Brain Targeting of Temozolomide via the Intranasal Route Using Lipid

Sep 23, 2016 - Fermish Clinical Technologies Private Limited, Noida 201301, Uttar Pradesh, India. ABSTRACT: The aim of the present work was to investi...
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Brain Targeting of Temozolomide via the Intranasal Route Using Lipid-Based Nanoparticles: Brain Pharmacokinetic and Scintigraphic Analyses Anam Khan,† Syed Sarim Imam,‡ Mohammed Aqil,*,† Abdul Ahad,§ Yasmin Sultana,† Asgar Ali,† and Khalid Khan∥ †

Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India Department of Pharmaceutics, Glocal School of Pharmacy, Glocal University, Saharanpur 247121, Uttar Pradesh, India § Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia ∥ Fermish Clinical Technologies Private Limited, Noida 201301, Uttar Pradesh, India ‡

ABSTRACT: The aim of the present work was to investigate the efficacy of temozolomide nanostructured lipid carriers (TMZ-NLCs) to enhance brain targeting via nasal route administration. The formulation was optimized by applying a four-factor, three-level Box−Behnken design. The developed formulations and the functional relationships between their independent and dependent variables were observed. The independent variables used in the formulation were gelucire (X1), liquid lipid/total lipid (X2), Tween 80 (X3), and sonication time (X4), and their effects were observed with regard to size (Y1), % drug release (Y2), and drug loading (Y3). The optimized TMZ-NLC was further evaluated for its surface morphology as well as ex vivo permeation and in vivo studies. All TMZ-NLC formulations showed sizes in the nanometer range, with high drug loading and prolonged drug release. The optimized formulation (TMZ-NLCopt) showed an entrapment efficiency of 81.64 ± 3.71%, zeta potential of 15.21 ± 3.11 mV, and polydispersity index of less than 0.2. The enhancement ratio was found to be 2.32-fold that of the control formulation (TMZdisp). In vivo studies in mice showed that the brain/blood ratio of TMZ-NLCopt was found to be significantly higher compared to that of TMZ-disp (intranasal, intravenous). Scintigraphy images of mouse brain showed the presence of a high concentration of TMZ. The AUC ratio of TMZ-NLCopt to TMZ-disp in the brain was the highest among the organs. The findings of this study substantiate the existence of a direct nose-to-brain delivery route for NLCs. KEYWORDS: temozolomide, brain targeting, NLCs, pharmacokinetic, gamma scintigraphy

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INTRODUCTION A major hurdle in the pharmacological management of aggressive malignant brain tumors is the presence of the blood−brain barrier (BBB).1 Among other routes of administration, the nasal route is gaining preference as it is the only route of drug administration that can target the brain while avoiding systemic side effects through bypassing the BBB by utilizing olfactory or trigeminal nerves.2−4 The neuronal link between the nasal mucosa and the brain provides a unique pathway for the noninvasive delivery of therapeutic agents. Temozolomide (TMZ) is an alkylating agent with the ability to cross the BBB, and it must be administered in high systemic doses due to its short half-life; only 20% of TMZ with respect to a systemic dose reaches the brain.2 TMZ was found to be the most effective antineoplastic agent for treating high-grade metastatic melanoma and glioma. It requires high systemic doses to reach therapeutic levels in the brain, which simultaneously brings about a number of side effects. Its various side effects include headache, nausea, vomiting, fatigue, bone marrow depression, and oral ulcerations.5 © 2016 American Chemical Society

Nanolipid carriers (e.g., NLCs) are considered to be a promising drug delivery vehicle that does not require any modification of the drug molecule. This delivery system was found to be ideal for brain targeting because of the rapid uptake, bioacceptability, and biodegradability of NLCs. The absence of a burst effect and the ease with which they can be scaled up make NLCs promising carriers for drug delivery.6 A nanostructured lipid carrier system is prepared by using a blend of solid lipids and liquid lipids, preferably at a ratio where the differences in the structures of the solid and liquid lipids result in the formation of a perfect crystal mixture. The mixture accommodates a drug in its molecular form or in amorphous clusters.7 NLCs also show high drug loading for both lipophilic and hydrophilic drugs.8,9 NLCs, by virtue of their lipophilic nature and low particle size, are being widely explored as a Received: Revised: Accepted: Published: 3773

June 29, 2016 September 3, 2016 September 23, 2016 September 23, 2016 DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

Article

Molecular Pharmaceutics

Table 1. Box−Behnken Design Based TMZ-NLCs Obtained Using Independent Variables and Their Effects on Dependent Responsesa Y1 (nm)

a

code

X1

X2

X3

X4

1 2 3 4 5 6 7 8 9 10 11 12 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27

5 5 5 5 5 5 2 8 5 8 5 2 2 8 2 5 5 5 5 5 5 2 2 8 8 5 8

0.2 0.05 0.35 0.2 0.2 0.05 0.2 0.2 0.2 0.35 0.05 0.2 0.2 0.2 0.35 0.05 0.35 0.2 0.2 0.35 0.35 0.2 0.05 0.05 0.2 0.2 0.2

1 1 7 4 1 4 4 4 7 4 7 7 4 1 4 4 4 4 7 4 1 1 4 4 4 4 7

2.5 5 5 5 7.5 7.5 7.5 7.5 2.5 5 5 5 2.5 5 5 2.5 7.5 5 7.5 2.5 5 5 5 5 2.5 5 5

actual 196.77 195.89 144.28 171.67 195.12 173.12 145.78 195.78 166.89 182.77 180.61 141.28 151.34 220.11 137.66 182.34 159.78 172.87 162.78 154.78 188.89 174.56 153.22 204.34 199.78 170.61 194.87

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Y2 (%) predicted

4.45 7.45 6.11 4.28 3.59 5.65 4.88 7.16 7.34 8.22 4.71 8.76 5.78 7.44 7.28 9.34 7.45 7.23 6.73 5.32 6.75 6.21 5.52 5.39 4.88 4.18 7.43

197.32 193.54 145.54 169.21 196.21 175.93 145.25 193.27 165.98 180.32 179.42 139.54 150.21 219.57 138.56 183.29 160.43 173.22 163.65 155.76 189.64 175.87 152.75 205.17 200.35 169.55 193.67

Y3 (%)

actual

predicted

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

55.45 55.21 76.39 73.11 60.48 67.43 67.32 62.87 68.11 72.44 58.36 65.89 62.98 50.54 73.17 65.34 68.43 72.65 71.67 65.43 56.34 61.23 69.78 45.11 59.21 74.12 53.64

56.28 54.24 75.33 72.57 59.27 66.54 66.87 63.88 69.45 73.45 60.66 64.44 61.53 51.22 71.33 63.72 70.27 71.45 69.45 64.43 55.48 59.66 71.56 44.44 58.13 72.38 55.28

5.78 4.45 7.32 5.22 4.29 5.77 4.39 6.43 6.23 6.98 5.48 4.65 5.89 6.76 5.11 4.78 5.28 5.38 6.21 4.28 5.71 5.38 4.81 4.26 5.25 4.28 3.22

actual

predicted

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.38 5.05 7.87 7.77 5.27 5.38 5.12 8.84 7.21 8.98 6.05 5.76 5.85 7.88 7.87 6.21 7.32 7.11 6.78 7.95 6.21 5.98 4.11 7.21 8.11 7.78 8.12

6.22 5.22 8.13 7.28 5.66 5.88 5.79 8.16 7.56 8.84 6.43 6.18 6.21 7.23 7.24 6.64 7.59 7.38 7.45 7.57 6.75 5.32 4.88 7.61 8.34 7.23 8.69

0.45 0.54 0.47 0.53 0.58 0.51 0.64 0.71 0.58 0.66 0.67 0.56 0.37 0.78 0.46 0.52 0.59 0.56 0.44 0.79 0.76 0.43 0.86 0.78 0.49 0.41 0.45

X1, gelucire (%); X2, liquid lipid/total lipid; X3, surfactant (%); X4, sonication time (min); Y1, size (nm); Y2, drug release (%); Y3, drug loading (%).

excipient, excess drug was added, mixed for 2 min, and sonicated to dissolve the drug. This mixture was kept on a shaker for 8−12 h to completely solubilize the drug in the lipids. The contents were then centrifuged at 5000 rpm for 15 min, and aliquots of the supernatant were diluted appropriately and analyzed using a UV spectrophotometer (Shimadzu 1800, Japan) at 330 nm. In the case of solid lipids, a known amount of drug was added to a measured quantity of lipid. The minimum amount of molten lipid required to solubilize the drug was noted visually within 24 h. The end point of the solubility study was the formation of a clear solution of molten lipid. Formulation Approach. High-pressure homogenization (HPH) (Heidolph, Diax 900, Schwabach, Germany) followed by ultrasonication (Altrasonics India, Mumbai, India) was used to prepare and optimize TMZ-NLCs.13 Briefly, Vit E (a liquid lipid) was mixed with melted gelucire (a solid lipid) at 5 °C above the melting temperature, and TMZ was added to this. The hot surfactant solution, which consisted of Tween 80/ transcutol (6:4), was dispersed above the lipid phase. The obtained primary emulsion was ultrasonicated for 10 min, homogenized, and cooled to form TMZ-NLCs (Table 1). The TMZ-NLCs obtained were subjected to freeze-drying (Martin Christ, Germany) at a pressure lower than 0.5 milibar to obtain the NLCs in a solid form. Prior to the drying process, the NLC dispersion was frozen for 4 h. The frozen samples were subjected to the freeze-drying process for 8−12 h. Mannitol (3%) was added as a lyoprotectant to avoid the lysis of the nanoparticles present in the suspension.14

delivery system to enhance the brain uptake of a number of drugs, where they have been investigated for the delivery of numerous therapeutic substances and demonstrated therapeutic effects in animal studies.10−12 The objectives of this study were to use a Box−Behnken design to establish the functional relationships among four operating variables, i.e., solid lipid concentration (X1), ratio of liquid lipid to total lipid (X2), surfactant concentration (X3), and sonication time (X4), with regard to size (Y1), drug release (Y2), and drug loading (Y3). Furthermore, the study was carried out to investigate the brain pharmacokinetics and scintgraphic imaging of TMZ after administering it through intranasal (i.n.) and intravenous (i.v.) routes in mice.

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MATERIALS AND METHODS Materials. TMZ was obtained as a gift from Natco Pharma Ltd. (Dehradun, India). Gelucire 44/14, labrafil, labrasol, labrafac, CapmulPG8, plurol, and transcutol were obtained as gifts from Gattefosse (Saint Priest, Cedex France). Vitamin E (DL-α tocopherol) was supplied by Merck, Mumbai. Polyoxyethylene sorbitan mono-oleate (Tween 80), HPLC-grade methanol, and acetonitrile were purchased from Merck, Mumbai, India. HPLC-grade water was obtained from a MilliQ water purification system (Millipoire, MA). All other chemicals and solvents were of analytical grade. Methods. Solubility Studies. The solubility of TMZ in various components (solid lipids, liquid lipids, and surfactants) used in the formulation was determined to select the most suitable one from each category. To a known volume of 3774

DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

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Molecular Pharmaceutics Experimental Design. The present work involved a fourfactor, three-level statistical optimization study to prepare TMZ-NLCs and explore their application for i.n. delivery. This design was used to explore quadratic response surfaces and construct second-order polynomial models using Design Expert (version 8.0.0, Stat-Ease Inc., Minneapolis, MN, USA). The polynomial equation for the experimental design is given as

EE(%) = amount of entrapped drug in NLC /amount of drug added × 100 DL(%) = amount of drug encapsulated /weight of dried NLC × 100

Compatibility Study. This study was performed to ascertain the compatibility of TMZ with the excipients. Thermograms of pure TMZ and lyophilized TMZ-NLCopt were recorded using a DSC6 (PerkinElmer, Uberlingen, Germany) for the identification of changes in the melting behavior. Five milligram samples were weighed, kept in standard aluminum pans, and sealed. The samples were heated at a scanning rate of 10 °C/ min over a temperature range between 10 and 350 °C, and an inert atmosphere was maintained by purging with nitrogen. An empty pan was used as the reference. In Vitro Release Effect. In vitro release studies of all developed TMZ-NLCs were performed using a dialysis membrane (Hi-media, Mumbai, India) with a molecular weight cutoff of 10 kDa.6 A volume containing a known amount of TMZ in TMZ-disp and TMZ-NLCs was placed in a dialysis bag, and both ends were tied to prevent any leakage. The bag was dipped into 250 mL of PBS (pH 6.8) as the release medium, and the medium was stirred continuously at 50 rpm, maintaining the temperature at 37 °C ± 0.5 °C. One milliliter of the sample was withdrawn at predetermined intervals (1, 2, 4, 6, 8, 12, 24 h) and immediately replenished with fresh medium. The samples were diluted, and drug release was analyzed using HPLC.16 Data for the optimized formulation was applied to kinetic models such as the zero-order, first-order, Higuchi, Peppas, Hixson−Crowell, and Weibull models.17 Ex Vivo Transport Study Across Nasal Mucosa. Ex vivo transport studies of both TMZ-NLCopt and TMZ-disp were performed on porcine nasal mucosa.18 The porcine mucosa was obtained from a local slaughterhouse and was immediately rinsed with PBS (pH 6.8). The adhering mucous and fat were wiped with isopropyl alcohol and cut to an appropriate size. The mucosa was mounted between the donor and receiver compartments (Logan diffusion cell, USA), with the mucosal and serosal surfaces facing the donor and receiver compartments, respectively, with a surface area of 0.6 cm2. The mucosa was stabilized through a 30 min preincubation period by filling both compartments with saline solution to obtain electrophysiological equilibrium at 37 ± 0.5 °C. After stabilization, the receiver compartment was filled with PBS (pH 6.8) and stirred continuously on a magnetic stirrer at 200 rpm. One milliliter of both formulations was placed in a donor compartment, and samples were withdrawn at predetermined intervals (0.5, 1, 2, 4, 6, 8, 12, 16, 24 h) from the receiver compartment. The sample was replenished with the same medium, and after appropriate dilution, samples were analyzed for drug permeation using HPLC.16 Animal Study Design. In vivo absorption and brain uptake studies were performed according to previously reported procedures.12,19 Healthy Wistar rats (body weight, 300−350 g) of either sex were used for the in vivo studies. The animals were obtained from the Central Animal Facility at Jamia Hamdard, and the study protocol was approved by the Institutional Animal Ethical Committee of Jamia Hamdard, New Delhi. The animals (3 groups comprising 12 animals in each group) were housed in cages with free access to standard chow pellets and water, under uniform housing and environ-

Y0 = b0 + b1X1 + b2X 2 + b3X3 + b12X1X 2 + b13X1X3 + b23X 2X3 + b11X12 + b22X 2 2 + b33X32

where Y0 is the dependent variable, b0 is the intercept, b1 to b33 are regression coefficients (computed from the observed experimental values) of Y, X1, X2, and X3 (coded levels), which are the independent variables, X2i (i = 1, 2, or 3) are the interaction and quadratic terms The software-generated amounts of gelucire (X1), liquid lipid/total lipid (X2), Tween 80 (X3), and sonication time (X4) were used to prepared different batches of NLCs, and the responses observed are size (Y1), % drug release (Y2), and drug loading (Y3), as shown in Table 1. Pharmaceutical Characterization. Particle Size, PDI, and Zeta Potential. The particle size, polydispersity index, and zeta potential of the formulations were determined by photon correlation spectroscopy (PCS) using a Zetasizer (Malvern 1000 HS, Malvern Instruments, UK). TMZ-NLCs (1 mL) were resuspended in distilled water and mixed thoroughly with vigorous shaking. Before taking measurements, batches were diluted with double-distilled water until the appropriate concentration of particles was achieved to avoid multiscattering events. Surface Morphology. The sample was prepared by placing a drop of TMZ-NLCs on a copper grid and air-drying for 1 min.15 Afterward, the grid was kept inverted, and a drop of a 2 M aqueous solution of phototungstic acid (PTA) was applied for contrast enhancement. The remaining PTA was removed by absorbtion using filter paper, and the morphology was studied using TEM (Morgani 268D, USA) operating at 200 kV. A combination of bright-field imaging at increasing magnifications and of diffraction modes was used to reveal the morphology of NLCs. Drug Loading and Encapsulation Efficiency. Drug loading (DL) of the NLCs was determined as the amount of drug loaded (as a percentage) in relation to the lipid phase (matrix lipid and drug). A known volume of TMZ-NLCs was transferred to centrifuge tubes fitted with an ultrafilter (Pall Life Sciences, Mumbai, India). The sample was centrifuged at 4000 rpm (Remi centrifuge, Mumbai, India) for 30 min, and the separated NLCs were solubilized in appropriate medium and filtered through a 0.45 μm PTFE (polytetrafluoroethylene) membrane filter. The aqueous and organic phases were diluted appropriately, and the amount of TMZ was estimated. The encapsulation efficiency (EE) of the drug in the NLC dispersions was estimated by taking a known volume of NLC dispersion, which was ultracentrifuged (Remi centrifuge IEC61010, Mumbai, India) at 15 000 rpm for 15 min. The supernatant containing free drug was solubilized in methanol and filtered through a 0.45 μm nylon membrane filter; after appropriate dilution, the drug content was spectrophotometrically measured at 330 nm. The difference between the total drug and free drug indicates the actual amount of drug encapsulated. DL and EE percentages were calculated according to the following equations 3775

DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

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Molecular Pharmaceutics

aqueous 99 mTcO4 (2 mCi/mL), and the whole suspension was incubated for 15 min. The amount of SnCl2, pH of the solution, and encapsulation period are the factors that affect the maximum labeling, so these were previously optimized. The labeling efficiency of the radiolabeled formulation was determined by paper chromatography using acetone as the mobile phase. Paper (stationary phase) was cut well above the spot of the formulation and the region below the solvent front. The piece of paper was then put in a gamma counter to check the radioactivity, which ensures that the formulation has been radiolabeled. Eighty microliters of the radiolabeled formulation at a dose of 5 mg/mL was administered through the i.n. route to Wistar rats (40 μL in each nostril). Rats were anesthetized using an intramuscular injection of 0.4 mL of ketamine (50 mg/ mL) and placed on the imaging board. A single photon emission computerized tomography (SPECT, LC 75-005, Diacam, Siemens AG, Erlanger, Germany) gamma camera was used to collect images. The scintigraphy images following i.n. administration of NLCs were recorded at 1, 2, 4, 24 h postadministration.

mentally controlled conditions. During i.n. administration, the animals were held from the back in a slanted position. Three animals for each formulation per time point were used for this study. Thirty-five microliters (equivalent to 5 mg/mL TMZ) of TMZ-NLCopt (Group 1) and TMZ-disp (Group 2) was administered in each nostril via 2 cm polyethylene tubing (outer diameter, 0.965 mm; inner diameter, 0.58 mm) attached to a microliter syringe. The process was performed gently, allowing the rats to inhale all of the loading formulation. A similar dose of TMZ-disp (Group 3) was injected through the tail vein of the rats. The TMZ concentration in the blood and brain was analyzed by a previously reported HPLC method.16 Pharmacokinetic and Brain Distribution Study. The plasma and brain samples were prepared in accordance with a reported method.2,20,21 After administration of the dose, all animals underwent ether anesthesia before being euthanized by cervical dislocation. Two milliliters of blood was collected at different time points (0.5, 2, 6, 24 h) from the retinoorbital veins present in the eye of treated rats and stored in prechilled and precoated EDTA tubes. Then, it was centrifuged at 5000 rpm for 15 min to separate the plasma from the blood. The separated plasma was then mixed with internal standard, i.e., caffeine (500 ng/mL), and acetonitrile (2 mL) was added to it to precipitate the proteins. The sample was vortexed for proper mixing, and diethyl ether (2 mL) was added to extract the drug. The samples were then centrifuged at 4000 rpm for 5 min, and the supernatant was pipetted out and evaporated at ambient temperature. The dried sample was reconstituted with the mobile phase and filtered with a 0.2 μm nylon membrane filter before analysis. For brain distribution studies, the skull was cut open and the brain was carefully excised. It was quickly rinsed with saline and blotted with filter paper to get rid of blood-taint and macroscopic blood vessels as much as possible. Brain samples were obtained after administration of doses by different routes. The brain samples so collected at different time points (0.5, 2, 6, 24 h) were placed in methanol and homogenized (Fisher Scientific, Bombay, India). The plasma concentration time profiles, brain concentration time profiles, and brain-to-plasma ratios after i.v. and i.n. deliveries were calculated by pharmacokinetic software (PK functions for Microsoft Excel, Pharsight Corporation, Mountain View, CA). The maximum plasma concentration (Cmax) and time to reach maximum plasma concentration (Tmax) were estimated directly from the software, and the area under the curve (AUC0−24) between 0 and 24 h was calculated by the linear trapezoidal method. The direct drug transport percentage (DTP) and drug targeting efficiency (DTE) in the brain were calculated according to the literature as follows22,23 DTE =

DTP =

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RESULTS TMZ-NLCs prepared by a probe-sonication technique showed a large particle size with a higher PDI for all tested formulations, which made them unacceptable. The formulations prepared by the HPH technique showed a lower particle size range (141.28−220.11 nm) and PDI (0.1−0.4). All formulations showed a high encapsulation efficiency and loading efficiency that might be attributed to the smaller particle size. Therefore, the technique chosen to prepare the NLCs was HPH. Equibilirium Solubility Study. Gelucire 44/14 as a solid lipid (50.43 ± 0.84 mg/mL), vit E (51.33 ± 0.5 mg/mL) as a liquid lipid, and their blend (124.83 ± 6.33 mg/mL) proved to be the best solubilizers for TMZ, as they showed the maximum solubility. This solubility result is also supported by another report demonstrating that the solubility of a drug in a liquid lipid is higher than in a solid lipid.24 The solubility of the drug in different solid lipids was found to be in a decreasing order: gelucire 44/14 > apifil > stearic acid > glycerylmonostearate > cetylpalmitate, whereas with liquid lipids the order was vit E > castor oil > acconon > oleic acid > plurol > CapmulPG8 > labrafac > isopropyl myristate > ethyl oleate (Figure 1). Different surfactants and cosurfactants were screened to check the emulsification efficiency. The results showed that Tween 80

[AUC brain /AUC blood ](i.n.) [AUC brain /AUC blood ](i.v.) (AUC brain )i.n. − (AUCX ) (AUC brain )i.n.

Gamma Scintigraphy Study. TMZ-NLCopt was radiolabeled with technetium (99mTc) by direct labeling using aqueous stannous chloride (SnCl2) as the reducing agent. SnCl2 solution (0.02 mL, 1 mg/mL) was added to 1 mL of a formulation, and the pH was adjusted to 6.8 with 50 mM sodium bicarbonate buffer solution. The resultant mixture was filtered (0.22 μm nylon-66 membrane). To this was added

Figure 1. Solubility profile of TMZ in various lipids (n = 3). 3776

DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

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release, but a further increase in the surfactant concentration decreases the drug release. Increasing the sonication time was found to have a nonsignificant effect (p > 0.05) on drug release. Changes in the total lipid concentration lead to a gradual increase in drug release. On the basis of the response of the various dependent variables with respect to the composition of the independent variables, a formulation (TMZ-NLCopt) was selected as the optimized formulation for this study. Effect of Dependent Variables on Loading Efficiency. The highest and lowest loadings were 8.69 ± 0.58 and 4.88 ± 0.45 for F27 and F23, respectively (Table 1). It was found that with an increase in the concentration of total lipid, the loading efficiency increased. Changes in the lipid content also reduced the escape of the drug into the external phase.25,26 An increased EE was observed with an increase in the amount of liquid lipid (Figure 2c). It was observed that an increase in the surfactant concentration first increases and then decreases the loading of the drug in the NLCs. Point Prediction Optimization. Four optimum check point batches were selected based on the criteria from the optimum formulation to validate the chosen experimental design and polynomial equations. The formulations corresponding to these checkpoints were prepared and evaluated for various response properties. Subsequently, the resultant experimental data for the response properties were quantitatively compared with their predicted values shown in Table 3. Among these four checkpoints, the optimized TMZ-loaded NLCs (TMZNLCopt) were selected based on their optimum particle size and maximum drug loading and release. The optimized TMZNLCopt formulation had a gelucire concentration of 3.3%, a liquid lipid/total lipid ratio of 0.25, a surfactant concentration of 4.89%, and a sonication time of 3.85 min. The optimized TMZ-NLCopt formulation showed a particle size of 131.58 nm, drug release of 73.91%, drug loading of 8.71%, a PDI of 0.177, a zeta potential of 15.21, and a flux of 9.81 ± 0.76 μg/ cm2/h. The reason for the high entrapment and loading of TMZ might be attributed to its highly lipophilic nature. The lower PDI values of TMZ-NLCopt indicate that the size distribution of the particles was quite narrow and had a uniform size. The lower zeta potential could be attributed to particle stability in the suspension through electrostatic repulsion between particles. The repulsive interactions will be larger between particles as the zeta potential increases or decreases, leading to the formation of more stable particles with a more uniform size distribution. Surface Morphology. The surface morphology of TMZNLCopt was studied, and it was found this NLC was roughly spherical in shape. The particles appeared dark with bright surroundings, and no aggregation of the particles was seen Compatibility Study. DSC thermograms of pure TMZ and TMZ-NLCopt are shown in Figure 3. The thermal curve of pure TMZ showed an endothermic peak at 172.2 °C, whereas the lyophilized TMZ-NLCopt formulation with vit E and gelucire 44/14 did not show any peak near the melting point of the drug. This indicated that TMZ was not in its crystalline state but rather present in an amorphous state and that the drug was completely entrapped within the nanoparticles. Furthermore, none of the formulation’s excipients showed any interference with the drug. In Vitro Release Effect. The in vitro release from TMZNLCs was compared with that of TMZ-disp and plotted against time. The drug release of TMZ-disp was found to be 94.67 ± 3.56% over 8 h, whereas the release from TMZ-NLCs was in

and transcutol were the most efficient and showed the maximum solubilizing power (55.33 mg/mL ± 3.06) with the drug. Besides its emulsification efficiency, Tween 80 is a nonionic surfactant and thus is less toxic. Optimization. The predicted responses given by the statistical design and the actual responses for all three variables are shown in Table 1. The results of both the actual and predicted responses were found to be very close to each other. The closeness between these two responses shows that the results from the experimental process were accurate. The observed responses were fit to different statistical models, and the best fit model for all three dependent variables was found to be a quadratic model with a coefficient of correlation nearly equal to 1 (Table 2). The value of the correlation coefficient Table 2. Summary of Statistical Parameters for Responses Y1 (Size), Y2 (Drug Release), and Y3 (Drug Loading) for Fitting to a Different Model statistical parameter 2

R adjusted R2 predicted R2 SD %CV model

size (nm)a

drug release (%)b

drug loading (%)c

0.9988 0.9974 0.9939 1.1 0.63 quadratic

0.9959 0.9911 0.9771 1.02 1.52 quadratic

0.9923 0.9833 0.9569 0.14 1.91 quadratic

Size = +171.72 + 24.5X1 − 10.11X2 − 15.07X3 − 1.63X4 − 1.5X1X2 + 1.97X1X3 + 0.39X1X4 − 7.33X2X3 + 3.55X2X4 − 0.61X3X4 + 1.73X12 − 3.77X22 + 9.3X32 − 0.45X42. bDrug release = +72.13 − 9.83X1 + 4.26X2 + 6.54X3 + 1.15X4 + 10.81X1X2 − 5.18X1X3 + 1.35X1X4 + 3.36X2X3 − 0.25X2X4 − 0.75X3X4 − 1.05X12 − 2.21X22 − 8.55X32 + 0.21X42. cDrug loading = +7.3 + 1.1X1 + 0.79X2 + 0.67X3 − 0.17X4 − 0.28X1X2 + 0.15X1X3 + 0.06X1X4 + 0.043X2X3 + 0.19X2X4 + 0.11X3X4 + 0.013 X12 − 0.2 X22 − 0.44X32 − 0.16X42. a

(R2; Table 2, equation 1 in footnote a) was found to be 0.9988, indicating a good fit. The predicted R2 of 0.9939 is in reasonable agreement with the adjusted R2 of 0.9934. Adequate precision was used to measure the signal-to-noise ratio and found to be 13.231, indicating an adequate signal for the size. The value of the correlation coefficient (R2; Table 2, eq 2 in footnote b) was found to be 0.9959, indicating a good fit. The predicted R2 of 0.9711 is in reasonable agreement with the adjusted R2 of 0.9911. The signal-to-noise ratio of 11.411 indicates an adequate signal for drug release. Table 2, equation 3 in footnote c, shows an R2 value of 0.9923, indicating a good fit of the model, and the predicted R2 of 0.9569 is in reasonable agreement with the adjusted R2 of 0.9833. The signal-to-noise ratio of 15.57 indicates an adequate signal for drug loading. Response Analysis for Optimization. Effect of Dependent Variables on Size. The particle size of the TMZ-NLCs was found to be in the range 137.66−220.28 nm (Table 1). The size of the particles increases with the increase in the solid lipid concentration, whereas with an increase in the amount of liquid lipid to total lipid, the particle size decreases (Figure 2A). The increase in the amount of liquid lipid reduces the particle size. An increase in the surfactant causes a significant decrease in size (p > 0.05). Effect of Dependent Variables on Drug Release. Drug release from all formulations was found to be between 54.33 and 71.56%. As shown in Figure 2b, increasing the amount of surfactant causes a significant decrease in drug release. The initial increase in surfactant concentration increases the drug 3777

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Figure 2. Three-dimensional response surface plots showing the influence of the independent variables on size (A−B), drug release (C−D), and drug loading (E−F) for TMZ-NLCs.

Table 3. Temzolomide NLC Formulation Batches with Their Actual and Predicted Values experimental value Y1 (size, nm)

dependent variables

Y2 (% drug release)

Y3 (% drug loading)

S. No.

X1 - gelucire (%)

X2 - liquid lipid:total lipid

X3 - surfactant (%)

X4 - sonication time (min)

actual

predicted

actual

predicted

actual

predicted

BI B2 B3 B4

3.3 3.8 2.8 4.3

0.25 0.35 0.15 0.25

4.89 3.27 2.5 5.67

3.85 4.55 2.35 5.25

131.58 129.56 138.32 121.45

133.43 130.53 137.87 122.45

73.91 70.56 74.12 67.91

72.22 68.32 75.23 65.89

8.71 7.79 8.11 8.55

8.98 7.39 8.17 7.98

Figure 3. Differential scanning calorimetry of pure TMZ and lyophilized TMZ-NLCopt.

the range of 44.44−71.56% over 24 h (Table 1). TMZ-NLCopt showed a release over 24 h, whereas TMZ-disp releases more

than 80% of the drug within 6 h (Figure 4), which proved the sustained release action of TMZ-NLCopt. The data obtained 3778

DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

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concentration was lower for TMZ-disp (i.n., i.v.) than TMZNLCopt because of its solubility in plasma, ensuring its rapid distribution, elimination, and slower release and leading to its lower clearance. The brain/plasma ratio of NLC was higher at all time points compared to that of the dispersion given by either the i.n. or i.v. routes, proving the direct nose-to-brain transport pathway of the drug via nasal administration (Table 5 and Figure 5). Gamma Scintigraphy Study. The accumulation of radioactivity from TMZ-NLCopt in the brain can be clearly seen in Figure 6 after 0.5, 2, and 6 h, respectively. After 0.5 h, radioactivity can be seen only in the nostrils. After 2 and 6 h, the accumulation of radioactivity in the brain is clearly visible. A good amount of activity was also noticed in the esophagus and in the abdominal region, which could be due to absorption of part of the formulation from the gastrointestinal tract.28,29 It can be clearly seen that the brain levels were higher while blood levels were lesser after i.n. administration compared with those from the i.v. route.

Figure 4. Comparative in vitro drug release profiles of TMZ-NLCopt and TMZ-disp.

from the in vitro release experiments was fitted to various release models, and the best fit was the Higuchi model (0.988), followed by the first-order release model (0.981). n-values were found for Fickian diffusion (0−0.5), non-Fickian (0.5−1), zeroorder (1), and super diffusion (>1) for spherical particles.27,32 The obtained value of n = 0.075 for the NLC showed that the release behavior was Fickian diffusion. Ex Vivo Transport Across Nasal Mucosa. The steadystate flux of TMZ-disp after 24 h was found to be 4.23 μg/cm2/ h, whereas from TMZ-NLCopt, it was 9.815 μg/cm2/h. The flux and permeability coefficient for NLC were almost double those obtained for TMZ-disp. The significantly high drug permeation effect of the NLC (P < 0.05) compared to that of the dispersion was due to the presence of surfactant on the surface of the NLC, which is well-known to function as a penetration enhancer. Pharmacokinetic and Brain Distribution Study. The brain and plasma concentrations after administration of TMZNLCopt (i.n.), TMZ-disp (i.n.), and TMZ-disp (i.v.) with their pharmacokinetic parameters are shown in Table 4. The mean concentration of TMZ at different time points in plasma and brain were estimated. The TMZ concentration from NLCopt was found to be high at all time points in comparison to that of TMZ-disp (i.n.). This might be attributed to the higher nasal retention effect of the NLC formulation at the target site. The brain targeting efficiency was increased, i.e., 457%, when TMZ was encapsulated in NLCs to enhance its permeation into the nasal mucosa as compared to TMZ-disp (i.n.), with an enhancement of only 169.7%. TMZ-disp distributed rapidly in the region, resulting in lower absorption compared to that of TMZ-NLC. The initial plasma concentration (0.5 h) was lower for TMZ-NLCopt than TMZ-disp (i.n., i.v.), possibly because TMZ was released slowly from TMZ-NLCopt for an extended period of time. The free drug was available for distribution only after its release from the dispersion and was comparatively high as compared to that of TMZ-disp (i.v.). After 1.5 h, the plasma

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DISCUSSION Optimization. TMZ-NLCs were formulated and optimized through a four-factor, three-level Box−Behnken statistical design. We evaluated different compositions and parameters, such as lipid concentration, lipid ratio, surfactant concentration, and sonication time, to obtain a small particle size, maximum loading, and maximum encapsulation efficiency with optimum drug release. Fitting the data from our observed responses to various models was attempted, and the best fit model for all three dependent variables was found to be the quadratic model (Table 2). With the increase in total lipid concentration, the particle size increased due to a decrease in the emulsifying efficiency of the surfactant and an increase in particle agglomeration. As there is an increase in the amount of liquid lipid, a reduction in the particle size was observed because of a decrease in the viscosity of the formulation and the surface tension inside the NLCs. An increase in total lipid concentration increased the loading efficiency significantly by providing more space for the accommodation of the drug as an increment of the lipid content and also reducing the escape of the drug into the external phase. Also, as the concentration of solid lipid (gelucire) increases with an increase in the total lipid concentration, it solubilizes the drug due to the presence of fatty acid mono-, di-, and triglycerides. A significant increase in EE was obtained as the liquid lipid concentration increased because more of the drug particles were entrapped inside the oil enriched lipid core. This can be explained due to the presence of more imperfections in the highly ordered solid lipid crystals due to the incorporation of a spatially incompatible liquid lipid. Increasing the amount of total lipid causes a significant decrease in the cumulative drug release percentage. This is due to the fact that increasing the lipid concentration increases the size of

Table 4. Pharmacokinetic Distribution Profiles of TMZ-NLC (i.n.) and TMZ-disp (i.n., i.v.) in Rats formulation

organ/plasma

TMZ-NLCopt (i.n.)

brain plasma brain plasma brain plasma

TMZ-disp (i.n.) TMZ-disp (i.v.)

0.5 h (ng/mL) ± SD 1815.6 1336.0 3479.6 2724.9 783.6 5590.2

± ± ± ± ± ±

2 h (ng/mL) ± SD

1.38 31.8 4.09 0.54 0.36 14.42

4606.3 1636.5 2122.4 2030.8 1916.9 2739.5 3779

± ± ± ± ± ±

1.46 5.22 15.07 2.45 1.52 4.11

6 h (ng/mL) ± SD 1432.4 667.8 365.6 726.8 180.8 177.1

± ± ± ± ± ±

1.70 6.20 15.14 38.75 0.80 15.98

24 h (ng/mL) ± SD 1189.1 85.1 263.1 444.6 2.96 5.88

± ± ± ± ± ±

19.23 17.24 2.00 33.43 0.82 2.15

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Table 5. Brain/Plasma Ratios and Absorption Profiles of TMZ at Different Time Intervals for TMZ-NLCopt (i.n.) and TMZdisp (i.n., i.v.) formulation

brain/plasma

Cmax (ng/mL)

Tmax (min)

AUC0−24h

elimination rate constant (Ke)

TMZ-NLCopt (i.n.)

brain plasma brain plasma brain plasma

4606 1633.4 3279.6 3419.3 1916.9 5590

1.5 1.5 0.5 0.5 0.5 0.5

37863.3 13684.2 13394 19559.8 6437.8 10652.2

0.03 0.124 0.08 0.06 0.25 0.26

TMZ-disp (i.n.) TMZ-disp (i.v.)

particulate system, which causes electrostatic repulsion among the nanoparticles, preventing the formation of aggregates. In Vitro Release Effect. The in vitro drug release profile of TMZ from TMZ-NLCs showed a sustained release of the drug from the formulation. The release from the NLCs was found to be fast over the initial time period, followed by prolonged release over a period of 24 h. The initial rapid release of the drug may be due to the release of TMZ from the NLCs surface, whereas at a later stage, TMZ may be constantly released from the core of NPs, which is responsible for the prolonged release. A kinetic analysis of the in vitro release profile of TMZ-NLCopt was done to ascertain the release order and was found to follow the Higuchi model. There are many factors that can influence the release of a drug from NLCs, including the solubility of the drug in the lipid, the lipid matrix and its concentration, and the size of the particles.31 Ex Vivo Transport Across Nasal Mucosa. The steadystate flux achieved from a control formulation was found to be 4.23 μg/cm2, whereas from the NLC formulation after 24 h of study, it was recorded as 9.815 μg/cm2/h. The higher flux for TMZ-NLCopt was due to the nanosized particles formed and the presence of surfactants on the surface of the NLCs. The enhancement ratio of TMZ-NLCopt was significantly higher than TMZ-disp over 24 h, which could be attributed to the lipophilic nature of the drug. The higher lipophilicity of TMZ gives greater permeation through the nasal mucosa.33 The increase in permeation shows the contribution of neuronal transport, bypassing the BBB. Pharmacokinetic and Brain Distribution Study. This effect is due to direct nose-to-brain drug delivery, as compared to an oral solution, which shows first-pass hepatic metabolism and has to cross the gastrointestinal barrier and BBB before finally reaching the brain. This may be due to the fact that nanoformulations show increased nose-to-brain drug delivery as compared to drug solution/dispersion of an equivalent dose, as shown earlier by in vitro permeation studies. Protection of the

Figure 5. Brain/plasma ratio in rats following administration of TMZNLCopt (i.n.), TMZ-disp (i.n.), or TMZ-disp (i.v.).

the nanoparticle, thereby decreasing the effective surface area available to interact with the releasing medium and hence decreasing the drug release. Surfactant concentration significantly affects the EE and drug release. It first increases the EE and then decreases the EE after its concentration increases. This decrease maybe attributed to the entrapment of surfactant molecules into the NLCs at higher concentrations of surfactant. An increase in the drug release characteristics until a particular concentration of surfactant is reached is due to its ability to decrease the particle size, and a further increase in the surfactant concentration causes a decrease in EE, which decreases the amount of drug released. It can be stabilized by two different mechanisms: steric stabilization and electrostatic repulsion. Tween 80 and poloxamer 188, being nonionic stabilizers, stabilize the system by steric stabilization due to the presence of dense hydrophobic tails, which do not allow particles to come close to each other; thus, particle agglomeration is prevented.30 These surfactants also impart a negative zeta potential of 11.75 ± 2.96 mV to the nano-

Figure 6. Gamma scintigraphic images of rat following intranasal administration of TMZ-NLCopt at different time points. 3780

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excellent plateform for brain targeting. Expert Opin. Drug Delivery 2013, 10 (7), 957−972. (4) Bahadur, S.; Pathak, K. Physicochemical and physiological considerations for efficient nose-to-brain targeting. Expert Opin. Drug Delivery 2012, 9, 19−31. (5) Trinh, A. V.; Patel, S. P.; Hwu, W. J. The safety of Temozolomide in the treatment of malignancies. Expert Opin. Drug Saf. 2009, 8, 493− 499. (6) Alam, M. I.; Baboota, S.; Ahuja, A.; Ali, M.; Ali, J.; Sahni, J. K. Nanostructured lipid carrier containing CNS Acting drug: formulation, optimization and evaluation. Curr. Nanosci. 2011, 7, 1014−1027. (7) Das, S.; Ng, W. K.; Tan, R. B. H. Are nanostructured lipid carriers (NLCs) better than solid lipid nanoparticles (SLNs): Development, characterization and comparative evaluations of clortrimazole-loaded SLNs and NLCs. Eur. J. Pharm. Sci. 2012, 47, 139−151. (8) Huang, G.; Zhang, N.; Bi, X.; Dou, M. Solid lipid nanoparticles: Potential reduction of cardial and nephric toxicity. Int. J. Pharm. 2008, 355, 314−320. (9) Illum, L. Nasal drug delivery-possibilities, problems and solutions. J. Controlled Release 2003, 87, 187−198. (10) Fazil, M.; Md, S.; Haque, S.; Baboota, S.; Ali, J. Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting. Eur. J. Pharm. Sci. 2012, 47, 6−15. (11) Seju, U.; Kumar, A.; Sawant, K. K. Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-to-brain delivery: In vitro and in vivo studies. Acta Biomater. 2011, 7, 4169−4176. (12) Eskandari, S.; Varshosaz, J.; Minaiyan, M.; Tabbakhian, M. Brain delivery of valporic acid via intranasal administration of nanostructured lipid carriers: in vivo pharmacodynamic studies using rat electroshock model. Int. J. Nanomed. 2011, 6, 363−371. (13) Alam, M. I.; Baboota, S.; Ahuja, A.; Ali, M.; Ali, J.; Sahni, J. K. Intranasal administration of nanostructured lipid carriers containing CNS acting drug: Pharmacodynamic studies and estimation in blood and brain. J. Psychiatr. Res. 2012, 46, 1133−1138. (14) Jia, L.; Zhang, D.; Li, Z.; Duan, C.; Wang, Y.; Feng, F.; Wang, F.; Liu, Y.; Zhang, Q. Nano-structured lipid carriers for parenteral delivery of silybin: biodistribution and pharmacokinetic studies. Colloids Surf., B 2010, 80, 213−218. (15) Bhaskar, K.; Anbu, J.; Ravichandiran, V.; Venkateswarlu, V.; Rao, Y. Lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in vitro, ex vivo and in vivo studies. Lipids Health Dis. 2009, 8, 6. (16) Jedynak, L.; Puchalska, M.; Zezula, M.; Laszcz, M.; Luniewski, W.; Zagrodzka, J. Stability of sample solution as a crucial point during HPLC determination of chemical purity of Temozolomide drug substance. J. Pharm. Biomed. Anal. 2013, 83, 19−27. (17) Costa, P.; Lobo, J. M. S. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 2001, 13, 123−133. (18) Wadell, C.; Bjork, E.; Camber, O. Permeability of porcine nasal mucosa correlated with human nasal absorption. Eur. J. Pharm. Sci. 2003, 18, 47−53. (19) Haque, S.; Md, S.; Fazil, M.; Ali, J.; Baboota, S. Venlafaxine loaded chitosan NPs for brain targeting: Pharmacokinetic and pharmacodynamic evaluation. Carbohydr. Polym. 2012, 89, 72−79. (20) Fazil, M.; Md, S.; Haque, S.; Kumar, M.; Baboota, S.; Sahni, J. K.; Ali, J. Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting. Eur. J. Pharm. Sci. 2012, 47, 6−15. (21) Md, S.; Haque, S.; Fazil, M.; Kumar, M.; Baboota, S.; Sahni, J. K.; Ali, J. Optimised nanoformulation of bromocriptine for direct nose-to-brain delivery: biodistribution, pharmacokinetic and dopamine estimation by ultra-HPLC/mass spectrometry method. Expert Opin. Drug Delivery 2014, 11 (6), 827−842. (22) Vyas, T. K.; Babbar, A. K.; Sharma, R. K.; Misra, A. Intranasal mucoadhesive microemulsion of zolmitriptan: Preliminary studies on brain-targeting. J. drug Target. 2005, 13, 317−324. (23) Zhang, Q.; Jiang, X.; Jiang, W.; Lu, W.; Su, L.; Shi, Z. Preparation of nimodipine-loaded microemulsion for intranasal delivery and evaluation on the targeting efficiency to the brain. Int. J. Pharm. 2004, 275, 85−96.

drug from efflux back into the intranasal cavity may also be the reason for this effect because solutions are rapidly cleared from the nasal cavity. The effect of the nanoformulation was maintained for 24 h due to its sustained drug release profile. Tween 80 also enhances the penetration of the drug through the nasal mucosa. P-gp expressed in olfactory epithelium causes drug efflux, which is prevented by poloxamer 188, resulting in a higher drug concentration in the brain after i.n. administration as compared to that from the dispersion.12 Hence, it shows the effectiveness of the developed formulation in controlling tonic hind limb extension even after 24 h. Gamma Scintigraphic Study. This study showed a significantly higher accumulation of TMZ from TMZ-NLCopt in rat brain. Radioactivity from TMZ-NLCopt after 4 h showed a longer retention time of TMZ in the brain. The higher level of radioactivity found in the brain following i.n. delivery proved that brain targeting can be achieved by this route. The images from this treatment were consistent with the biodistribution and pharmacokinetic data. TMZ-NLCopt was found to be effective in increasing the TMZ concentration in brain. Moreover, i.n. administration further improved the brain uptake of the drug as compared with the i.v. route due to direct nose-to-brain delivery. A significant amount was also present in the gastrointestinal tract because of intragastric ingestion of the formulation by way of the throat.26

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CONCLUSIONS TMZ-loaded NLCs were developed using Box−Behnken statistical design, which gave the optimum concentrations of lipids and surfactants in the formulation to obtain the minimum size and maximum drug loading and release. The release of TMZ was found to follow zero-order kinetics with Fickian diffusion. The present results clearly showed that i.n. administration of TMZ-NLCopt in rats is efficient in maintaining the effect of TMZ. This effect was achieved due to its direct brain targeting and the increased residence time of TMZ in the brain as compared to that with the i.n. dispersion. The higher bioavailability in the brain of the NLCs compared to the dispersion was seen with lower doses, indicating that this provides a remarkable delivery route for targeting the brain through the design of an appropriate dosage form.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-9811798725. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank AIMS and INMAAS, New Delhi, India, for providing the TEM and scintigraphy studies. REFERENCES

(1) van Woensel, M.; Wauthoz, N.; Rosiere, R.; Amighi, K.; Mathieu, V.; Lefranc, F.; van Gool, S.; de Vleeschouwer, S. Formulation for intranasal delivery of pharmacological agents to combat brain diseases: a new opportunity to tackle GBM. Cancers 2013, 5, 1020−1048. (2) Portnow, J.; Badie, B.; Chen, M.; Liu, A.; Blanchard, S.; Synold, T. W. The neuropharmacokinetics of Temozolomide in patients with resectable brain tumors: Potential Implications for the current approach to chemoradiation. Clin. Cancer Res. 2009, 15, 7092−7098. (3) Pardeshi, V.; Belgamwar, S. Direct nose to brain delivery via integrated nerve pathways bypassing the blood-brain barrier: an 3781

DOI: 10.1021/acs.molpharmaceut.6b00586 Mol. Pharmaceutics 2016, 13, 3773−3782

Article

Molecular Pharmaceutics (24) Muller, R. H.; Radtke, M.; Wissing, S. A. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetics and dermatological preparations. Adv. Drug Delivery Rev. 2002, 54, 131−155. (25) Subedi, R. K.; Kang, K. W.; Choi, H. K. Preparation and characterization of solid lipid nanoparticles loaded with doxorubicin. Eur. J. Pharm. Sci. 2009, 37, 508−13. (26) Shah, K. A.; Date, A. A.; Joshi, M. D.; Patravale, V. Solid lipid nanoparticles (SLN) of tretinoin:potential in topical delivery. Int. J. Pharm. 2007, 345, 163−171. (27) Neupane, Y. R.; Srivastava, M.; Ahmad, N.; Kumar, N.; Bhatnagar, A.; Kohli, K. Lipid based nanocarrier system for the potential oral delivery of decitabine: formulation design, characterization, ex vivo, and in vivo assessment. Int. J. Pharm. 2014, 477 (1−2), 601−612. (28) Wang, D.; Gao, Y.; Yun, L. Study on brain targeting of raltitrexed following intranasal administration in rats. Cancer Chemother. Pharmacol. 2006, 57, 97−104. (29) Kumar, M.; Misra, A.; Babbar, A. K.; Mishra, A. K.; Mishra, P.; Pathak, K. Intranasal nanoemulsion based brain targeting drug delivery system of resperidone. Int. J. Pharm. 2008, 358, 285−291. (30) Wu, L.; Zhang, J.; Watanabe, W. Physical and chemical stability of drug nanoparticles. Adv. Drug Delivery Rev. 2011, 63 (6), 456−469. (31) Müller, R. H.; Runge, S.; Ravelli, V.; Mehnert, W.; Thünemann, A. F.; Souto, E. B. Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN) versus drug nanocrystals. Int. J. Pharm. 2006, 317 (1), 82−89. (32) Peppas, N. A. Analysis of Fickian and non-Fickian drug release from polymers. Pharmac Acta Helv. 1985, 60, 110−111. (33) Misra, A.; Ganesh, S.; Shahiwala, A. Drug delivery to the central nervous system. J. Pharm. Pharm. Sci. 2003, 6, 252−273.

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