug–lipid melt, BCT characteristic maxima for Form I was not observed. Moreover, the absence of maxima at 1430 cm−1 , 1496 cm−1 , and 1700 cm−1 ruled out the existence of BCT crystalline Form II in the melt sample [38]. This established the presence of amorphous BCT in the melt sample. These results supported the findings of DSC and PXRD analysis.

standard TMS (trimethyl silane). The spectrum revealed intense signals at 10.38 ppm, 8.42 ppm, 8.21 ppm, 7.96–7.89 ppm, 7.37 ppm, 6.43 ppm, 3.93 ppm, 3.63 ppm and 1.40 ppm which can be attributed to broad singlet, 1H, N H; singlet, 1H, Ar H; doublet, 1H, Ar H; multiplet, 3H, Ar H; multiplet, 2H, Ar H; broad singlet, 1H, HO C CO; doublet, 1H, SO CHH C; doublet, 1H, SO CHH C; and singlet, 3H, CH3 C CO, respectively. These are the characteristic proton signals of BCT [41]. Besides, additional proton signals were noted in the spectrum at 3.32 ppm, 2.5 ppm, 2.03 ppm, and 0.9 ppm. The sharp signals recorded at 3.32 ppm and 2.5 ppm was attributed to the solvent DMSO-d6. The proton signal recorded at 0.9 ppm and 2.03 ppm could be attributed to CH3 and OCH3 protons of Precirol® ATO 5 and triacetin respectively [42,43]. Thus, the 1 H NMR spectrum of NLC-2 clearly demonstrated, well-resolved, characteristic proton signals of the drug, the solid lipid, and the oil. This clearly indicated the lack of interaction between the drug molecule and the lipid carrier and thus ensured compatibility of BCT with the carrier lipid.

3.10. Proton nuclear magnetic resonance (1 H NMR) study

3.11. In vitro release kinetic study

1 H NMR spectrum of NLC-2 in the solvent DMSO-d6 is depicted in Fig. 8. Peak at 0 ppm was assigned to the internal

Drug release profiles of the optimized NLC-2 dispersion and the pure BCT suspension from cellophane membrane (molecular

40

D.D. Kumbhar, V.B. Pokharkar / Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

Fig. 8.

1

H NMR spectrum of lyophilized NLC-2 in solvent DMSO-d6.

weight cut off 12 000–14 000 Da) are shown in Fig. 9. NLC-2 had shown drug release of 62.08% at the end of 24 h while pure drug suspension (used as reference) released 21.99% of the drug at the end of 24 h. The observed difference in the release pattern strongly indicated the influence of lipid excipients on the dissolution rate enhancement from the BCT-NLC. The release data was fitted into various kinetic models like zero order, first order, Higuchi-matrix, Korsmeyer-Peppas, and Hixson-Crowell using PCP-DISSO V3 software in order to establish the mechanism of drug release from NLC-2. The correlation coefficients (r), the rate constants (k) and a release exponent (n) were determined and summarized in Table 3. NLC-2 was found to follow Peppas release kinetics (r = 0.9844) and demonstrated the Fickian diffusion (n < 0.5) as the drug release mechanism. The release pattern (Fig. 9) indicated that using this delivery system, a loading dose of around 30–35% drug could be made available within 4 h of its administration while the remaining

would support the maintenance dose for a considerable duration. This may prevent excessive fluctuations in the drug plasma level. Precirol® ATO 5 owing to its high lipophilicity (HLB-2) could result in a slow release of the encapsulated BCT molecule. However, the presence of hydrophilic surfactant such as pluronic F-127 (HLB 1823) has been suggested to modulate drug release from the Precirol® ATO 5 matrices [44]. 3.12. Stability study The stability studies of NLC-2 dispersion revealed insignificant changes in the stability parameters (p-value < 0.05). The particle sizes were found to be 162 ± 1.8 nm, 164 ± 3 nm, and 160 ± 1.4 at 25 ◦ C/60%RH, 40 ◦ C/75%RH, and 4 ◦ C respectively at the end of six months. The percent drug entrapments of 96.70 ± 1.2%, 95 ± 1.8%, and 98.46 ± 0.5% were noted at 25 ◦ C/60%RH, 40 ◦ C/75%RH and 4 ◦ C

Table 3 Release kinetic parameters.a Batch code

Drug release models Zero order

NLC-2 BCT-suspension

First order

r

k0

r

0.9382 0.8784

0.8490 0.4094

0.9491 0.8834

k1 −0.012 0.005

Higuchi–Matrix

Peppas–Korsmeyer

Hixon–crowell

r

km

r

kp

n

r

0.9721 0.9271

6.20 3.04

0.9844 0.9520

15.69 9.103

0.32 0.29

0.9456 0.8818

kh −0.0037 −0.0016

a “r” indicates correlation coefficients, k0 , k1 , km , kp , and kh indicate rate constants for zero order, first order, Higuchi-Matrix, Peppas–Korsmeyer and Hixon–Crowell respectively. “n” indicates release exponent.

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41

References

Fig. 9. Comparative in vitro drug release profiles from optimized batch (NLC-2) and BCT-suspension using 1% SLS as dissolution medium.

respectively at the end of six months. This ruled out the possibility of drug leakage from the NLC-2 and ensured its potential to retain the drug for a longer period. As mentioned earlier, pluronics are capable to provide steric stabilization to the lipid nanoparticles. Besides, sodium taurocholate, used as a co-surfactant, can provide electrostatic stability [45]. In case of combined electrostatic and steric stabilization, a zeta potential of about −20 mV can be sufficient for physical stability [46]. This was reflected through the observed stability of developed NLC-2. Thus, both, the steric stabilization by pluronic F-127 and the electrostatic stabilization by sodium taurocholate might speculate about a long-term stability of the NLC-2 dispersion.

4. Conclusion In this work, bicalutamide loaded nanostructured lipid carrier (BCT-NLC) was successfully prepared using the hot high-pressure homogenization technique. The optimized batch (NLC-2) revealed a spherical morphology and achieved high drug encapsulation efficiency. Investigations on the drug–lipid interaction using FT-IR and 1 H NMR spectroscopy confirmed the compatibility of BCT with the carrier lipid. Extensive studies involving DSC, XRD and Raman analysis confirmed the existence of amorphous BCT in drug–lipid melt and the most stable; BCT crystalline Form I in BCT loaded NLC. The high-pressure homogenization and the elevated processing temperature were identified as mechanical activators for BCT phase transition during the NLC processing. Drug release study indicated significance of the hydrophilic surfactants to modulate BCT release from the NLC-2. The investigation on crystallinity index (CI) was well in agreement with the observations on zeta potential and particle size and reflected through the stability of NLC-2. Finally, NLCs made of Precirol® ATO 5 (solid lipid) and triacetin (oil) posses the potential to entrap the poorly water-soluble drug, bicalutamide and the system can be tailor-made to meet the desired drug release. Thus, the developed lipid nanocarrier may provide better prospects for the oral delivery of bicalutamide.

Acknowledgements Authors wish to acknowledge Indian Institute of Technology (IIT), Mumbai, India, for availing Raman analysis facility. University of Pune, Pune, India for providing SEM and 1 H NMR facility.

[1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA Cancer J. Clin. 61 (2011) 69–90. [2] A. Heidenreich, G. Aus, M. Bolla, S. Joniau, V.B. Matveev, H.P. Schmid, F. Zattoni, EAU guidelines on prostate cancer, Eur. Urol. 53 (2008) 68–80. [3] A.K. Singh, A. Chaurasiya, G.K. Jain, A. Awasthi, D. Asati, G. Mishra, R.K. Khar, R. Mukherjee, High performance liquid chromatography method for the pharmacokinetic study of bicalutamide SMEDDS and suspension formulations after oral administration to rats, Talanta 78 (2009) 1310–1314. [4] G.J. Kolvenbag, A. Nash, Bicalutamide dosages used in the treatment of prostate cancer, Prostate 39 (1999) 47–53. [5] F. Ren, Q. Jing, Y. Tang, Y. Shen, J. Chen, F. Gao, J. Cui, Characteristics of bicalutamide solid dispersions and improvement of the dissolution, Drug Dev. Ind. Pharm. 32 (2006) 967–972. [6] M.V. Srikanth, G.V. Murali Mohan Babu, N.S. Rao, S.A. Sunil, S. Balaji, K.V. Ramanamurthy, Dissolution rate enhancement of poorly soluble bicalutamide using ␤-cyclodextrin inclusion complexation, Int. J. Pharm. Pharm. Sci. 2 (2010) 191–198. [7] C. Li, C. Li, Y. Le, J.F. Chen, Formation of bicalutamide nanodispersion for dissolution rate enhancement, Int. J. Pharm. 404 (2011) 257–263. [8] S.S. Rane, B.D. Anderson, What determines drug solubility in lipid vehicles: is it predictable? Adv. Drug Deliv. Rev. 60 (2008) 638–656. [9] C.J.H. Porter, C.W. Pouton, J.F. Cuine, W.N. Charman, Enhancing intestinal drug solubilization using lipid-based delivery systems, Adv. Drug Deliv. Rev. 60 (2008) 673–691. [10] Y. Luo, D. Chen, L. Ren, X. Zhao, J. Qin, Solid lipid nanoparticles for enhancing vinpocetine’s oral bioavailability, J. Control. Release 114 (2006) 53–59. [11] A.C. Silva, E. Gonzalez-Mira, M.L. García, M.A. Egea, J. Fonseca, R. Silva, D. Santos, E.B. Souto, D. Ferreira, Preparation, characterization and biocompatibility studies on risperidone-loaded solid lipid nanoparticles (SLN): high-pressure homogenization versus ultrasound, Colloids Surf. B 86 (2011) 158–165. [12] R.H. Muller, S. Runge, V. Ravelli, W. Mehnert, A.F. Thunemann, E.B. Souto, Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN® ) versus drug nanocrystals, Int. J. Pharm. 317 (2006) 82–89. [13] H. Li, X. Zhao, Y. Ma, G. Zhai, L. Li, H. Lou, Enhancement of gastrointestinal absorption of quercetin by solid lipid nanoparticles, J. Control. Release 133 (2009) 238–244. [14] S. Doktorovova, E.B. Souto, Nanostructured lipid carrier-based hydrogel formulations for drug delivery: a comprehensive review, Expert Opin. Drug Deliv. 6 (2009) 165–176. [15] V. Jenning, A.F. Thunemann, S.H. Gohla, Characterization of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids, Int. J. Pharm. 199 (2000) 167–177. [16] E.B. Souto, S.A. Wissing, C.M. Barbosa, Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery, Int. J. Pharm. 278 (2004) 71–77. [17] D.R. Vega, G. Polla, A. Martinez, E. Mendioroz, M. Reinoso, Conformational polymorphism in bicalutamide, Int. J. Pharm. 328 (2007) 112–118. [18] S.R. Vippagunta, H.G. Brittain, Crystalline solids, Adv. Drug Deliv. Rev. 48 (2001) 3–26. [19] I. Kanfer, Report on the international workshop on the biopharmaceutics classification system (BCS): scientific and regulatory aspects in practice, J. Pharm. Pharm. Sci. 5 (2000) 1–4. [20] G.P. Andrews, O.A. Abudiak, D.S. Jones, Physicochemical characterization of hot melt extruded bicalutamide-polyvinylpyrrolidone solid dispersions, J. Pharm. Sci. 99 (2010) 1322–1335. [21] M. Joshi, V. Patravale, Nanostructured lipid carrier (NLC) based gel of celecoxib, Int. J. Pharm. 346 (2008) 124–132. [22] H. Shen, M. Zhong, Preparation and evaluation of self-microemulsifying drug delivery systems (SMEDDS) containing atorvastatin, J. Pharm. Pharmacol. 58 (2006) 1183–1191. [23] V. Jenning, A. Lippacher, S.H. Gohla, Medium scale production of solid lipid nanoparticles (SLN) by high-pressure homogenization, J. Microencapsul. 19 (2002) 1–10. [24] 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. [25] M.N.V. Padamwar, B. Pokharkar, Development of vitamin loaded topical liposomal formulation using factorial design approach: drug deposition and stability, Int. J. Pharm. 320 (2006) 37–44. [26] D.W. Fry, J.C. White, I.D. Goldman, Rapid separation of low molecular weight solutes from liposome without dilution, Anal. Biochem. 90 (1978) 809–815. [27] F. Han, S. Li, R. Yin, H. Liu, L. Xu, Effect of surfactants on the formation and characterization of a new type of colloidal drug delivery system: Nanostructured lipid carriers, Colloids Surf. A: Physicochem. Eng. Aspects 315 (2008) 210–216. [28] R.H. Muller, C.M. Keck, Challenges and solutions for the delivery of biotech drugs – a review of drug nanocrystal technology and lipid nanoparticles, J. Biotechnol. 113 (2004) 151–170. [29] K. Kostarelos, P.F. Luckham, Th.F. Tadros, Effect of the addition of block copolymers on the formation and stability of vesicles (liposomes) prepared using soybean lecithin, Progr. Colloid Polym. Sci. 98 (1995) 69–74. [30] M.A. Schubert, C.C. Muller-Goymann, Solvent injection as a new approach for manufacturing lipid nanoparticles—evaluation of the method and process parameters, Eur. J. Pharm. Biopharm. 55 (2003) 125–131.

42

D.D. Kumbhar, V.B. Pokharkar / Colloids and Surfaces A: Physicochem. Eng. Aspects 416 (2013) 32–42

[31] P. Walstra, Emulsion stability, in: P. Becher (Ed.), Encyclopedia of Emulsion Technology, Marcel Dekker, New York, 1996, pp. 1–62. [32] M.L. Adams, A. Lavasanifar, G.S. Kwon, Amphiphilic block copolymers for drug delivery, J. Pharm. Sci. 92 (2003) 1343–1355. [33] J. Hamdani, A.J. Moës, K. Amighi, Physical and thermal characterization of Precirol® and Compritol® as lipophilic glycerides used for the preparation of controlled-release matrix pellets, Int. J. Pharm. 260 (2003) 47–57. [34] W. Zhao, Y. Su, C. Li, Q. Shi, X. Ning, Z. Jiang, Fabrication of antifouling polyethersulfone ultrafiltration membranes using Pluronic F-127 as both surface modifier and pore-forming agent, J. Membr. Sci. 318 (2008) 405–412. [35] W. Abdelwahed, G. Degobert, S. Stainmesse, H. Fessi, Freeze-drying of nanoparticles: formulation, process, and storage considerations, Adv. Drug Deliv. Rev. 58 (2006) 1688–1713. [36] Z. Nemet, J. Sztatisz, A. Demeter, Polymorph transitions of bicalutamide: a remarkable example of mechanical activation, J. Pharm. Sci. 97 (2008) 3222–3232. [37] D.Z. Hou, C.S. Xie, K.J. Huang, C.H. Zhu, The production and characteristics of solid lipid nanoparticles (SLNs), Biomaterials 24 (2003) 1781–1785. [38] O.A. Abu-diak, D.S. Jones, G.P. Andrews, Understanding the performance of melt-extruded poly(ethylene oxide)-bicalutamide solid dispersions: characterization of microstructural properties using thermal, spectroscopic and drug release methods, J. Pharm. Sci. 101 (2012) 200–213.

[39] B. Albertini, N. Passerini, M.L. Gonzalez-Rodriguez, B. Perissutti, L. Rodriguez, Effect of Aerosil® on the properties of lipid controlled release microparticles, J. Control. Release 100 (2004) 233–246. [40] D. Tromans, J.A. Meech, Enhanced dissolution of minerals: stored energy, amorphism, and mechanical activation, Miner. Eng. 14 (2001) 1359–1377. [41] R.N. Rao, A.N. Raju, R. Narsimha, Isolation and characterization of process related impurities and degradation products of bicalutamide and development of RP-HPLC method for impurity profile study, J. Pharmaceut. Biomed. Anal. 46 (2008) 505–519. [42] V. Jenning, K. Mader, S.H. Gohla, Solid lipid nanoparticles (SLNTM ) based on binary mixtures of liquid and solid lipids: a 1 H NMR study, Int. J. Pharm. 205 (2000) 15–21. [43] N. Yuksel, M. Baykara, H. Shirinzade, S. Suzen, Investigation of triacetin effect on indomethacin release from poly (methyl methacrylate) microspheres: evaluation of interactions using FTIR and NMR spectroscopies, Int. J. Pharm. 404 (2011) 102–109. [44] V. Jannin, E. Pochard, O. Chambin, Influence of poloxamers on the dissolution performance and stability of controlled-release formulations containing Precirol® ATO 5, Int. J. Pharm. 309 (2006) 6–15. [45] A.J. Fillery-Travis, L.H. Foster, M.M. Robins, Stability of emulsions stabilized by two physiological surfactants: l-alpha-phosphatidylcholine and sodium taurocholate, Biophys. Chem. 54 (1995) 253–260. [46] C. Jacobs, R.H. Muller, Production and characterization of a budesonide nanosuspension for pulmonary administration, Pharm. Res. 19 (2002) 189–194.