Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting

European Journal of Pharmaceutical Sciences 47 (2012) 6–15

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Development and evaluation of rivastigmine loaded chitosan nanoparticles for brain targeting Mohammad Fazil a, Shadab Md a, Shadabul Haque a, Manish Kumar b, Sanjula Baboota a, Jasjeet kaur Sahni a, Javed Ali a,⇑ a b

Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi, India

a r t i c l e

i n f o

Article history: Received 12 January 2012 Received in revised form 22 March 2012 Accepted 13 April 2012 Available online 27 April 2012 Keywords: Biodistribution Brain targeting Confocal microscopy Intranasal route Nanoparticles Rivastigmine

a b s t r a c t The rivastigmine (RHT) loaded chitosan nanoparticles (CS-RHT NPs) were prepared by ionic gelation method to improve the bioavailability and enhance the uptake of RHT to the brain via intranasal (i.n.) delivery. CS-RHT NPs were characterized for particles size, particle size distribution (PDI), encapsulation efficiency, zeta potential and in vitro release study. Nose-to-brain delivery of placebo nanoparticles (CSNPs) was investigated by confocal laser scanning microscopy technique using rhodamine-123 as a marker. The brain/blood ratio of RHT for different formulations were 0.235, 0.790 and 1.712 of RHT (i.v.), RHT (i.n.), and CS-RHT NPs (i.n.) respectively at 30 min are indicative of direct nose to brain transport bypassing the BBB. The brain concentration achieved from i.n. administration of CS-NPs (966 ± 20.66 ng ml1; tmax 60 min) was significantly higher than those achieved after i.v. administration of RHT sol (387 ± 29.51 ng ml1; tmax 30 min), and i.n. administration of RHT solution (508.66 ± 22.50 ng ml1; tmax 60 min). The higher drug transport efficiency (355 ± 13.52%) and direct transport percentage (71.80 ± 6.71%) were found with CS-RHT NPs as compared to other formulation. These results suggest that CS-RHT NPs have better brain targeting efficiency and are a promising approach for i.n. delivery of RHT for the treatment and prevention of Alzheimer’s disease (AD). Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system (CNS) and is the most common cause of dementia in the elderly population (Ray and Lahiri, 2009) in which there is a progressive deterioration of intellectual and social functions, memory loss, personality changes and inability for self care (Kosasa et al., 1999). The exact pathogenesis of the neuronal degeneration and cognitive impairment in AD still remains to unclear. AD is multisystemic in nature and this presents numerous difficulties for the potential treatment of this disorder. Various factors hinder the discovery and development of effective drug delivery systems (DDS) for the delay, treatment, and prevention of AD, the inability to deliver drugs effectively to the brain due to the numerous protective barriers surrounding the CNS i.e., blood–brain barrier (BBB) being a major concern (Lockman et al., 2002). Many strategies which include development of chemical delivery systems, magnetic drug targeting, or drug carrier systems such as antibodies, liposomes, or nanoparticles (NPs) have been ⇑ Corresponding author. Tel.: +91 9811055385/9811312247; fax: +91 011 26059633. E-mail addresses: [email protected], [email protected] (J. Ali). 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.04.013

developed to overcome these problems. Among the various DDS developed polymeric NPs have attracted great attention as potential drug delivery systems in the CNS because they have the ability to deliver a wide range of drugs to targeting areas of the body for controlled drug release and site-specific drug targeting (Sahni et al., 2011). Many authors have delivered a variety of drugs such as hydrophilic drugs, hydrophobic drugs, proteins, vaccines, and biological macromolecules using NPs (NPs) as carriers (Hans and Lowman, 2002). NPs have a further advantage over larger bulk materials, because they have a higher surface-to-volume ratio and therefore the dose and frequency of administration is reduced hence increasing patient compliance, thereby making them better suited for intranasal (i.n.) delivery (Wilson et al., 2010). CNS drug delivery strategy that has received relatively little attention is the administration of drug by i.n. route. Drugs delivered intranasally are transported along olfactory sensory neurons to yield significant concentrations in the CSF and olfactory bulb. Recent evidence of direct nose-to-brain transport and direct access to CSF of neuropeptides bypassing the bloodstream has been shown in human trials, despite the inherent difficulties in delivery (Misra et al., 2003). i.n. delivery is non-invasive, essentially painless, does not require sterile preparation, and can be easily and readily administered by the patients themselves or by a physician,

M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15

e.g., in an emergency setting. Drug delivery systems are designed with rationale of promoting the therapeutic effect of a drug and minimizing its toxic side effects, which is achieved by optimizing the amount and duration of the drug in the vicinity of the target cells while reducing the drug exposure to non-target cells. There are many approaches for the treatment of AD, but cholinergic hypothesis has special attention. Rivastigmine (RHT) was chosen as a drug candidate for AD as it is inhibitor of both acetylcholinestrase (AChE) and butrylcholinestrase (BuChE) enzyme and is 4–17 times more specific for inhibiting AChE in brain as compared to heart and blood. However limitation with its oral drug delivery is its restricted entry into brain due to its hydrophilicity, thereby necessitating frequent dosing resulting in severe cholinergic side effects. Other investigators have worked on RHT loaded poly(n-butyl cyanoacrylate) NPs for brain targeting through i.v. route. The results showed that surfactant coated poly(n-butylcyanoacrylate) NPs significantly transported the drug RHT in comparison with the free drug to the brain. The high concentrations of RHT achieved in the brain may be a significant improvement for treating AD. But the approach is also associated with several disadvantages like possibility of systemic side effects, patient non-compliance as it is a painful techniques beside possibility of distribution to non targeted site. The present investigation was aimed to formulate nanoparticulate system of RHT that will be targeted to brain through nasal route to avoid first pass metabolism and avoid the distribution to non-targeted sites thus leading to decrease peripheral side effects. The mucoadhesive polymeric NPs of RHT so developed are also expected to offer many advantages over conventional nasal dosage forms, like increased nasal residence and possibility of drug release at slow and constant rate. 2. Materials Chitosan with medium molecular weight (Mw = 750,000 Da) and degree of deacetylation about 85% and sodium tripolyphosphates (TPP) were purchased from Sigma–Aldrich (Bangalore, India). RHT having molecular weight (Mw = 400.43 g/mol) was received as a gift sample from Torrent Pharmaceuticals Ltd. (Himachal Pradesh, India) having molecular weight. Potassium dihydrogen phosphate, Methanol, sodium hydroxide (NaOH) and 1-Octanol were all purchased from S.D. Fine Chemicals, Ltd. (Mumbai, India). Glacial acetic acid was purchased from IOL Chemical Ltd. (Mumbai, India). Methanol HPLC grade, Acetonitrile HPLC grade and Ammonia solution AR grade also procured from S.D. Fine Chemicals, Ltd. (Mumbai, India). Dialysis sacs (mol. wt. cut-off: 12000 Da, flat with 25 mm, diameter of 16 mm, capacity 60 ml ft) was purchased from Sigma Aldrich Chemicals, Missouri, USA. All reagents were of analytical grade.

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(Remi, Delhi, India) at 15,000 rpm for 45 min at 4 °C and the supernatant was used to determine encapsulation efficiency (EE) and loading capacity (LC). 4. Physiochemical characterization of chitosan NPs The surface morphology of the prepared NPs was determined for by using transmission electron microscopy (TEM). The nanosuspension samples were prepared by dispersing a small amount of NPs into distilled water. A drop of nanosuspension was placed on a paraffin sheet and carbon coated grid was placed on sample and left for 1 min to allow CS-NPs to adhere on the carbon substrate. The remaining suspension was removed by adsorbing the drop with the corner of a piece of filter paper. Then the grid was placed on a drop of phosphotungstate for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and the sample was air dried. The sample was examined by TEM (Morgagni 268D TEM, Massachusetts, USA). The particle size, particle size distribution, Polydispersity index, and zeta potential were determined by Zetasizer Nano ZS, (Malvern Instruments Ltd, Worcestershire, UK). Measurements were performed using Standard laser 4 mW He–Ne, 633 nm, room temperature 25 °C at fixed angle of 90°. The sample volume used for the analysis was kept constant i.e., 1 ml. The instrument is equipped with appropriate software for analysis of particle size and polydispersity index. 4.1. Determination of the loading capacity, encapsulation efficiency and process yield of NPs The EE and LC of NPs were determined by separation of NPs from the aqueous medium containing non-associated RHT by centrifugation at 15,000 rpm at 4 °C for 45 min. The amount of free RHT in the supernatant was measured by UV spectrophotometer at 261 nm. The EE and LC of CS-RHT NPs were calculated as per equations given below with all the measurements were performed in triplicate and averaged

EE ¼

total drug  free drug  100; total drug

LC ¼

Total drug  free drug  100 Nano particles weight

The process yield was calculated from the weight of dried NPs recovered (W1) and the sum of the initial dry weight of starting materials (W2) using the following formula:-

Process Yield ð%Þ ¼ W1=W2  100 4.2. In vitro release study

3. Preparation of chitosan NPs CS-NPs were prepared according to the ionic gelation process (Calvo et al., 1997; Vila et al., 2002; Aktas et al., 2005). CS-NPs were obtained upon the addition of a TPP aqueous solution (2 mg/ml) to a CS solution (1.75 mg/ml) stirred at room temperature. The formation of NPs was a result of the ionic interaction between the positively charged amino groups of chitosan and negative groups of TPP. The ratio of chitosan/TPP was established according to the preliminary studies. CS-RHT NPs were obtained according to the same procedure and the ratio of chitosan/TPP remained unchanged. Variable ratio of RHT was incorporated to the chitosan solution prior to the formation of NPs in order to investigate the effect of the initial RHT concentration on the NP characteristics and in vitro release profiles. CS-NPs were collected by centrifugation

The in vitro release profile of RHT from CS-RHT NPs and RHT solution was performed using dialysis sacs. The drug loaded CS-RHT NPs (containing about 4.275 mg of drug) were placed in pretreated dialysis sacs which were immersed into 100 ml of phosphate buffer solution, pH 7.4, at 37 °C and magnetically stirred at 50 rpm. At selected time intervals, aliquots were withdrawn from the release medium and replaced with the same amount of phosphate buffer. The samples were analyzed in triplicate using HPLC. The HPLC determination was performed using reverse phase Phenomenex C18 column (25 cm  4.6 i.d., 5 lm). The mobile phase consisted of 10 mM ammonium hydroxide (pH 10.50):acetonitrile (30:70 v/v%) at a flow rate of 1 ml/min. The peak detection was performed at 215 nm (LaPorte and Wu, 2007). The data obtained from in vitro drug release studies was fitted to various release models like zero order,

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M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15

first order, Higuchi, and Korsemeyer Peppas model (Ge et al., 2002) to understand the mechanism of drug release from the NPs. 4.3. Differential scanning calorimetry (DSC) study DSC analysis of pure RHT, pure CS, physical mixture (CS + RHT), freezed dried CS-NPs and freeze dried CS-RHT NPs were carried out using Perkin ElmerÒ 7 DSC (Massachusetts, USA) calibrated with indium. Sample (5 mg) was placed onto a standard aluminum pan, crimped and heated from 40–400 °C at a heating rate of 5 °C/min with continuous purging of nitrogen (20 ml/min). An empty sealed pan was used as reference. All samples were run in triplicate. 5. In vitro permeability studies Fresh nasal tissues were carefully removed from the nasal cavity of porcine obtained from the local slaughter house. The tissue samples were fixed in Logan instrument (Logan Instrument Carporation, NJ, USA) cells displaying a permeation area of 0.785 cm2. Twenty milliliters of phosphate buffer saline (PBS) pH 6.4 was added to the receptor chamber. To ensure oxygenation and agitation, a mixture of 95% O2 and 5% CO2 was bubbled through the system. The temperature was maintained at 37 °C. After a pre-incubation time of 20 min, pure drug solution and formulation equivalent to 10.0 mg of RHT were placed in the donor chamber in each case. At predetermined time points, 2 ml samples were withdrawn from the receptor chamber. The withdrawn samples were replaced with an equivalent volume of PBS pH 6.4 after each sampling, for a period of 24 h. The samples withdrawn were filtered and used for analysis. Blank samples (without RHT) were run simultaneously throughout the experiment to check for any interference. The amount of permeated drug was determined using UV–VIS spectrophotometer. 6. In vivo study Wistar rats (aged, 4–5 months) of either sex weighing between 200 and 250 g were selected for the study of biodistribution and pharmacokinetic studies. The protocol for animal studies was approved by the Institutional Animal Ethical Committee of Jamia Hamdard, New Delhi, India, and study was carried out in accordance with principles of laboratory animal care and the approved protocol. 6.1. Biodistribution studies by confocal laser scanning microscopy (CLSM) Rhodamine-123 (ROD-123) a fluorescent dye was used to determine the biodistribution of CS NPs for qualitative analysis. ROD123 is widely used due to its less ability to cross BBB even if given intravenously. For the preparation of ROD-123 loaded CS NPs, ROD-123 was first dissolved in ethanol (20 mg/ml) and then mixed in 10 ml of CS solution (0.175% w/v) and cross linked with 4 ml of 0.2% w/v TPP solution at room temperature with constant stirring at 1000 rpm for 30 min. The resultant NPs were concentrated by centrifugation at 15000 rpm at 4 °C for 45 min. The supernatant was used for estimating the ROD-123 encapsulated in NPs. For biodistribution studies, animals were divided into three groups, each composed of 3 animals. Group 1 received i.v. ROD123 loaded NPs dissolved in 1 ml of sterile isotonic solution by tail vein, group 2 received i.n. ROD-123 loaded NPs dissolved in 100 ll of normal saline solution through nasal route and group 3 received ROD-123 solution through nasal route. The animals were sacrificed at fixed time point of 30 min to localize the NPs in different vital organs (i.e. brain, liver and lung). Isolated brain of each rat was washed with Ringer’s solution and cerebellum was separated after

dissection of right, left and frontal encephalon. Each tissue was cut by a microtome into 5 lm in its thickness. The slides were then fixed in 4% (w/v) formaldehyde solution and stored at 4 °C before CLSM studies. In order to stress the cellular structures, tissue slides were stained for 10 min with 50 ll (250 ng/ml) of DAPI (40 -6diamidino-2-phenylindole; Sigma Aldrich, Mumbai, India) solution, which is known to form fluorescent complexes with natural double-stranded DNA (Tosi et al., 2007), and observed using a fluorescence microscope (Olympus FluoView™ FV 1000, CA, USA) with double band, for DAPI and ROD-123. The red fluorescent spots due to the fluorescent dye ROD-123 were considered as the visible markers of the drug embedded into NPs. 6.2. Pharmacokinetic parameters and Brain targeting study The rats were housed six per cage at 20–24 °C with free access to food and water with a 12-h light–dark cycle. The rats were divided into three groups, one for control RHT nasal solution; the second for the CS-RHT NPs and the third for the i.v. RHT solution. Three rats for each formulation per time point were used in the study. The rats were anaesthetized using diethyl ether. A sufficient dose was given by inhalational route, to keep the rats sedated for a short period of 3 min during instillation of formulations to prevent sneezing. CS-RHT NPs previously suspended in saline buffer, were administered equivalent to 0.0648 mg of RHT in each rat. Dosing was given as shown in the Tables 4 and 5. The rats were sacrificed humanely by cervical dislocation method at different time intervals and the blood was collected using retino-orbital vein. Subsequently, the brain was dissected, washed twice using normal saline, made free from adhering tissue/fluid and weighed. Finally the drug was quantified using validated HPLC method. Before injecting the sample, brain was crushed and vortexed. Then fluids and supernatant were separated using cooling centrifuge, supernatants were separated. The brain was weighed and an aliquot (1:10) of ice cold saline solution was added. The organs were then homogenized on ice. An aliquot of brain homogenates (100–500 ll) was extracted with acetonitrile (liquid–liquid extraction) and vortexed for 1 min. After centrifugation (4 °C, 4000 rpm, 20 min), the supernatant was separated, and directly injected into HPLC for analysis (Aminia and Ahmadiani, 2010). The time intervals used for the study were 10, 30, 60, 120, 240 and 480 min. Animals were divided into three groups of three animals each. The group A had positive control (RHT solution i.n), group B had NPs formulation (RHT i.n) and group C had RHT solution (i.v). The pharmacokinetic analysis was done using pharmacokinetic software (PK Functions for Microsoft Excel, Pharsight Corporation, Mountain view, CA) and various parameters like AUC, Tmax, and Cmax were calculated. The drug targeting efficiency (DTE%) and direct nose to brain drug transport (direct transport percentage, DTP%) were calculated. The maximum plasma concentration of RHT (Cmax) and the time required to reach the maximum concentration (Tmax) were obtained directly from the actual plasma profiles. The area under the curve between 0 and 480 min was calculated by the linear trapezoidal method. The brain targeting efficiency was calculated using the following equations (Kumar et al., 2008):Drug targeting efficiency (DTE %) that represents time average partitioning ratio was calculated as follows:

DTE% ¼

  ðAUCbrain =AUCblood Þi n   100 ðAUCbrain =AUCblood Þi v 

Nose to brain direct transport percentage (DTP %) was calculated as follows

DTP% ¼

  Bi n   Bx  100 Bi n 

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M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15

where Bx = (Bi.v./Pi.v.)  Pi.n., Bx is the brain AUC fraction contributed by systemic circulation through the BBB following intranasal administration. Bi.v. is the AUC0–480 (brain) following intravenous administration. Pi.v. is the AUC0–480 (blood) following intravenous administration. Bi.n. is the AUC0–480 (brain) following intranasal administration. Pi.n. is the AUC0–480 (blood) following intranasal administration.

(38.4 ± 2.85 mV), LC (43.37 ± 3.9) and EE (85.3 ± 3.5). The mean particle sizes of optimized formulation F21D after PCS was found to be 183.7 nm as shown in Fig 1B. The optimized formulation F21D was also characterized by TEM. The mean particle sizes of optimized formulation F21D after TEM was found to be 133.49 to 189.45 as shown in Fig. 2B. 8.2. In vitro release study

8. Results

The cumulative percentage release of RHT from CS-RHT NPs (drug: Polymer ratio 1:1) was 89.27 ± 2.672 over a period of 24 h as shown in Fig. 3. The percentage drug release of RHT from RHT drug solution was 92.54 ± 3.512% after 2 h whereas 47.4 ± 6.163% drug release was observed from CS-RHT NPs after 2 h. The co-efficient of correlation (R2) of zero order, first order, Higuchi and Korsemeyer and Peppas model for CS-RHT NPs was found to be 0.7845, 0.9275, 0.9446 and 0.934, respectively. Since the Co-efficient of correlation (R2) for Higuchi model was nearer to unity i.e., (0.9446), was selected as the best fit model.

8.1. Preparation and characterization of chitosan NPs

8.3. Differential scanning calorimetry (DSC) study

Different concentrations of CS and TPP were used to optimize the best CS/TPP ratio on the basis of particle size, polydispersity index (PDI), zeta potential and process yield. The mean particle size, PDI and process yield of different formulations of CS-NPs are shown in Table 1. The process yield of different formulation of CS-NPs ranged between 40.56 ± 2.30% and 88.42 ± 3.25%. The mean particle size and PDI varied from 143.1 ± 9.2 to 3300 ± 7.0 nm and 0.424 ± 0.012 to 0.985 ± 0.032 respectively depending upon the CS and TPP ratio used. Out of all the CS-NP formulations shown in Table 1, F4C having CS/TPP ratio 2.19/1 was selected as best formulation due to its optimum particle size, PDI, zeta potential and process yield of 163.7 ± 7.6 nm, 0.422 ± 0.065, 45.30 ± 6.21 mV and 77.68 ± 3.12% respectively. The mean particle sizes of optimized CS-NPs containing 1.75 mg/ml CS and 2 mg/ml TPP with PCS was found to be 162.6 nm as shown in Fig. 1. The particle size of optimized CS-NPs was also characterized with TEM and the result was found to be in the range 82.63 to 171.43 nm as shown in Fig. 2. Hence, the optimized CS-NPs (F4C) were selected for further optimization studies after incorporating the drug. The CS-RHT NPs were prepared and evaluated for process yield, mean particle size, PDI, LC and EE. The results are shown in Table 2. The process yield, mean particle size and PDI of CS-RHT NPs with different drug polymer ratio ranged between 79.42 ± 4.62 to 86.12%, 185.4 ± 8.4 to 341.3 ± 4.7 and 0.391 ± 0.065 to 0.462 ± 0.064 respectively. The LC and EE varied from 43.37 ± 3.9% to 58.19 ± 2.3% and 85.3 ± 3.5% to 75.18 ± 3.8% which depended upon the drug polymer ratio used. Out of these formulations F21D was selected as the best formulation due to its optimum particle size (185.4 ± 8.4) PDI (0.391 ± 0.065), process yield (79.42 ± 4.62), zeta potential

DSC thermograms of polymer, RHT, physical mixture and freeze-dried CS-RHT NPs are shown in Table 3. CS-RHT NPs showed exothermic peak at 243.991 °C, the polymer showed endothermic peak at 224.112 °C, drug had endothermic peak at 126.053 °C and physical mixture showed both peaks of drug as well as polymer. The peak at 126.053 °C exhibited by RHT was not visible in the CS-RHT NPs, indicating that RHT was encapsulated by the polymer in the NPs (Joshi et al., 2010).

7. Statistical analysis All data are reported as mean ± S.E.M. and the differences between the groups were tested using Student’s t-test at the level of P < 0.05. More than two groups were compared using ANOVA and the difference greater at P < 0.05 was considered significant.

8.4. In vitro permeability study The cumulative percentage of drug permeated through nasal mucosa from CS-RHT NPs was 70.1% where as only 20.3% was found to permeate from pure drug solution in 24 h. The steadystate flux and permeability coefficient of drug solution through the nasal mucosa was 1.06  102 lg cm2h1 and 1.06  105 cm2h1 respectively whereas steady-state flux and permeability coefficient of CS-RHT NPs was 1.25  102 lg cm2h1 and 1.25  105 cm2h1 respectively. The two tailed P-value was 0.0073, which suggested that the difference was statistically very significant with P < 0.05. 8.5. In vivo study Biodistribution studies of CS-NPs and RHT solution were done by visualizing the intrinsic fluorescence of ROD-123 using confocal laser scanning fluorescence microscopy (CLSM). For qualitative localization and biodistribution studies the ROD-123 loaded CS NPs showed mean particle size of 165.4 ± 4.98 nm. These NPs

Table 1 Optimization of placebo nanoparticles (CS-NPs) on the basis of chitosan and TPP ratio.

a

Formulation code

Concentration of CS (mg/ml)

Concentration of TPP (mg/ml)

a

F-1C F-1D F-2C F-2D F-3A F-3B F-4B F-4C F-5C F-6B

0.5 0.5 1 1 1.5 1.5 1.75 1.75 2 2.25

2 3 2 3 1 1.5 1.5 2 2 1.5

2146 ± 8.0 3300 ± 7.0 580.5 ± 5.6 1645 ± 4.0 468.3 ± 6.6 143.1 ± 9.2 452.4 ± 7.2 163.7 ± 7.6 234.2 ± 8.6 356.6 ± 5.8

Mean particle size(nm) ± (S.D)

a

Mean PDI ± (S.D)

0.918 ± 0.045 0.985 ± 0.032 0.588 ± 0.022 0.775 ± 0.013 0.501 ± 0.035 0.424 ± 0.012 0.466 ± 0.023 0.422 ± 0.065 0.611 ± 0.023 0.552±.018

PDI, polydispersity index. All formulation codes were the CS-NPs samples. Values were expressed as mean ± standard deviation.

a

Process Yield ± (S.D)

40.56 ± 2.30 42.61 ± 3.46 47.64 ± 5.13 49.89 ± 2.24 54.68 ± 3.56 61.45 ± 2.50 70.52 ± 3.64 77.68 ± 3.12 82.23 ± 2.37 88.42 ± 3.25

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Fig. 1. PCS photographs of (A) CS NPs (B) CS-RHT NPs containing 1.75 mg/ml CS and 2 mg/ml TPP.

Fig. 2. TEM photographs of (A) CS NPs (B) CS-RHT NP particles containing 1.75 mg/ml CS and 2 mg/ml TPP.

Table 2 Optimization of drug polymer ratio of CS-RHT NPs on the basis of Particle size, PDI, zeta potential, process yield, LC and EE%. Formulation code

Drug:Polymer ratio

Mean particle sizea(nm) ± (S.D)

Mean PDIa ± (S.D)

Process Yielda i.v. (S.D)

Mean Zeta potential(mV)

LCa (%)

EEa (%)

F-21D F-22D F-23D

1:1 2:1 3:1

185.4 ± 8.4 255.4 ± 5.6 341.3 ± 4.7

0.391 ± 0.065 0.424 ± 0.045 0.462 ± 0.064

79.42 ± 4.62 82.21 ± 5.13 86.12 ± 4.68

38.40 ± 2.85 31.35 ± 3.15 27.29 ± 3.65

43.37 ± 3.9 54.89 ± 2.1 58.18 ± 2.3

85.3 ± 3.5 81.6 ± 4.2 75.1 ± 3.8

a PDI, polydispersity index. F21D, F22D and F3, F23D were the CS-RHT NPs samples with CS/TPP ratio was fixed at 2.19/1. Values were expressed as mean ± standard deviation

Fig. 3. In vitro release profile of CS-RHT NPs and pure drug solution.

had an average EE of 73.6 ± 3.28% and also showed biphasic release pattern having 24.2% drug release in 2 h followed by slow release of ROD-123 (83.56 ± 7.32) over a period of 24 in phosphate buffer (pH 7.4) which is quite similar to CS-RHT NPs. The results revealed

that ROD-123 was not released promptly. Three different formulations were administered via i.n. and i.v. to see the biodistribution after 30 min. The confocal microscopic images of brain, liver and lungs shown in Figs. 4–6 correlate with the result of qualitative biodistribution study. When ROD loaded CS NPs were administered i.n. in rats there was more intensity of fluorescent dye ROD-123 in brain as compared to liver and lungs as shown in Fig. 4. It means that the prepared NPs reached to brain via I.N route. When ROD-123 solution was administered i.n. there was less intensity of ROD-123 in brain region but more intensity in liver and lungs as shown in Fig. 5. When ROD-123 loaded CS NPs was administered i.v. there was more intensity of ROD-123 in liver and lungs. There was no intensity of dye in brain as shown in Fig. 6. Brain-blood ratio of RHT formulations following i.n. (RHT solution and CS-RHT NPs) and i.v. administration (RHT solution) in Wistar rats was calculated and the concentration was estimated at different intervals up to 8 h. The RHT concentration in brain following the intranasal (i.n.) of CS-RHT NPs) was found to be significantly higher at all the time points compared to both RHT solution i.v and RHT solution i.n. as shown in Table 4 and Fig. 7. The RHT concentration in plasma following the i.n. of CS-RHT NPs was

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M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15 Table 3 DSC thermograms of CS, RHT, physical mixtures, CS-NPs and CS-RHT NPs. S.No.

Sample

Endothermic Peak (°C)

Inference

1. 2. 3. 4. 5.

CS polymer RHT Physical mixture CS-NPs CS-RHT NPs

224.112 126.053 126.053 (Drug), 224.112 (Polymer) 220.215–229.025 243.991 (Exothermic)

Sharp peak Sharp peak Both peak was sharp Broad and diminished peak Diminished peak

Fig. 4. Qualitative biodistribution studies ROD-123 loaded CS NPs (i.n.) in brain, liver and lungs by CLSM.

Fig. 5. Qualitative biodistribution studies ROD-123 solution (i.n.) in brain, liver and lungs by CLSM.

Fig. 6. Qualitative biodistribution studies ROD-123 loaded CS NPs (i.v.) in brain, liver and lungs by CLSM.

found to be significantly lower at all the time points compared to RHT (i.v.) as shown in Fig. 8. The brain/ blood ratios of RHT for different formulations were 0.235, 0.790 and 1.712 for RHT i.v, RHT i.n., and CS-RHT NPs (i.n.), respectively at 30 min which are indicative of direct nose to brain transport bypassing the BBB as shown in Table 4. Plasma concentration–time profiles of RHT after i.v and i.n. delivery were evaluated by pharmacokinetic software (PK Functions for Microsoft Excel, Pharsight Corporation, Mountain view, CA). Selected pharmacokinetic parameters after i.n. and i.v.

administration were calculated from the individual brain and plasma concentration–time profile experiments and shown in the Table 5 where values of Cmax, tmax, AUC, t1/2 and Ke can be found. The brain concentration achieved after i.n. administration of CSNPs (966 ± 20.66 ng ml1; tmax 60 min) was significantly higher than that achieved after i.v. administration of RHT solution (387 ± 29.51 ng ml1; tmax 30 min), and i.n. administration of RHT sol (508.66 ± 22.50 ng ml1; tmax 60 min). The brain concentration of RHT solution after i.n. administration was slightly higher than that after i.v. administration of RHT solution at all the time points

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Table 4 Compartmental distribution of RHT CS NPs (i.n.), RHT solution (i.n.) and RHT solution i.v. at different time interval in normal Wistar rats. Formulation

Organ/ Tissue

10 min(ng/ ml) ± SD

30 min(ng/ ml) ± SD

60 min(ng/ ml) ± SD

120 min(ng/ ml) ± SD

240 minng/ ml) ± SD

480 min(ng/ ml) ± SD

RHT (IV)

Brain Blood Brain Blood Brain Blood Brain/blood Brain/blood Brain/blood

127.33 ± 23.79 2050.67 ± 15.13 169 ± 37.16 243.67 ± 33.84 174 ± 21.07 128.33 ± 18.87 0.062 0.695 1.35

387 ± 29.51 1640 ± 40.73 374 ± 29.05 473.33 ± 25.71 545 ± 41.58 292 ± 29.05 0.235 0.790 1.712

342 ± 30 1167 ± 43.13 508.66 ± 22.50 361 ± 31.51 966 ± 20.66 638 ± 46.7 0.293 1.40 2.159

203 ± 29.05 541.33 ± 27.22 357.66 ± 29.02 309 ± 31.09 746 ± 34.51 777.33 ± 20.51 0.375 1.157 1.601

140.66 ± 28.91 244.66 ± 35.53 184.66 ± 24.63 169 ± 30.19 575.66 ± 66.30 414.5 ± 31.04 0.574 1.09 1.388

87.66 ± 25.54 – 105.88 ± 23.03 101.33 ± 25.17 266 ± 32.04 218.33 ± 25.77 – 1.04 1.216

RHT (IN) RHT CS-NPs (IN) RHT (i.v.) RHT (i.n.) RHT CSNPs(i.n.)

Table 5 Pharamacokinetic parameters of CS-RHT NPs after i.n. and i.v. administration to rats at the dose of 0.068 mg kg1 in brain and plasma (n = 6, mean ± SD). Administration

Brain and Plasma samples

Cmax (ng/ml)

Tmax (min)

t1/2 (min)

Ke (min1)

AUC 0–1(ng min/ml)

CS-NP (i.n.)

Brain Plasma Brain Plasma Brain Plasma

966 847.33 508.66 573.33 387 2050.66

60 120 60 30 30 10

2441.9 729.66 483.6 232.1 290.462 74.25

0.00095 0.00028 0.001433 0.002986 0.002386 0.009335

525636.97 1037912 236613.5 133719.1 79584.33 232990.7

RHT sol (i.n.) RHT sol (i.n.)

9. Discussion 9.1. Preparation and characterization of CS-RHT NPs

Fig. 7. Graph of RHT conc. in brain at different time intervals.

Fig. 8. Graph of RHT conc. in rat plasma at different time intervals.

except at the time point of 30 min. Significant differences between i.n. and i.v. administration were found (t-test) for the calculated pharmacokinetic parameters (P < 0.05). The DTP% and DTE% represent the percentage of drug directly transported to the brain via the olfactory pathway. RHT from two different formulation i.e., CS-RHT NPs was administered i.n. and RHT solution i.v. in the rats and DTP% and DTE% were calculated using formula given above. It was recorded that RHT CS NPs showed the highest DTE% and DTP% of (355 ± 13.52%) and (71.80 ± 6.71%) respectively as compared to RHT solution administered i.v.

The chitosan/TPP particles prepared with different concentrations of chitosan or TPP were studied. The results indicated that with a increase in the concentration of either CS or TPP particle size increased. Fan and coworker found that the formation of chitosan/ TPP nanoparticles was only possible for some specific concentrations of CS and TPP (Fan et al., 2012). On the basis of experimental data analysis it was observed that the ratio between CS and TPP 2.19/1 to 2.5/1 was found optimum in which the particle size was found below the range of 200 nm. Above or below this range a large variation in size was found. This fact was also observed in our study that when concentration of CS and TPP was below 2.19/1 to 2.5/1 micro-particles were formed as shown in Table 1. It means that the concentration of CS and TPP may not be in stoichiometric ratio (Papadimitriou et al., 2008). Among the selected formulations, F-4C was having more process yield than F-4B. However F-5C was having more process yield than F-4C but the particle size was above 200 nm which is not a feasible option for brain targeting via i.n. route. The possible reason is that with increase in the concentration of CS and TPP more NPs are formed which may be due to more binding sites available for ionic cross linking of molecules as a result of which more and larger sized particles are formed (Dudhani and Kosaraju, 2010). The effects of drug concentration on particle size and PDI of CS-RHT NPs are summarized in Table 2. When the drug: polymer ratio increased from 1 to 3 the average sizes of CS-RHT NPs were increased from 183.7 ± 8.4 to 341.3 ± 4.7 nm. Increasing the drug proportion in solution caused reduction of CS/TPP interaction, which lead to an increase in size of the NPs. The increase in drug concentration also slightly increased the PDI value. From comparison of two TEM photographs (Fig. 2A and B) of CS-NPs and CS-RHT NPs it can be concluded that the size of NPs increased when RHT was encapsulated because some RHT could also be absorbed on the surface of NPs (Wu et al., 2005). The mean particle size of CS-RHT NPs by PCS was found to be more than that obtained after TEM analysis. This apparent discrepancy between PCS and TEM results can be explained by the dehydration of CS-RHT NPs during sample

M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15

preparation for TEM imaging. PCS measures the apparent particles size of NPs, including hydrodynamic layers that form around the hydrophilic particles leading to an overestimation of size of NPs (Motwani et al., 2008). The value of PDI can range from 0.00 (for mono-dispersed systems) to 1.00 (for highly-dispersed systems), where PDI value greater than 0.50 indicate a relatively broader size distribution (Avadi et al., 2010). The formulation codes F-4C and F21D have low PDI as compared to other formulations which means that particles are homogenous, unimodal and with narrow particle size distribution. From the result of TEM and PCS it can be concluded that the optimized concentrations of CS and TPP for formation of NPs were 1.75 and 2 mg/ml respectively and the ratio is 2.19/1–2.5:1 (Hu et al., 2002). Fig. 2(A and B) show TEM images of CS NPs and CS-RHT NPs respectively. The TEM images of CS NPs and CS-RHT NPs prepared using the ionic gelation method were uniform and roughly spherical and subspherical in shape, separated from each other, suggesting possible stabilisation of the NPs due to positive surface charges. CS-RHT NPs also shows the some pores on the surface which suggests possibility of release of drugs from the pores. The zeta potential is a measure of the charge of the particles. As such larger the absolute value of the zeta potential larger is the amount of charge of the surface. Thus, the zeta potential represents an index for particle stability. A physically stable nanosuspension solely stabilized by electrostatic repulsion will have a minimum zeta potential of ±30 mV (muller et al., 2001). The mean zeta potential of CS NPs and CS-RHT NPs are more than 30 mV and are all positively charged which is a typical characteristic of CS NPs. This can be due to presence of residual amino groups which are not neutralized by their interaction with negative charge of TPP molecules (Wang et al., 2008). 9.2. Loading capacity and Encapsulation efficiency of CS-RHT NPs The EE increased from 75.1% to 85.3% depending upon the drug polymer ratio as shown in Table 2. From the above data it is clear that 1:1 drug polymer ratio shows better EE. The LC increased from 43.37% to 58.18% depending upon the drug: polymer ratio used. With the increase of initial CS concentration during the entrapment process, more protonized CS (–NH3+) were available in the system, evidenced by increased surface charge, leading to a stronger electrostatic attraction between RHT and CS due to negative charge on RHT. This high amount of polymer concentration led to an increase in the binding sites for cross linker which led to high EE. But when we increased the drug: polymer ratio, the interaction between the free amino groups and TPP carrying negative charge is decreased as the drug creates a barrier, this leads to lower encapsulation of drug and large free chain of CS gave larger size particles. Thus, the EE of RHT decreased with the increase of drug concentration. This can be attributed to the experimentally determined fact that the higher the drug concentration, less is the EE and more is the LC (Mohanraj and Chen, 2006).

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which releases more than 92.54 ± 3.512% of drug. The rapid release of RHT from drug solution may be due to rapid dissolution of the RHT upon dilution under sink conditions. The graph of the release and subsequent diffusion of the drug from the NPs becomes similar to the diffusion of drug solution from the dialysis sacs as shown in Fig 3. The kinetic analysis of the in vitro release profile of the optimized CS-NPs was done to ascertain release order. Since the coefficient of correlation (R2) for Higuchi model was nearer to unity i.e., (0.9446) for CS-RHT NPs, therefore the best fit model for CS-NPs was Higuchi model. When the release data were analyzed using the Korsmeyer-Peppas equation, the value of release exponent n was between 0.43 and 0.85 an indication of both diffusion controlled and swelling controlled drug release i.e., anomalous transport (Ritger and Peppas, 1987). 9.4. Differential scanning calorimetry (DSC) study DSC thermograms of polymer, RHT, physical mixture, CS NPs and freeze-dried CS-RHT NPs are shown in Table 3. The pure CS shows sharp endothermic peak at 224.12 °C and CS-NPs (Placebo) show broad and diminished endothermic peak in the range of 220.215–229.025 which means that the crystalline nature of polymer could be converting into amorphous form. The physical mixture of CS and RHT shows independent sharp endothermic peak at 126.053 and 224.112 °C, respectively, which indicated that the drug and polymer are not interacting with each other. CS-RHT NPs showed broad and diminished peak near 126.053 °C which could be indicative of amorphous nature of CS-NPs. DSC thermograms showed that RHT was dispersed as amorphous state in CSRHT NPs (Joshi et al., 2010). The CS-RHT NPs also showed small peak at 243.991 °C which could be due to some impurities present in the formulation. 9.5. In vitro permeability studies The CS-RHT NPs showed more permeation as compared to the pure drug solution as given in above result, this might be due to the permeation enhancing activity of chitosan (Porporatto et al., 2005). The increase in permeation of RHT could be attributed to an interaction of a positively charged amino group on the C-2 position of chitosan with negatively charged sites on the cell membranes and tight junctions of the mucosal epithelial cells to allow opening of the tight junctions (Borchard et al., 1996). It has been demonstrated that chitosan when applied to confluent cell cultures, is able to transiently open the tight junctions between the cells (Dodane et al., 1999). Another reason for lesser permeation of pure drug solution may be the hydrophilic nature of drug. For permeation through nasal mucosa drug should be lipophilic (Richter and Keipert, 2004). Thus, NPs overcome this problem regarding drug nature also. 9.6. Biodistribution studies

9.3. In-vitro release studies The release profiles of RHT from CS-RHT NPs and drug solution are shown in Fig 3. The percentage drug release profile of RHT from CS-RHT NPs showed the sustained release of the drug from the formulation. It was apparent that RHT released from the formulation in vitro showed a rapid initial release for 1 h (more than 30%) followed by slow drug release over a period of 24 h (Zhou et al., 2001). The initial rapid release of drug may be due to release of RHT from the NPs surface while at a later stage RHT may be constantly released from the core of NPs as a consequence of CS hydration and swelling which is responsible for the prolonged release which is desired for sustained action (Sadeghi et al., 2008). The release of drug from drug solution showed a burst release for 2 h

The distribution of ROD-123 in different tissue like brain, liver and lungs collected at 0.5 h after i.n. administration was done by taking microscopic images to give qualitative results of biodistribution study. Figs. 4–6 show the concentration of ROD-123 in different organs after the administration of ROD-123 solution i.n., ROD loaded CS-NPs i.n. and ROD loaded CS-NPs i.v. From the results of the present investigation it was observed that the tissue concentration in the form of intensity of fluorescent dye of ROD-123 was higher in the brain with the CS-NPs administered i.n. in comparison to the ROD-123 loaded CS-NPs given i.v. due to the existence of direct nose to brain transport bypassing the BBB (Tosi et al., 2007; Vergoni et al., 2009). The concentration of ROD-123 CS-NPs was also higher in the brain as compared to ROD-123 solution i.n.

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M. Fazil et al. / European Journal of Pharmaceutical Sciences 47 (2012) 6–15

because the mucoadhesive nature of the formulation decreased the mucociliary clearance, which under normal circumstances rapidly clears the instilled formulation. Another reason is that it has been already reported that ROD-123 is a substrate of p-glycoprotein (Pgp) which is present in nose-brain interface which effluxes out the ROD 123 solution. When ROD-123 was encapsulated into CS NPs, it overcome the P-gp efflux pump operating at the interface and CS NPs could be easily detected in brain. The concentrations of ROD-123 loaded CS-NPs in the liver when administered as i.v. was higher as compared to ROD-123 loaded CS-NPs i.n. and ROD123 solution i.n. due to the presence of reticuloendothelial system (RES). It is known that NPs drug carriers are passively taken up by the RES, particularly by the Küffer cells of the liver, after i.v. administration (Zhou et al., 2001). A similar pattern of ROD-123 loaded CS-NPs distribution was also obtained in the lungs. The higher concentrations of ROD-123 achieved in the highly perfused organs, such as liver and lungs are probably due to the combined activity of the circulating blood passing through the organs as well as due to the particle uptake by cells of the endothelial system of these organs. Similar findings were reported for other drugs loaded to the NP formulations (Lobenberg et al., 1998). 9.7. Pharmacokinetic parameters and Brain targeting study The RHT concentrations in brain following the i.n. of CS-RHT NPs were found to be significantly higher at all the time points compared to both RHT (i.n.) and RHT (i.v.). The RHT concentration in plasma following the i.n. of CS-RHT NPs were found to be significantly lower at all the time points compared to RHT (i.v.). The brain/blood ratios of 0.235, 0.790, and 1.712 of RHT (i.v), RHT (i.n), and CS- RHT NPs (i.n.) respectively, at 30 min are indicative of the direct nose to brain transport bypassing the BBB, hence proving the superiority of nose to brain delivery of RHT by CSNPs (Kumar et al., 2008). When the pharmacokinetic parameters for the RHT formulations were calculated the lower Tmax value for brain (1 h) was found when compared to blood (2 h) which may also be attributed to the preferential nose to brain transport following i.n. administration. When the Cmax and AUC of brain concentrations of RHT (i.n.), RHT (i.v.) and CS-RHT NPs (i.n.) were compared, the Cmax (966 ± 20.66) and AUC (247730 ng. min./ml) of CS-RHT NPs were found to be significantly higher because of the direct transport of drug through olfactory route by bypassing the BBB. In addition to this, another reason is the mucoadhesive nature of CS that decreased the mucociliary clearance, which under normal circumstances rapidly clears the instilled formulation. When we compared the AUC in brain of CS-NPs (i.n.) and RHT solution (i.v.) it was found to be nearly 3.11 times higher with CS-NPs (i.n.) where as 1.917 times higher AUC was found with RHT solution (i.n.). This result reveals that the drug uptake into the brain from the nasal mucosa mainly occurs via two different pathways. One is the systemic pathway by which some of the drug is absorbed into the systemic circulation and subsequently reaches the brain by crossing the BBB. The other is the olfactory pathway by which the drug partly travels from the nasal cavity to CSF and/or brain tissue (Khan et al., 2010., Ghananeem et al., 2010). The DTE% and DTP% represent the percentage of drug directly transported to the brain via the olfactory pathway. The higher DTE (355 ± 13.52%) and DTP (71.80 ± 6.71%) was found with RHT CS-NPs. Higher DTE% and DTP% suggest that CS-NPs have better brain targeting efficiency. Similar types of results have also been reported by Qizhi and coworkers (Qizhi et al., 2004). 10. Conclusion The present research work proposed a novel nanoparticulate formulation for the intranasal delivery of RHT. The effect of

different variables on NPs preparation was investigated. The encapsulation efficiency, typically 85.3 ± 3.5%, and the reproducibility of the preparations were satisfactory. The in vitro release found to be 89.27 ± 2.672 over 24 h indicated a controlled and sustained release profile of CS-RHT NPs. An enhanced brain uptake of CS–RHT NPs was clearly observed following nose to brain delivery. The biodistribution and pharmacokinetic study in Wistar rats, in which the DTE% and DTP% are more indicative of direct nose to brain transport bypassing the BBB thereby proving the superiority of CS-RHT NPs over RHT solution i.v. The qualitative biodistribution study by CLSM also demonstrated the existence of nose to brain transport of CS-RHT NPs as compared to RHT solution i.v. and i.n. On the basis of these research findings, it was concluded that CSRHT NPs could be a novel colloidal drug delivery system for the treatment of Alzheimer’s disease via nose to brain. However, clinical data is needed to evaluate the risk/benefit ratio. Acknowledgements The authors are grateful to University Grant Commission (UGC), Government of India for providing fellowship to Mohammad Fazil as financial assistance and are also grateful to Department of Science and Technology (DST), New Delhi for providing DST INSPIRE Fellowship to Shadab Md as financial assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2012.04.013. References Aktas, Y., Andrieux, K., Alonso, M.J., Calvo, P., Gürsoy, R.N., Couvreur, P., Capan, Y., 2005. Preparation and in vitro evaluation of chitosan NPs containing a caspase inhibitor. Int. J. Pharm. 298, 378–383. Aminia, H., Ahmadiani, A., 2010. High-performance liquid chromatographic determination of rivastigmine in human plasma for application in pharmacokinetic studies. Iranian. J. Pharm. Res. 9, 115–121. Avadi, M.R., Sadeghi, A.M.M., Mohammadpour, N., Abedin, S., Atyabi, F., Dinarvand, R., Rafiee-Tehrani, M.M., 2010. Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method. Nanomed: Nanotech. Biol. Med. 6, 58–63. Borchard, G., Luessen, H.L., deBoer, A.G., Verhoef, J.C., Lehr, C.M., Junginger, H.E., 1996. The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III effects of chitosanglutamate and carbomer on epithelial tight junctions in vitro. J. Control Release 39, 131–138. Calvo, P., Remunan-Lopez, C., Vila-Jata, J.L., Alonso, M.J., 1997. Chitosan and chitosan: ethylene oxide-propylene oxide block copolymer NPs as novel carriers for proteins and vaccines. Pharm. Res. 14, 1431–1436. Dodane, V., Khan, M.A., Merwin, J.R., 1999. Effect of chitosan on epithelial permeability and structure. Int. J. Pharm. 182, 21–32. Dudhani, A.R., Kosaraju, S.L., 2010. Bioadhesive chitosan NPs: Preparation and characterization. Carbohyd. Polym. 81, 243–251. Fan, W., Yan, W., Xu, Z., Ni, H., 2012. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf., B 90, 21–27. Ge, H., Hu, Y., Jiang, X., Cheng, D., Yuan, Y., Bi, H., Yang, C., 2002. Preparation, characterization, and drug release behaviors of drug nimodipine-loaded poly(ecaprolactone)–poly(ethylene oxide)–poly(e-caprolactone) amphiphilic triblock copolymer micelles. J. Pharm. Sci. 91, 1463–1473. Ghananeem, A.M., Saeed, H., Florence, R., Yokel, R.A., Malkawi, A.H., 2010. Intranasal drug delivery of didanosine-loaded chitosan nanoparticles for brain targeting; an attractive route against infections caused by aids viruses. J. Drug. Target 18, 381–388. Hans, M.L., Lowman, A.M., 2002. Biodegradable NPs for drug delivery and targeting. Curr. Opin. Solid State Mater. Sci. 6 (4), 319–327. Hu, Y., Jiang, X., Ding, Y., Ge, H., Yuan, Y., Yang, C., 2002. Synthesis and characterization of chitosan–poly(acrylic acid) NPs. Biomaterials 23 (15), 3193–3201. Joshi, S.A., Chavhan, S.S., Sawant, K.K., 2010. Rivastigmine-loaded PLGA and PBCA NPs: Preparation, optimization, characterization, in vitro and pharmacodynamic studies. Eur. J. Pharm. Biopharm. 76, 189–199. Khan, S., Patil, K., Bobade, N., Yeole, P., Gaikwad, R., 2010. Formulation of intranasal mucoadhesive temperature-mediated in situ gel containing ropinirole and evaluation of brain targeting efficiency in rats. J. Drug. Target 18, 223–234.

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