Brain Targeting of Temozolomide via the Intranasal Route Using Lipid

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Brain Targeting of Temozolomide via the Intranasal Route Using lipid based nanoparticle: Brain Pharmacokinetic and Scintigraphic Analysis Anam Khan, Syed Sarim Imam, Mohammed Aqil, Abdul Ahad, Yasmin Sultana, Asgar Ali, and Khalid Khan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00586 • Publication Date (Web): 23 Sep 2016 Downloaded from http://pubs.acs.org on September 25, 2016

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Molecular Pharmaceutics

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Brain Targeting of Temozolomide via the Intranasal Route Using lipid based nanoparticle: Brain Pharmacokinetic and Scintigraphic Analysis Anam Khana, Syed Sarim Imamb, Abdul Ahadc, Mohammed Aqila*, Yasmin Sultanaa, Asgar Alia, Khalid Khand.

a. Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India. b. Department of Pharmaceutics, Glocal School of Pharmacy, Glocal university, Saharanpur, 247121, Uttar Pradesh, India. c. Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451. Saudi Arabia. d. Fermish Clinical Technologies Private. Limited, Noida, Uttar Pradesh, India.

*Corresponding Author: Dr. M. Aqil Sr. Assistant Professor Department of Pharmaceutics, Faculty of Pharmacy Hamdard University, New Delhi-110 062, India Tel: +91-9811798725 E-mail: [email protected]

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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. The formulation was optimized by applying four factor three levels Box Behnken design. The developed formulations and their functional relationships between independent variables on dependent variables were observed. The independent variables used in the formulation were gelucire (X1), liquid lipid:total lipid (X2) tween 80 (X3), sonication time (X4) and their effect were observed on size (Y1), % drug release (Y2) and drug loading (Y3). The optimized TMZ-NLC was further evaluated for surface morphology, ex-vivo permeation study and in vivo study. The all TMZ-NLC formulations showed size in nanometer range with high drug load and prolonged drug release. The optimized formulation (TMZ-NLCopt) showed entrapment efficiency (81.64 ± 3.71%), zeta potential (15.21± 3.11mV) and poly dispersity index (less than 0.2). The enhancement ratio was found to be 2.32 times in comparison to control formulation (TMZ-disp). The in-vivo study in mice showed brain/blood ratio of TMZ-NLCopt was found to be significantly higher as compared with TMZ disp (i.n, i.v.). The scintigraphy image of mice brain showed presence of high concentration of TMZ. The AUC ratio of TMZ-NLCopt to TMZ-disp in the brain was the highest among the organs. The finding of this study substantiates the existence of direct nose-tobrain delivery route for NLCs.

Key words- Temozolomide, Brain targeting, NLCs, Pharmacokinetic, Gamma scintigraphy


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Introduction The major hurdle for pharmacological management of aggressive malignant brain tumor is the presence of blood brain barrier [1]. Among the other routes, nasal route has gained one more step of preference as it is the only route of drug administration that can target the brain with avoidance of systemic side effects by passing blood brain barrier utilizing olfactory or trigeminal nerves [2-4]. The neuronal link between the nasal mucosa and brain provides a unique pathway for the non-invasive delivery of the therapeutic agents. TMZ is an alkylating agent having capability of crossing BBB and must be administered in systemic high doses due to short halflife, only 20% of TMZ with respect to systemic dose reaches to the brain [2]. TMZ was found to be the most effective antineoplastic agents for treating high-grade metastatic melanoma and glioma. It required high systematic doses to reach therapeutic brain levels, which simultaneously brings about number of side effects. The various side effects includes headache, nausea, vomiting, fatigue, bone marrow depression and oral ulcerations [5]. Nano lipid carriers (e.g., NLC) are considered to be a promising dosage forms to deliver thee drug without any modification to the drug molecule. This delivery system was found to be ideal for brain targeting because of their rapid uptake, bioacceptability and biodegradability. The absence of burst effect and easy scale up technique make them promising carriers for drug delivery [6]. Nanostructured lipid carrier system is prepared by using the blend of solid lipids and liquid lipids preferably in a ratio, the differences in the structures of the solid and liquid lipids, there is formation of a perfect crystal mixture. The mixture accommodates the drugs in molecular form or in amorphous clusters [7]. NLCs also show high drug loading for both lipophilic and hydrophilic drugs [8,9]. NLC by virtue of their lipophilic nature and low particle size are widely explored as a delivery system to enhance brain uptake for a number of drugs. It is prepared by using the blend of solid lipids and liquid lipids preferably in a ratio, the differences in the structures of the solid and liquid lipids, there is formation of a perfect crystal mixture. The mixture accommodates the drugs in molecular form or in amorphous clusters [4]. NLC have been investigated for the delivery of numerous therapeutic substances and shown effective therapeutic effects in the studies performed in animals [10-12]. Objectives of this study were to use Box-Behnken design to establish the functional relationships between four operating variables i.e. solid lipid concentration (X1), ratio of liquid lipid to total lipid (X2), surfactant concentration (X3) and sonication time (X4) on responses of



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particle size (Y1), drug release (Y2) and drug loading (Y3). Further, the study was carried out to investigate the brain pharmacokinetics and scintgraphic imaging of TMZ after administering through intranasal (i.n) and intravenous (i.v) routes in mice. Materials and Methods Materials TMZ was obtained as a gift sample from Natco Pharma Ltd. (Dehradun, India). Gelucire 44/14, Labrafil, Labrasol, Labrafac, CapmulPG8, Plurol and Transcutol® were obtained as gift samples from Gattefosse (Saint Priest, Cedex France). Vitamin E (DL-alpha tocopherol) was supplied by Merck, Mumbai. Poly-oxyethylene sorbitan mono-oleate (Tween 80), HPLC-grade methanol and acetonitrile were purchased from Merck, Mumbai, India. HPLC-grade water was obtained from Milli-Q 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) to be used in the formulation was determined to select the most suitable one out of each category. To a known volume of excipient, excess drug was added, mixed for 2 min and sonicated to dissolve the drug. This mixture was kept on shaker for 8–12 h to complete solubilize the drug in lipids. The contents were then centrifuged at 5000 rpm for 15 min. and aliquots of supernatant were diluted appropriately and analyzed using a UV spectrophotometer (Shimadzu 1800, Japan) at 330 nm. In 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 clear solution of molten lipid. Formulation approach The high pressure homogenization (HPH) technique (Heidolph, Diax 900, Schwabach, Germany) followed by ultrasonication (Altrasonics India, Mumbai, India) was used to prepare and optimize TMZ-NLCs [13]. Briefly, Vit. E (as liquid lipid) mixed with melted Gelucire (as solid lipid) at 5ºC above melting temperature and TMZ was added in it. The hot surfactant solution which consisted of tween 80:transcutol (6:4) was dispersed in above TMZ added lipid phase. The obtained primary emulsion was ultrasonicated for 10 min and homogenized, further it


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was cooled down to form TMZ-NLCs (Table 1). TMZ-NLCs obtained were subjected to freeze drying using a freeze dryer (Martin Christ, Germany) at a pressure lower than 0.5 milibar to obtain solid form of the NLCs. Prior to the drying process the NLC dispersion was frozen in a freezer 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]. Experimental design The present work involved 4 factor 3 level statistical optimization study to prepare temozolomide NLCs and explore its application for intranasal delivery. The design was used to explore quadratic response surfaces and construct second order polynomial models with Design Expert® (Version 8.0.0, Stat-Ease Inc., Minneapolis, MN, USA). The polynomial equation of experimental design generated is given as: Y0= b0+ b1X1+ b2X2+b3X3+b12X1X2+b13X1X3+b23X2X3+b11X12+b22X22+b33X32. where, Y0 is the dependent variable, b0 is the intercept, b1 to b33 is the regression coefficients (computed from observed experimental values of Y, X1, X2 and X3 (coded levels) is the independent variables, X2i (i = 1, 2 or 3) = interaction and quadratic terms The software generated amount of the gelucire (X1), liquid lipid:total lipid (X2), tween 80 (X3) and sonication time (X4) were used to prepared different batches of the NLCs and their observed responses are taken as size (Y1), % drug release (Y2) and drug loading (Y3) shown in Table I. Pharmaceutical characterization Particle size, PDI and zeta potential. The particle size, polydispersity index and zeta potential of the formulations was determined by Photon correlation spectroscopy (PCS) using a zetasizer (Malvern 1000 HS, Malvern Instruments, UK). One mL of TMZ-NLCs was resuspended in the distilled water and mixed thoroughly with vigorous shaking. Before measurement, 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 dropping a drop of TMZ-NLC on copper grid and air dried for 1 min [15]. Afterwards, the grid was kept inverted and a drop of 2M aqueous solution of phototungstic acid (PTA) was applied for contrast enhancement. The remaining PTA was



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removed by absorbing on a filter paper and the morphology was studied using TEM (Morgani 268D, USA) operating at 200 KV. Combination of bright field imaging at increasing magnification and of diffraction modes was used to reveal the morphology of NLC. Drug-loading and encapsulation efficiency Drug loading (DL) of NLC was determined as amount of loaded drug in percent related to the lipid phase (matrix lipid and drug). A known volume of TMZ- NLC was transferred to the 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., separated NLC was solubilized

in

appropriate

medium

and

was

filtered

through

0.45

µm

PTFE

(polytetrafluoroethylene) membrane filter. The aqueous and organic phase was diluted appropriately and the amount of TMZ was estimated. The Encapsulation efficiency (EE) of drug in NLC dispersion was estimated by taking a known volume of NLC dispersion which was centrifuged usingultracentrifuge (Remi centrifuge IEC-61010, Mumbai, India) at 15000 rpm for 15 min. The supernatant containing free drug was solubilized in methanol, filtered through 0.45µm nylon membrane filter and after appropriate dilution, the drug content was measured at 330nm UV spectrophotometrically. The difference between total drug and free drug indicates the actual amount of drug encapsulated. DL and EE percentage were calculated according to following equation: 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 by means of DSC6 (Perkin Elmer, Uberlingen, Germany) for the identification of changing in melting behavior. 5 mg 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 10–350◦C and inert atmosphere was maintained by purging with nitrogen. An empty pan was used as reference. In-vitro release effect In-vitro release study of all developed TMZ-NLCs were performed using dialysis membrane (Himedia, Mumbai, India) with molecular weight cut off 10k Dalton [6]. A volume containing known amount of TMZ in TMZ-disp and TMZ-NLCs were placed in a dialysis bag and both


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ends were closely tied to prevent any leakage. The bag was dipped into 250ml of PBS (pH-6.8) as release medium and stirred continuously at 50 rpm maintaining the temperature at 37°C±0.5°C. One mL of the sample were withdrawn at predetermined intervals (1, 2, 4, 6, 8, 12, 24 hrs.) and immediately replenished with the fresh medium. The samples were diluted and drug release was analyzed using HPLC [16]. Data so obtained for the optimized formulation was treated to kinetic models such as zero order, first order, Higuchi model, Peppas model, HixsonCrowell model, and Weibull model [17]. Ex-vivo transport study across nasal mucosa The ex-vivo transport study of the both TMZ-NLCopt and TMZ-disp were performed on porcine nasal mucosa [18]. The porcine mucosa was obtained from a local slaughter house and was immediately rinsed with PBS (pH-6.8). The adhering mucous and fat was wiped with isopropyl alcohol and cut to an appropriate size. The mucosa was mounted between the donor and receiver compartment (Logan Diffusion cell, USA) with the mucosal and serosal surfaces facing the donor and receiver compartment respectively, with surface area of 0.6 cm2. The mucosa was stabilized though a 30 min pre-incubation period by filling both the compartments with saline solution for electrophysiological equilibrium at 37 ± 0.5°C. After stabilization, the receiver compartment was filled with the PBS (pH-6.8) and stirred continuously on a magnetic stirrer at 200 rpm. One ml of both formulations were placed in a donor compartment and samples were withdrawn at predetermined intervals (0.5, 1, 2, 4, 6, 8, 12, 16, 24 hrs) from receptor compartment. The sample replenished with the same medium and after appropriate dilutions, samples were analyzed for drug permeation using HPLC [16]. Animal Study design In vivo absorption and brain uptake studies were performed according to the procedure reported [12,19]. Healthy Wistar rats (body weight- 300-350 gm.) of either sex were used for the in vivo studies. The animals were obtained from the Central Animal Facility at Jamia Hamdard, and 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 in the environmentally controlled conditions. The animals during intranasal administration were held from the back in slanted position. Three animals for each formulation per time point were used for this study. Thirty five µL (equivalent to 5mg/ml of TMZ) of TMZ-NLCopt (Group 1), and



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TMZ-disp (Group 2) were administered in each nostril via 2cm polyethylene tubing (outer diameter: 0.965mm, inner diameter: 0.58mm) attached to a microlitre syringe. The process was performed gently allowing the rats to inhale all the loading formulation. Similar dose of TMZdisp (Group 3) was injected through the tail vein of rats. The presence of TMZ concentration in blood and brain were analyzed by reported HPLC method [16]. Pharmacokinetic and brain distribution study The plasma and brain samples were prepared in accordance with the reported method [2,20,21]. After administration of the dose, all the animals underwent ether anesthesia before their sacrifice by cervical dislocation method. Two milliliters of blood was collected at different time points (0.5, 2, 6, 24 hrs) from 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 blood. The above separated plasma was then mixed with the internal standard i.e caffeine (500ng/ml) and acetonitrile (2ml) was added to it to precipitate the proteins. The sample was vortexed for proper mixing and diethyl ether (2ml) was added to extract the drug out. The samples were then centrifuged at 4000 rpm for 5 min and supernatant was pipette out and evaporated at ambient temperature. The dried sample was reconstituted with the mobile phase, filtered with 0.2µm nylon membrane filter before analysis. While for brain distribution study, the skull was cut open and 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. The brain samples were obtained after administration of dose by different routes. The brain samples so collected at different time points (0.5, 2, 6, 24 hr) were placed in methanol and homogenized (Fisher Scientific, Bombay, India). Plasma concentration time profile, brain concentration time profile and brain to plasma ratio after i.v and i.n delivery 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 area under curve (AUC0-24) between 0 to 24hr was calculated by the linear trapezoidal method. The direct drug transport percentage (DTP) and drug targeting efficiency (DTE) in brain were calculated according to the reported literature as following [22,23]. (i . n )

[AUCBrain / AUCBlood ] DTE = [AUCBrain / AUCBlood ]

(i . v )


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DTP =

( AUC Brain )i.n − ( AUC X ) ( AUC Brain )i.n

Gamma scintigraphy study The TMZ-NLCopt was radiolabeled with technetium (99mTc) by direct labeling method using aqueous stannous chloride (SnCl2) as reducing agent. 0.02 ml of stannous chloride solution (1mg/ml) was added in one mL of formulation and pH was adjusted to 6.8 with 50mM sodium bicarbonate buffer solution. The resultant mixture was filtered (0.22µm nylon 66 membrane) and to this, aqueous 99mTcO4 (2mci/ml) was added and the whole suspension was incubated for 15 min. The amount of stannous chloride, pH of the solution and encapsulation period are the factors which affect maximum labeling so all these were previously optimized. The labeling efficiency of radiolabelled formulation was determined by paper chromatography using acetone as mobile phase. Paper (stationary phase) was cut well above the spot of formulation and the region below the solvent front. The piece of paper was then put in gamma counter to check the radioactivity which ensures that the formulation has been radiolabelled. 80µl of radiolabelled formulation at a dose of 5mg/ml was administered through intranasal route to wistar rat (40µl in each nostril). The rat was anaesthetized using intramuscular injection of 0.4 ml ketamine (50 mg/ml) and placed on the imaging board. 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 intranasal administration of NLC were recorded at 1, 2, 4, 24 hr post administration. Results The TMZ -NLCs were prepared by probe-sonication technique showed large particle size with higher PDI for all tested formulation which could not be accepted due to large particle size and high PDI. The prepared formulations by HPH technique showed lower particle size range (141.28-220.11nm) and PDI (0.1-0.4). All the formulations showed high encapsulation efficiency and loading efficiency that might be attributed to the smaller particle size of the formulation. Therefore, the final technique chosen for preparation of NLC was HPH. Equibilirium solubility study Gelucire 44/14 as solid lipid (50.43±0.84mg/ml) and Vit. E (51.33±0.5mg/ml) as liquid lipid and their blend (124.83±6.33mg/ml) proved to be the best solubilizers for TMZ, as they showed the maximum solubility. This result of solubility is also supported by another report that the



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solubility of drug in liquid lipid is higher than solid lipid [24]. The solubility of drug in different solid lipids was found to be in a decreasing order: Gelucire 44/14>Apifil> Stearic acid>Glycerylmonostearate> Cetylpalmitate while with liquid lipids, it was like Vit E> Castor oil > Acconon> Oleic acid > plurol > CapmulPG8 >Labrafac > Isopropyl myristate > Ethyl oleate (Fig. 1). Different surfactants and co-surfactants were screened to check the emulsification efficiency. The results showed that Tween 80 and transcutol were the most efficient and showed maximum solubilizing power (55.33 mg/ml ±3.06) with drug. Apart from emulsification efficiency, Tween 80 is non-ionic surfactant and thus, less toxic. Optimization The predicted given by design expert statistical design and actual response for all the three variables were shown in Table I. The results of both actual and predicted responses were found to be very close to each other. The closeness between these two responses shows that result found from experimental process was accurate. The observed responses were fit to different statistical models and observed that best fit model for all the three dependent variables was found to be quadratic model with coefficient of correlation nearly equal to one (Table 2). The value of correlation coefficient (R2) of Eq. (1) was found to be 0.9988, indicating good fit. The “Predicted R-Squared” of 0.9939 is in reasonable agreement with the “Adjusted R-Squared” of 0.9934. “Adequate Precision” measures the signal to noise ratio was found to be 13.231, indicates an adequate signal for size. The value of the correlation coefficient (R2) of Eq. (2) was found to be 0.9959, indicating good fit. The “Predicted R-Squared” of 0.9711 is in reasonable agreement with the “Adjusted R-Squared” of 0.9911. The signal to noise ratio of 11.411 indicates an adequate signal for drug release. The equation 3 showed R-squared value of 0.9923, indicating good fit model and “Predicted R-Squared” of 0.9569 is in reasonable agreement with “Adjusted R-Squared” 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 variable on size The particle size of TMZ-NLCs was found to be in range of 137.66 to 220.28nm (Table 1). The size of particle increases with increase in solid lipid concentration whereas, with increase in the amount of liquid lipid to total lipid concentration particle size decreases (Fig. 2A). The increase


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in the amount of liquid lipid reduces particle size. Increase in surfactant causes a significant decrease in size (p>0.05). Effect of dependent variable on drug release The drug release from all formulations was found to be between 54.33 -71.56%. As shown in Fig. 2b, increase in the amount of surfactant causes significant decrease in % drug release. Initial Increase in surfactant concentration increases drug release, a further increase in surfactant concentration decreases the drug release. Increase in sonication time was found to have nonsignificant effect (p>0.05) on drug release. The increment in total lipid concentration leads to gradual increase in drug release. On the basis of the response of various dependent variables with respect to composition of independent variables, formulation was selected as optimized formulation for the study. Effect of dependent variable on loading efficiency Highest and lowest loading was 8.69±0.58 and 4.88±0.45 for F27 and F23 respectively (Table 1). It was found that with increase in concentration of total lipid, loading efficiency increases. Increment of the lipid content also reduces the escaping of drug into the external phase [25,26]. An increased EE was observed with the increase in the amount of liquid lipid (Fig. 2c). It was observed that increase in surfactant concentration first increases and then decreases the loading of drug in NLC. 3.3.4. Point prediction optimization The four optimum checkpoints batches were selected based on the criteria from 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 of response properties were quantitatively compared with that of their predicted values shown in Table 3. Among these four check points the optimized Temozolomide loaded Nanolipid carrier (TMZ-NLCopt) was selected based on optimum particle size and maximum drug loading and release. The optimized formulation TMZ-NLCopt had gelucire (3.3 %), liquid lipid: total lipid (0.25), surfactant 4.89 % and sonication time 3.85min. The optimized formulation TMZ-NLCopt has shown particle size (131.58nm), drug release (73.91%), drug loading (8.71%), PDI value (0.177), zeta potential 15.21 and flux 9.81±0.76µg/cm2/h, respectively. The reason of high entrapment and loading of TMZ might be attributed to its highly lipophilic nature. The lower polydispersity index values



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TMZ-NLCopt indicating that size distribution of the particles was quite narrow and had a uniform size. The lower zeta potential could be attributed to particle stability in suspension through the 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 Surface morphology of the TMZ-NLCopt was studied and it was found that NLC was roughly spherical in shape. The particles appeared dark with bright surroundings and no aggregation of the particles were seen Compatibility study DSC thermograms of pure TMZ and TMZ-NLCopt formulation are shown in Fig. 3. The thermal curve of pure TMZ showed endothermic peak at 172.2°C, whereas lyophilized TMZ-NLCopt formulation of vit E and gelucire 44/14 does not showed any peak near the melting point of drug. This indicated that Temozolomide was not in crystalline state but rather present in amorphous state and drug was completely entrapped within the nanoparticles. And further none of the formulation excipients had interference with the drug. In-vitro release effect The in-vitro release study of TMZ-NLCs were compared with TMZ-disp and plotted against the time. The % drug release of TMZ-disp was found to be 94.67±3.56 over 8 hrs, while the release from developed TMZ-NLCs were in the range of 44.44-71.56% over 24hrs (Table 1). The TMZNLCopt showed release for 24 hrs, whereas TMZ-disp releases more than 80% drug in 6 hr. (Fig. 4), which proved the sustained release action of TMZ-NLCopt. The data obtained by in vitro release experiment was fitted to various release models and the observed best fit model was higuchi model (0.988) followed by first order release model (0.981). The n-values were found for fickian diffusion (0–0.5), non Fickian (0.5–1), Zero order (1) and super diffusion (>1) respectively for spherical particles [27,32]. The obtained value of n = 0.075 for NLC showed that the release behavior was fickian diffusion. Ex-vivo transport study across nasal mucosa The steady state flux after 24 hrs of study achieved from TMZ-disp 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 than those obtained for TMZ-disp. The significant high


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drug permeation effect of NLC (P< 0.05) compared to dispersion was due to the presence of surfactant on the surface of NLC, which are well known as penetration enhancers. Pharmacokinetic and brain distribution study The brain and blood concentration after intranasal administration of TMZ-NLCopt, 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. TMZ concentration from NLCopt was found to be high at all time-points in comparision to TMZ-disp (i.n.). This might be attributed to the higher nasal retention effect of NLC formulation at target site. Brain targeting efficiency was increased i.e. 457% when TMZ was encapsulated in NLC to enhance permeation into the nasal mucosa as compared to TMZ-disp (i.n.) with enhancement of 169.7% only. TMZ-disp distributed rapidly in the region due to which lesser absorption was achieved in compare to TMZ- NLC. The initial plasma concentration (0.5 h) was lower for the TMZ-NLCopt than TMZ-disp (i.n), (i.v.) possibly because TMZ was released slowly from TMZNLCopt for extended period of time. The free drug was available for distribution only after its release from dispersion and was comparatively high as compared to TMZ-disp (i.v). After 1.5 hr, the plasma concentration was lower for the TMZ-disp (i.n) and (i.v) than that of the TMZNLCopt because of its solubility in plasma ensuring rapid distribution, elimination and slower release leading to lower clearance. Brain/plasma ratio of NLC was higher at all time-points as compared to dispersion either given by intranasal or intravenous route that proves the direct nose to brain transport pathway of the drug via nasal administration (Table 5 and Fig. 5). Gamma scintigraphy study Radioactivity accumulation of TMZ- NLCopt in brain can be clearly seen in Fig. 6 (i), (ii) and (iii) after 0.5h, 2 h and 6 h of study, respectively. After 0.5h, radioactivity can only be seen in the nostrils. After 2h and 6h radioactivity accumulation in the brain is clearly visible. A good amount of activity was also noticed in oesophagus and in the abdominal region which could be due to absorption of a part of the formulation from gastrointestinal tract [28,29]. It can be clearly seen that the brain levels were higher while blood levels were lesser after intra-nasal administration as compared with intravenous route.



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Discussion Optimization TMZ-NLCs were formulated and optimized by 4 factors 3 levels Box- Behnken statistical design. We have tried different composition and parameters such as lipids concentration, lipid ratio, surfactant concentration and sonication time to obtain small particle size, maximum loading and encapsulation efficiency with optimum drug release. Fitting of the data for observed responses to various models was attempted and the best fit model for all the three dependent variables were found to be the quadratic model (Table 2). With the increase in total lipid concentration an increase in particle size was observed due to decrease in emulsifying efficiency of surfactant and increase in particle agglomeration. As there is increase in the amount of liquid lipid, a reduction in particle size was observed because of decrease in the viscosity of formulation and surface tension inside NLC. An increase in total lipid concentration increases loading efficiency significantly by providing more space for the accommodation of drug particle as increment of the lipid content also reduces the escaping of drug into the external phase. Also as concentration of solid lipid (Gelucire) increases with increase in total lipid concentration, it solubilizes the drug due to presence of mono, di and triglycerides of fatty acids. A significant increase in EE was obtained with increased liquid lipid concentration as more of the drug particles were entrapped inside the oil enriched lipid core. This can be explained due to more imperfection in highly ordered solid lipid crystal due to incorporation of spatially incompatible liquid lipid. Increase in the amount of total lipid causes significant decrease in cumulative % drug release. This is due to the fact that increase in lipid concentration increases the size of the nanoparticle, thereby decreasing the effective surface area available to interact with the releasing medium and hence decrease in drug release. Surfactant concentration significantly affects the EE and drug release. It first increases the EE and then decrease in the EE was observed after further rise in surfactant concentration. This decrease maybe attributed due to entrapment of surfactant molecule itself into the NLC at higher concentration of surfactant. An increase in the drug release characteristics till particular concentration of surfactant is due to its ability to decrease the particle size, a further increase in surfactant concentration causes decrease in EE which decreases the amount of drug release. It can be stabilized by two different mechanisms: steric stabilization and electrostatic repulsion. Tween 80 and poloxamer 188 being non-ionic stabilizers stabilize the system by steric stabilization due to presence of dense hydrophobic tail, which does not allow


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particles to come closer with each other and thus particle agglomeration is prevented [30]. These surfactants also impart negative zeta potential of 11.75±2.96 mV to the nanoparticulate system which causes electrostatic repulsion of nanoparticles and they are prevented to form aggregate. In-vitro release effect The invitro drug release profile of TMZ from TMZ-NLCs showed the sustained release of the drug from the formulation. The release from the NLCs were found to be fast for initial time period followed by prolonged release over a period of 24 h. The initial rapid release of drug may be due to release of TMZ from the NLCs surface while at a later stage TMZ may be constantly released from the core of NPs which is responsible for the prolonged release. The kinetic analysis of the in vitro release profile of the TMZ-NLCopt was done to ascertain release order and found to be higuchi model. There are many factors which can influence the release of drug from NLC are solubility of drug in the lipid, lipid matrix and its concentration and size of particle [31]. Ex vivo permeation studies The steady state flux achieved from control formulation was found to be 4.23µg/cm2, whereas from NLCs formulation after 24 hrs of study, it was recorded as 9.815 µg/cm2/h. The higher flux for TMZ-NLCopt was due to nano size particle of formulation and presence of surfactants on the surface of NLC. The enhancement ratio from TMZ-NLCopt was significantly higher than TMZdisp over 24 hrs that could be attributed to lipophilic nature of the drug. The higher lipophilicity of the TMZ gives greater permeation through the nasal mucosa [33]. The increase in permeation shows contribution of neuronal transport bypassing BBB. Pharmacokinetic and brain distribution study This effect is due to direct nose to brain drug delivery as compared to oral solution which shows hepatic first pass metabolism and has to cross gastrointestinal barrier and blood brain barrier before finally reaching to the brain. This may be due to the fact that nano formulations increases nose to brain drug delivery as compared to drug solution/dispersion of equivalent dose as shown earlier by in-vitro permeation study. Protection of the drug from efflux back into the intranasal cavity may also be the reason for this effect of nanoparticles as solutions are rapidly cleared from the nasal cavity. The effect of nano formulation maintained till 24 h was due to its sustained drug release profile. Tween 80 also enhances the penetration of drug through nasal mucosa. P-gp expressed in olfactory epithelium causes drug efflux which is prevented by poloxamer 188



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resulting in higher drug concentration in brain after i.n. administration as compared to dispersion [12]. Hence it shows the effectiveness of the developed formulation in controlling tonic hind limb extension even after 24 h. Gamma scintigraphic study The study showed significant higher accumulation of TMZ from TMZ-NLCopt in rat brain. Radioactivity of TMZ-NLCopt after 4 h showed longer retention time of TMZ in brain. The higher level of radioactivity was found in brain by intranasal route proved that brain target can be achieved by this route. The images of treatment were consistent with the biodistribution and pharmacokinetic data. TMZ-NLCopt was found to effective in increasing TMZ concentration in brain. Moreover, intranasal administration further improved the brain uptake of the drug as compared with intravenous route due to direct nose-to-brain delivery. A significant amount was also present in GIT because of intragastric ingestion of formulation by way of throat [26]. Conclusion Optimized TMZ loaded NLC was developed using Box- Behnken statistical design which gives the optimum concentration of lipids and surfactants in formulation to get the minimum size and maximum drug loading and release. The release of TMZ was found to follow zero order kinetics with fickian’s law of diffusion. The present results clearly showed that intranasal administration of TMZ-NLCopt in rats is efficient to maintain the effect of TMZ with much higher brain concentration due to direct brain targeting and increased residence time of drug in brain as compared to intranasal dispersion. The higher bioavailability in brain by NLCs compared to dispersion was seen with lower doses indicating a remarkable delivery route for targeting brain with an appropriate dosage form design. Acknowlegement Authors are thankfull to AIMS and INMAAS, New Delhi, India for providing the TEM and scintigraphy study.

Conflict of interest All authors have approved the final manuscript and the authors declare that they have no conflicts of interest to disclose. References


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3. Pardeshi, V.; Belgamwar, S. Direct nose to brain delivery via integrated nerve pathways bypassing the blood-brain barrier: an excellent plateform for brain targeting. Expert Opin in Drug Del. 2013, 10(7), 957-972.

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13. Alam, M.I.; Baboota, S.; Ahuja, A. Intranasal administration of nanostructured lipid carriers containing CNS acting drug: Pharmacodynamic studies and estimation in blood and brain, J. of Psych. Res. 2013, 46, 1133-1138.

14. Jia, L.; Zhang, D.; Li, Z. Nano-structured lipid carriers for parenteral delivery of silybin: biodistribution and pharmacokinetic studies. Colloid Surf. and Bioint. 2010, 80 13-18. 15. Bhaskar, K.; Anbu, J.; Ravichandiran, V. Lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in vitro, ex vivo and in vivo studies. Lipid in Heal. Dis. 2009, 8, 1-15. 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 Pharmac. Sci. 2001, 13, 123-133

18. Wadell, C.; E. Bjork E.; Camber, O. Permeability of porcine nasal mucosa correlated with human nasal absorption. Eur. J. of 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. Carb. Poly. 2012, 89, 72-79. 20. Fazil M, Md S, Haque S, Kumar M, Baboota S, Sahni JK, Ali J. Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting. European Journal of Pharmaceutical Sciences 47 (2012) 6–15. 21. Md S, Haque S, Fazil M, Kumar M, Baboota S, Sahni JK, 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 Deliv. (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. Preparation of nimodipine-loaded microemulsion for intranasal delivery and evaluation on the targeting efficiency to the brain. Int. J. Pharm. 2004, 275, 85-96. 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 Del. Rev. 2002, 54, 131-155.


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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. Solid lipid nanoparticles (SLN) of tretinoin:potential in topical delivery. Int. J. Pharm. 2007, 345, 163-71. 27. Neupane YR, 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, 30;477 (1-2): 601-12. 28. Wang, D.; Gao, Y.; Yun, L. Study on brain targeting of raltitrexed following intranasal administration in rats. Cancer Chem. 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 Del Rev. 2011, 63(6), 456-469.

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Table Legends

Table 1 Box–Behnken Design matrix based TMZ-NLCs obtained using independent variables (X1- Gelucire; X2- Liquid Lipid: Total Lipid; X3- Surfactant; X4- Sonication Time) and their effects on responses. Table 2 Summary of statistical parameter for responses Y1 (size in nm), Y2 (drug release in %), and Y3 (drug loading in %) for fitting to different model. Table 3 Check Point Batches of Temzolomide NLCs formulation with their actual and predicted value. Table 4 Pharmacokinetic distribution profile of TMZ-NLC (i.n) and TMZ-disp (i.n, i.v) in rats. Table 5 Brain/blood ratio and absorption profile of TMZ at different time intervals TMZNLCopt (i.n), TMZ-disp (i.n, i.v).

Figure legends Fig. 1 Solubility profile of TMZ in various lipids (n=3) Fig. 2 3D-Response surface plots showing the influence of independent variables on size (A-C), drug release (D-F) and drug loading (G-I) for the TMZ NLCs. Fig. 3 Differential Scanning Calorimetry image pure TMZ and lyophilized TMZ-NLCopt. Fig. 4 Comparative in-vitro drug release profile of TMZ-NLCopt and TMZ-disp. Fig. 5 Blood Plasma ratio of rat following intranasal administration TMZ-NLCopt, TMZ-disp (i.n) and TMZ-disp(i.v). Fig. 6 Gamma scintigraphic images of rat following intranasal administration of TMZ-NLCopt at different time points.


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Table. 1 Box–Behnken Design matrix based TMZ-NLCs obtained using independent variables (X1- Gelucire; X2- Liquid Lipid: Total Lipid; X3- Surfactant; X4- Sonication Time) and their effects on responses. Code X 1 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

X2

X3

X4

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

Y1 (nm)

Y2 (%)

Y3 (%)

Actual

Predicted

Actual

Predicted

Actual

Predicted

196.77±4.45 195.89±7.45 144.28±6.11 171.67±4.28 195.12±3.59 173.12±5.65 145.78±4.88 195.78±7.16 166.89±7.34 182.77±8.22 180.61±4.71 141.28±8.76 151.34±5.78 220.11±7.44 137.66±7.28 182.34±9.34 159.78±7.45 172.87±7.23 162.78±6.73 154.78±5.32 188.89±6.75 174.56±6.21 153.22±5.52 204.34±5.39 199.78±4.88 170.61±4.18 194.87±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

56.28±5.78 54.24±4.45 75.33±7.32 72.57±5.22 59.27±4.29 66.54±5.77 66.87±4.39 63.88±6.43 69.45±6.23 73.45±6.98 60.66±5.48 64.44±4.65 61.53±5.89 51.22±6.76 71.33±5.11 63.72±4.78 70.27±5.28 71.45±5.38 69.45±6.21 64.43±4.28 55.48±5.71 59.66±5.38 71.56±4.81 44.44±4.26 58.13±5.25 72.38±4.28 55.28±3.22

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

6.22±0.45 5.22±0.54 8.13±0.47 7.28±0.53 5.66±0.58 5.88±0.51 5.79±0.64 8.16±0.71 7.56±0.58 8.84±0.66 6.43±0.67 6.18±0.56 6.21±0.37 7.23±0.78 7.24±0.46 6.64±0.52 7.59±0.59 7.38±0.56 7.45±0.44 7.57±0.79 6.75±0.76 5.32±0.43 4.88±0.86 7.61±0.78 8.34±0.49 7.23±0.41 8.69±0.45

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

X1- Gelucire (%); X2- Liquid Lipid: Total Lipid, X3- Surfactant (%), X4- Sonication Time (min); Y1- Size (nm); Y2- Drug release(%); Y3- Drug loading(%).



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Table-2 Summary of statistical parameter for responses Y1 (size in nm), Y2 (drug release in %), and Y3 (drug loading in %) for fitting to different model. Statistical parameter Size Drug release Drug loading R2 0.9988 0.9959 0.9923 2 Adjusted R 0.9974 0.9911 0.9833 Predicted R2 0.9939 0.9771 0.9569 SD 1.1 1.02 0.14 %CV 0.63 1.52 1.91 Model Quadratic Quadratic Quadratic Size= +171.72 + 24.5X1 - 10.11X2 - 15.07X3 – 1.63X4 -1.5X1X2 + 1.97 X1X3 + 0.39 X1X4 – 7.33 X2X3 + 3.55 X2X4 – 0.61 X3X4 + 1.73X12 – 3.77 X22 + 9.3 X32 – 0.45 X42 Drug release = + 72.13 – 9.83X1 + 4.26X2 + 6.54X3 + 1.15X4 + 10.81 X1X2 – 5.18X1X3 + 1.35X1X4 + 3.36X2X3 – 0.25X2X4 – 0.75X3X4 – 1.05X12 - 2.21X22 – 8.55X32 + 0.21X42 Drug Loading= +7.3 + 1.1X1 + 0.79X2 + 0.67X3 - 0.17 X4 - 0.28 X1X2 + 0.15X1X3 +0.06X1X4 + 0.043X2X3 + 0.19X2 X4 + 0.11X3X4 + 0.013 X12 – 0.2 X22 – 0.44 X32 – 0.16 X42


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Table 3 Check Point Batches of Temzolomide NLCs formulation with their actual and predicted value. S. No.

Dependent variables X1

X2

X3

BI

Y2 % Drug Y3 % Drug release loading Actual Predicted Actual Predicted Actual Predicted 3.3 0.25 4.89 3.85 131.58 133.43 73.91 72.22 8.71 8.98

B2

3.8 0.35 3.27 4.55 129.56

130.53

70.56

68.32

7.79

7.39

B3

2.8 0.15

2.35 138.32

137.87

74.12

75.23

8.11

8.17

B4

4.3 0.25 5.67 5.25 121.45

122.45

67.91

65.89

8.55

7.98

2.5

X4

Experimental value Y1 - Size (nm)



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Table 4 Pharmacokinetic distribution profile of TMZ-NLC (i.n) and TMZ-disp (i.n, i.v) in rats. Formulations

Organs/ Plasma TMZ- NLCopt (i.n) Brain Plasma TMZ-disp (i.n) Brain Plasma TMZ-disp (i.v) Brain Plasma

0.5 hr (ng/ml)± SD 1815.6±1.38 1336.0±31.8 3479.6±4.09 2724.9±0.54 783.6±0.36 5590.2±14.42

2 hr (ng/ml) ± SD 4606.3±1.46 1636.5±5.22 2122.4±15.07 2030.8±2.45 1916.9±1.52 2739.5±4.11


6 hr (ng/ml) ± SD 1432.4±1.70 667.8±6.20 365.6±15.14 726.8±38.75 180.8±0.80 177.1±15.98

24 hr (ng/ml) ± SD 1189.1±19.23 85.1±17.24 263.1±2.00 444.6±33.43 2.96±0.82 5.88±2.15

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Table 5 Brain/blood ratio and absorption profile of TMZ at different time intervals TMZNLCopt (i.n), TMZ-disp (i.n, i.v). Formulations

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

Organ/ Blood

Cmax Tmax (ng/ml) (min)

AUC0-

Brain Blood Brain Blood Brain Blood

4606 1633.4 3279.6 3419.3 1916.9 5590

37863.3 13684.2 13394 19559.8 6437.8 10652.2

1.5 1.5 0.5 0.5 0.5 0.5

24hr


Elimination rate constant (Ke) 0.03 0.124 0.08 0.06 0.25 0.26


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


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Fig. 2 3D-Response surface plots showing the influence of independent variables on size (A-C), drug release (D-F) and drug loading (G-I) for the TMZ NLCs.



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Fig. 3 Differential Scanning Calorimetry image pure TMZ and lyophilized TMZ-NLCopt.


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Fig. 4 Comparative in-vitro drug release profile of TMZ-NLCopt and TMZ-disp.



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Fig. 5 Blood Plasma ratio of rat following intranasal administration TMZ-NLCopt, TMZ-disp (i.n) and TMZ-disp(i.v).


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



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Brain and plasma Distribution and Scintigraphic image of TMZ-NLCopt 33x23mm (300 x 300 DPI)


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Brain Targeting of Temozolomide via the Intranasal Route Using Lipid

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