Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early Online: 1–13 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.998824

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Pharmacological evaluation of nasal delivery of selegiline hydrochlorideloaded thiolated chitosan nanoparticles for the treatment of depression Devendra Singh1, Muzamil Rashid1, Supandeep Singh Hallan1, Neelesh Kumar Mehra1, Atish Prakash2,3 & Neeraj Mishra1 1Department of Pharmaceutics, I.S.F. College of Pharmacy, Moga, Punjab, India, 2Department of Pharmacology, I.S.F. College

of Pharmacy, Moga, Punjab, India, and 3Brain Research Laboratory, Department of Pharmacology, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam, Malaysia

to be dose-dependent, for example at a lower dose, it inhibits the MAO B only, but at a higher dose, it also inhibits the MAO A, widely prescribed for its anti-depressant activity. Selegiline is also reported for its neuroprotective effects, and the increment in life span of animals has already been welldocumented. Currently, selegiline hydrochloride is widely prescribed in more than 62 countries. The conventional formulation of selegiline had some drawbacks, for example high first pass metabolism and low bioavailability, along with severe side effects (Shimazu et al. 2005, Hawkins et al. 1978). Compared with traditional drug-delivery approaches, intranasal drug delivery has significant multiple advantages. First, large-sized drugs can be delivered to the brain by bypassing the blood-brain barrier (BBB); second, potential side effects of the drugs on the peripheral system can be minimized; third, smaller amounts of drug are required to produce the desired concentration of drugs in the CNS, which can reduce treatment cost; fourth, the non-invasiveness of intranasal drug delivery can minimize the pain patients suffer; and fifth, intranasal drug delivery may be conducted by patients themselves or other nonprofessionals, which could minimize the delay in medical treatment. The intranasal drug administration could enable drugs to directly enter the CNS through the olfactory pathways. A number of studies on both animals and human subjects have also demonstrated that intranasal delivery of various types of molecules can produce beneficial effects on the brain. These studies have suggested the great potential of intranasal drug delivery for treating various CNS diseases (Bhatt et  al. 2014, Dhanda et al. 2005, Zhang et al. 2006). Chitosan (CS), a linear polysaccharide derived from chitin obtained from crustacean shells, has emerged as a valuable drug delivery matrix because of it polycationic nature, biodegradability, biocompatibility, mucoadhesiveness, and ease of physical and chemical alteration (Lee et al. 2004). The interaction between cationic amino groups on CS and anionic

Abstract The aim of the present study was to investigate the propensity of thiolated chitosan nanoparticles (TCNs) to enhance the nasal delivery of selegiline hydrochloride. TCNs were synthesized by the ionic gelation method. The particle size distribution (PDI), entrapment efficiency (EE), and zeta potential of modified chitosan (CS) nanoparticles were found to be 215 ± 34.71 nm, 70 ± 2.71%, and + 17.06 mV, respectively. The forced swim and the tail suspension tests were used to evaluate the anti-depressant activity, in which elevated immobility time was found to reduce on treatment. TCNs seem to be promising candidates for noseto-brain delivery in the evaluation of antidepressant activity. Keywords: depression, nasal delivery, selegiline hydrochloride, thiolated chitosan nanoparticles

Introduction Depression is one of the most prevalent psychiatric disorders with significant lifetime prevalence, and produces major disability, generally manifested by a loss of interest in pleasure, feelings of guilt, depressed mood, disruption of sleep, low energy, poor concentration, and suicidal ideation and attempts (Skelin et al. 2011). The neurochemical alterations in depression, like deficiency of dopaminergic and/or serotonergic functions, are the part of depression so called the monoamine theory of depression (Byerley and Risch 1985). In research studies, the etiology of depression has been correlated to stress, which is involved in the pathogenesis of depression (Nestler et al. 2002). Depression is a severe psychiatric condition that affects about 16% of the population during their lifetime, and is predicted to become the second leading cause of disability by the year 2020 (Andlin-Sobocki et al. 2005). Selegiline hydrochloride is a monoamine oxidase (MAO) inhibitor; the inhibition pattern of selegiline was observed

Correspondence: Dr. Neeraj Mishra, Associate Professor, Department of Pharmaceutics, ISF College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga, Punjab 142001, India. Tel: +91636-324200. Fax: +91636-239515. E-mail: [email protected] (Received 5 November 2014; revised 4 December 2014; accepted 6 December 2014)

1

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2  D. Singh et al. moieties such as sialic and sulfonic acids on the mucus layer is responsible for its mucoadhesiveness (BernkopSchnurch et  al. 2003). In addition, CS enhances epithelial permeability through the opening of tight junctions between epithelial cells (Fernandez-Urrusuno et al. 1999). Recently, it was reported that the covalent attachment of thiol groups to polymers greatly increases their mucoadhesiveness and permeation properties, without affecting biodegradability. The derivatization of the primary amino groups of CS with coupling reagents bearing thiol functions leads to the formation of thiolated chitosan (TCS). The immobilization of the thiol group results in strong mucoadhesion and excellent permeation-enhancing properties, along with the in situ gelling features. The disulfide bond has been discovered as the reason for the covalent adhesion of polymer with the mucus gel layer. Thiolated nanoparticles are also known for the prolonged and controlled release of the entrapped drug (Kast et al. 2002). In 1995, Thorne et al. reported the first quantitative study indicating that intranasal administration could deliver large-sized molecules into the brain by bypassing the BBB (Thorne et al. 1995). Intranasal administration of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) led to a significant presence of WGA-HRP in the olfactory bulb of rats, while there was no detectable amount of WGA-HRP in the olfactory bulb after intravenous injection of the same concentration of WGA-HRP. Since 1997, many studies have indicated that intranasal administration can enable largesized molecules, such as IGF-1, FGF-2, TGFß1, erythropoietin, IFNß,HIV-1,Tat, insulin, and leptin, to be transported into the CNS at least partially through direct nose-to-brain routes (Illum 2000, Ma et al. 2007, Thorne et al. 2004, Reger et al. 2006, Pulliam et al. 2007). Selegiline hydrochloride is an excellent candidate for nasal administration, especially for those patients who use medication chronically. Selegiline hydrochloride has low oral bioavailability due to high first pass metabolism, and food reduces its systemic concentration. All these reasons result in the poor efficacy of the model drug selegiline hydrochloride, and in order to eliminate the route-associated problems, the nasal route was found to be the route of choice. In recent years, the nasal route has received the greatest deal of attention as a more convenient and reliable method for the systemic as well as CNS administration of drugs. Various studies in animal models have reported that in nose- to-brain-delivery, concentration of drugs is higher than by the intravenous (i.v.) injection. The

current study aims to enhance the efficacy of the selegiline hydrochloride by using the advantages of the excellent features of thiolated chitosan nanoparticles (TCNs), which favor the nose- to-CNS delivery.

Material and methods Chemicals Selegiline hydrochloride, EDAC (5,5’-dithiobis(2-nitrobenzoic acid), CS, thioglycolic acid, Schiff reagent (HiMedia Laboratory, Mumbai, India); sodium tripolyphosphate and all other chemicals were purchased from (Sigma, St. Louis, MO, USA).

Synthesis of thiolated chitosan TCS was prepared as described by Lee et al. (2006). Briefly, CS (500 mg) was dissolved in 50 mL of 1.0% acetic acid. In order to facilitate reaction with thioglycolic acid (TGA), 100 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added to the CS solution. After EDC was dissolved, 30 mL of TGA was added and the pH was adjusted to 5.0 with 3 N NaOH, and continuously stirred for 3 h at room temperature (RT). To eliminate the unbound TGA and to isolate the polymer conjugates, the reaction mixture was dialyzed against 5 mM HCl five times (molecular weight cut-off 10 kDa) over a period of 3 days in a dark condition, then two times against 5 mM HCl containing 1.0% NaCl, to reduce ionic interactions between the cationic polymer and the anionic sulfhydryl compound (Figure 1).

Thiol content determination The degree of thiol group substitution in the modified polymers was determined spectrophotometrically using Elman’s reagent (DTNB). DTNB is a symmetric aryl disulfide, very sensitive to the reaction of the thiol-disulfide interchange with a free thiol. Initially, 5 mg of each of the conjugates and controls were dissolved in double distilled water (DDW) to prepare a 2 mg/mL solution. Then, 2.5 mL aliquots were added to 2.5 mL of 0.5 M phosphate buffer (pH 8.0) and to 5 mL of Elman’s reagent (0.4 mg/mL of DTNB in 0.5 mol/L phosphate buffer; pH 8.0). The sample was incubated at room temperature (RT) and shielded from light for 3 h. The resulting solution was then centrifuged at 18,000  g for 10 min. After that, 3 mL of the supernatant was transferred to a microtitration plate and the absorbance was measured

Figure 1. Schematic representation of TCS synthesis. Covalent attachment was achieved by the formation of amide bonds between the primary amino groups of CS and the carboxylic acid groups of TGA-mediated EDC chemistry.

Selegiline hydrochloride-loaded TCNs  3 at a wavelength of 450 nm. A blank control was created with non-modified CS (Kast et al. 2003).

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Preparation of thiolated chitosan nanoparticles TCNs were prepared by the method already reported, after minor modifications by Zhu and coworkers. TCNs were prepared by dissolving CS in 1% w/v aqueous acetic acid solution to achieve the concentration of 1 mg/mL, 2 mg/mL (pH 5), and the drug selegiline (4 to 16 mg) was added into the above solution; the sodium tripolyphosphate solution was prepared at a concentration in the range of 1 mg/mL. After, that 7 mL of sodium tripolyphosphate was added to the 18 mL of already prepared CS solution, and subsequent stirring was maintained throughout the process of nanoparticle formation (Zhu et al. 2012).

Mucin adsorption study of nanoparticles Mucoadhesiveness was calculated as the amount of mucin adsorbed by CS nanoparticles (CNPs) (400 mg/80 mg) in a certain time period. Nanoparticle suspensions were mixed with type I-S mucin solutions (100 mg/mL), vortexed, and incubated at 37°C for 2, 4, and 6 h. After adsorption, the suspensions were centrifuged at 20,000 g for 30 min, and free mucin concentration was measured in the supernatant by a colorimetric method using PAS staining, as reported previously (He et al. 1998).

Animals Male Wistar rats weighing approximately 180–250 g (3–5 months old), were obtained from the Central Animal House facility, I.S.F. College of Pharmacy, Moga, Punjab, India. The animals were housed in groups of three, in polypropylene cages with husk bedding, under standard conditions of light and dark cycles, with food and water ad libitum. They were acclimatized to laboratory conditions prior to the test. All the behavioral assessments were carried out between 9:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and carried out in accordance with the guidelines of the Indian National Science Academy (INSA) for the use and care of the experimental animals.

Forced swim and tail suspension test The forced swim and the tail suspension tests were carried out with slight modifications, to induce and simulate a depression-like condition in the Wistar rats. The forced swimming model to test for anti-depressant activity was developed by Porsolt et al. (1977a, 1977b). The model used in the present study was similar to the original method described. The animals were forced to swim in a plastic cylinder measuring 30  30 cm containing water upto 20 cm depth, at room temperature. After an initial 2-min period of vigorous activity, each animal assumed a typical immobile posture. The mouse was considered immobile when it remained floating in the water without struggling, making only minimum movements of its limbs as necessary to keep its head above water for a period of 30 min, in which immobility time was also measured (Porsolt et al. 1977a, 1977b).

The next method was similar to that described by Steru et al. (1985). The animals were suspended upside down on a metal rod at a height of 55 cm from the ground, with the help of an adhesive tape placed approximately 1 cm from the tip of the tail. Initially, the animals tried to escape by making vigorous movements, but when unable to escape, became immobile. The animal was considered immobile when it did not show any movement and hung passively. The immobility displayed by rodents when subjected to this kind of unavoidable and inescapable stress has been hypothesized to reflect behavioral despair, which in turn may reflect depressive disorders in humans (Steru et al. 1985).

Optimization of nanoparticles For optimization of the TCNs developed, various process variables were considered, including particle size distribution, PDI, and EE etc. for both thiolate nanoparticles (TCNs) and unmodified nanoparticles (CNPs). In the preparation of nanoparticles, the homogenization time, speed, polymer/ sodium tripolyphosphate ratio, and the drug entrapment efficiency were optimized, for both TCS and CS.

Characterization Particle size, polydispersity index (PDI), and zeta potential Particle size, PDI, and zeta potential of the developed nanoparticles were measured by Zetasizer, Delsa Nano C (Malvern Instruments, Ltd., Malvern, UK).

Entrapment efficiency The EE was determined by measuring the concentration of free drug (unentrapped) in an aqueous medium, as reported by (Manjunath and Venkateswarlu 2005). Nanoparticles were taken in the centrifugation tube and centrifuged at 20,000 rpm for 20 min. Then, the supernatant was measured for the unentrapped drug, and EE was calculated (Manjunath and Venkateswarlu 2005).

In vitro drug release study The in vitro drug release was performed using the dialysis bag diffusion technique. A total of 3 mL of the formulation was taken into a dialysis bag (10 kDa molecular weight cutoff), and placed in a beaker containing 200 mL of phosphate buffer (PBS; pH 5.5) at 37  0.5°C, with continuous shaking on the incubator shaker (Lab Tech, New Delhi, India). An aliquot of 0.5 mL of the dissolution medium was removed at a series of various points. Samples were withdrawn, and the absorbance at 258 nm was measured against a reagent blank, using the UV spectrophotometer (PerkinElmer, UV/VIS spectrometer Lambda 25, Singapore). Sink conditions were maintained throughout the experiment. The aliquots were diluted and analyzed by UV spectrophotometry. All the experiments were performed in triplicate, and reported as mean  SD.

Drug treatment schedule Lyophilized nanoparticles were suitably dispersed in PBS buffer and administered through the nasal route, by means of a polyethylene tube connected to the Hamilton syringe, respectively. All the drugs and the vehicles were

4  D. Singh et al. Table I. Experimental protocol design for forced swim- and tail suspension-induced depression. Force swim (depressive condition) treatment period

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1st day 3rd day 5th day 7th day [FX] 14th day Alternate days of force swim Behavioural assessment Tail suspension Biochemical estimation Brain tissue drug concentration

administered from day-7 to day-14. The animals were randomly divided into various groups comprising six animals in each group, and the study was conducted in multiple phases. Behavioral parameters were assessed on days-1, 3, 5, 7, and 14, and biochemical parameters were measured at the end of the study (Table I). Group I: Disease control vehicle-treated Group II: Selegiline plain 10 mg/kg Group III: TCNs (low Dose) 5 mg/kg Group IV: TCNs (high Dose) 10 mg/kg Group VI: CNPs 10 mg/kg

Behavioral assessment Immobility period The forced swim test was performed as described (Porsolt et al. 1977b). One day prior to the test, a rat was placed for conditioning in a clear plastic tank (45 cm  635 cm  660 cm) containing 30 cm of water (2460.5uC) for 15 min (pretest session). Twenty-four hours later (test session), the total immobility period within a 5-min session was recorded as the immobility score (in sec). A rat was judged to be immobile, when its hind legs were no longer moving and the rat was hunched forward (a floating position). The immobility time was recorded manually by an observer who was blind to the drug treatment.

Sucrose preference test The rats were tested for sucrose consumption, as described earlier. Animals were housed individually throughout the duration of the test, and were presented two bottles simultaneously in the home cage, one containing a 1% w/v sucrose solution, and the other containing standard drinking water, during the 48-h training session. To prevent the preference to position, the location of the two bottles was varied during this period. After an 18-h period of food and water deprivation, an 8-h test session was conducted. The amount of liquid remaining in each bottle was measured at the end of the testing period. The sucrose preference score was expressed as a percent of the total liquid intake. Sucrose preference SP was calculated according to the following equation. SP  [(SI ÷ (SI  WI)] × 100 where SI  sucrose intake and WI  water intake in grams (Rinwa et al. 2013).

Locomotor activity The locomotor activity was monitored using an actophotometer (Medicraft, INCO, Ambala, Haryana, INDIA), which operates on photoelectric cells connected in circuit with a digital counter. Before subjecting the animals to a cognitive

task, all animals were individually placed in the activity meter and the total activity count was measured for 10 min. The locomotor activity was expressed in terms of total photo beam counts per 10 min per animal (Kalonia et al. 2009).

Measurement of drug concentration in the brain On the 15th day, all the animals were sacrificed by decapitation immediately after behavioral assessment. The brains were removed, and the forebrain was dissected out. Whole brains were used, and 10% (w/v) tissue homogenate was prepared in 0.1 mol/L phosphate buffer (pH 7.4). The homogenate was centrifuged for 20 min at 20 000 rpm and the supernatant was further stored and assayed for the biochemical parameters. The concentration of drug in the brain was also measured and the linearity was plotted.

Biochemical estimation Biochemical tests were conducted 24 h after the last behavioral test. The animals were sacrificed by decapitation. Their brains were removed and rinsed with ice-cold isotonic saline. The brain was homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10% w/v) was then centrifuged at 10 000 g for 15 min and the supernatant so formed was used for the biochemical estimations.

Estimation of protein The protein content was estimated by the Biuret method (Rinwa et al. 2013) using bovine serum albumin (BSA) as a standard.

Nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Greiss reagent [0.1%N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulfanilamide and 5% phosphoric acid]. Equal volumes of the supernatant and Greiss reagent were mixed, and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using the PerkinElmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve (Green et al. 1982).

Lipid peroxidation The extent of lipid peroxidation in the brain was determined quantitatively by the method reported earlier (Wills 1966). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using the PerkinElmer Lambda 20 spectrophotometer. The values were calculated using the molar extinction coefficient of chromophores (1.56  105 (mol/l) 1 cm 1).

Reduced glutathione Reduced glutathione (GSH) was estimated according to the method described by Ellman et  al. (1961). Firstly, 1 mL of the supernatant was precipitated with 1 mL of 4% sulfosalicylic acid and cold-digested for 1 h at 4°C. The samples were then centrifuged at 1200  g for 15 min at 4°C. To 1 mL of

Selegiline hydrochloride-loaded TCNs  5 the supernatant obtained, 2.7 mL of phosphate buffer (0.1 mmol/L, pH 8) and 0.2 mL of 5,50 –di thio bis (2-nitrobenzoic acid) (DTNB) was added. The yellow color developed was measured at 412 nm using the PerkinElmer Lambda 20 spectrophotometer. Results were calculated using the molar extinction coefficient of chromophores (1.36  104 (mol/l) 1cm 1).

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Catalase Catalase activity was assessed following the method reported by Bergmeyer, 1965, wherein the breakdown of H2O2 is measured. Briefly, the assay mixture consists of 3 mL of H2O2 phosphate buffer and 0.05 mL of the supernatant of the tissue homogenate. The change in absorbance was recorded for 2 min at 30 s intervals at 240 nm, using the PerkinElmer Lambda 20 spectrophotometer. The results were expressed as micromoles of hydrogen peroxide decomposed per min per mg of protein (Bergmeyer 1965).

Mitochondrial enzyme complex estimation The second set of animals was used for the assay of mitochondrial enzyme complex activities as described by Berman and Hastings (1999). The whole brain was homogenized in isolated buffer. The homogenates were centrifuged at 13,000 g for 5 min at 4uC. Pellets were resuspended in the isolation buffer with EGTA, and spun again at 13,000 g at 4uC for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA, and again spun at 13,000 g at 4uC for 10 min. Pellets containing purified mitochondria were resuspended in the isolation buffer, without EGTA.

Complex I (NADH dehydrogenase activity) Complex I was measured spectrophotometrically by the method described by King and Howard (King and Howard 1967). The method involves catalytic oxidation of NADH to NAD, with subsequent reduction in cytochrome c. The reaction mixture contained 0.2 M glycylglycine buffer of pH 8.5, 6 mm of NADH in 2 mm of glycylglycine buffer and 10.5 mm of cytochrome c. The reaction was initiated by the addition of the requisite amount of solubilized mitochondrial sample, and the absorbance change at 550 nm was followed for 2 min.

Complex II (Succinate dehydrogenase activity)

mitochondrial respiratory chain in isolated mitochondria (Liu et al. 1997b). Briefly, 100 mL of mitochondrial samples were incubated with 10 mL of MTT for 3 h at 37uC. The blue formazan crystals were solubilized with dimethylsulfoxide and measured by an ELISA reader with a 580 nm filter.

Complex IV (cytochrome c oxidase) Cytochrome oxidase activity was assayed in brain mitochondria according to the method described by Sottocasa et  al. (1967). The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample and the changes in absorbance were recorded at 550 nm for 2 min.    

Statistical analysis All the results were expressed as mean   standard deviation (mean  SD). The treated groups were compared with control by applying the analysis of variance (ANOVA). The statistical analysis was carried out using Prism software from GraphPad Software Inc. (San Diego, CA). The value of probability p  0.05 was considered significant.

Results and discussion In the current scenario, T CS is widely used in the delivery of bioactives through the nasal route. Thus, the thiolation of CS plays a pivotal role in enhancing mucoadhesion and anti-inflammatory properties. The increased mucoadhesion of TCS is well-documented in literature (Lee et al. 2006, Zhu et al. 2012, Milloti et al. 2014, Boateng and Ayensu 2014).The TCS was prepared as described by Lee et al. (2006). The chemical modification of CS and freeze-dried TCS resulted in a fibrous white powder easily soluble in water. In the conjugate developed, the degree of thiolation was found to be 135 m mol/g. TCNs were prepared by the method reported earlier by Zhu and coworkers, with slight modifications (Zhu et al. 2012). In mucoadhesion studies, CNPs were observed to adsorb more amount of mucin at the initial phase, in comparison with thiolated nanoparticles, but after 5 h, the mucoadhesion potential of TCS was found to be more than that of CNPs, which was nearly constant for 12–15 h.

Characterization of TCS by FT-IR

Complex II was measured spectrophotometrically according to the previously reported method (King 1967). The method involves oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer of pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of the mitochondrial sample, and the absorbance change at 420 nm was followed for 2 min.

The synthesis of the conjugate is based on the amide bond formation between the amino functional group (NH2) of chitosan (CHI) and the carboxylic acid (–COOH) of thioglycolic acid. In this, TCS shows the three prominent characteristic peaks at 2934, 2554, 1574, 1237 cm 1, which correspond C–H bonding, vibration of the S–C bond, the C  O double bonds of the amido bond, and the H–S bond, respectively (Figure 2).

Complex III (MTT activity)

The use of complexation between oppositely charged macromolecules to prepare CS microspheres has attracted much attention, because the process is very simple and mild (Liu et  al. 1997a). TPP is a poly anion, which interacts with the cationic CS by electrostatic forces (Kawashima et al. 1985).

The MTT assay is based on the reduction of (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl- H-tetrazolium bromide by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the

Optimization of TCNs

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6  D. Singh et al.

Figure 2. FT-IR spectrum of TCS.

Due to the complexation between oppositely charged species, CS undergoes ionic gelation and precipitates to form spherical particles (Agnihotri et  al. 2004). In the optimization of TCNs, different process variables were evaluated, and the polymer conjugate/TPP ratio was considered as one of the most important factors, which directly affects the particle size and particle size distribution. We observed that upon increasing the ratio of the polymer conjugate/TPP from 2.5/1 to 10/1 (F1 to F4), the increment in particle size was observed from 105  21.47 to 690  45.21 (Table II), and the chitosan/ TPP ratio of 5/1 (F2) was found to be optimal, considering the particle size and PDI which were found to be 186  21.45 nm and 0.214  0.041, respectively. On increasing the ratio of polymer as well as TPP above this ratio, increment in particle size was found. If we compare the CS and TCS, in the case of TCS, pH adjustment is one of the requirements for the preparation of nanoparticles. The pH values of 5 and 5.5 were

found to be desirable, and above this pH, undesirable white aggregates were observed; these aggregates were found to disappear on reducing the pH of the media. From the practical point of view, drop-wise addition of TPP into the polymer solution was considered best, keeping all other variables constant. With the change in the flow of TPP solution and the droplet size of the TPP solution, changes in the particle size, along with drastic changes in PDI, were observed. A smaller droplet size was found to favor the desired PDI and average particle size. Homogenization directly affects the average particle size, and the degree and extent of the force of homogenization directly affect the particle size and PDI. For the optimization of homogenization speed and speed range, the range of 5000 to 13,000 rpm was considered. These values were considered on the basis of a review of literature and a trial batch carried out without drug. In optimizing the

Table II. Optimization of polymer/TPP ratio, homogenization speed, homogenization time, and drug optimization. Formulation Polymer conjugate Homogenization Homogenization Avg. particle code TPP ratio speed time Drug EE size F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17

2.5:1 5:1 7.5:1 10:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1 5:1

5000 5000 5000 5000 5000 6000 7000 8000 6000 6000 6000 6000 6000 6000 6000 6000 6000

10 10 10 10 10 10 10 10 8 9 10 11 12 11 11 11 11

10 10 10 10 10 10 10 10 10 10 10 10 10 4 8 12 16

– – – – – – – – – – – – – 54  2.41 61  3.31 70 ± 2.71 69  3.14

105  21.47 186 ± 21.45 454  19.54 690  45.21 129  15.24 179 ± 12.37 245  19.14 451  15.17 287  16.91 260  23.54 245  20.78 216 ± 15.61 397  31.84 140  24.34 173  24.34 215 ± 34.71 359  29.85

PDI 0.328  0.048 0.214 ± 0.041 0.319  0.041 0.327  0.015 0.341  0.014 0.217 ± 0.016 0.364  0.014 0.045  0.042 0.251  0.043 0.264  0.041 0.287  0.041 0.284 ± 0.051 0.213  0.0519 0.341  0.0457 0.214  0.049 0.214 ± 0.0421 0.341  0.0271

The bold values under “Average particle size” and “PDI” were found to be optimum at optimized polymer conjugate TPP ratio (5:1), optimized homogenized speed (6000 rpm), optimized homogenization time (910 min) and optimum drug concentration (12 mg).

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Selegiline hydrochloride-loaded TCNs  7 homogenization speed at 5000 rpm, the size of the nanoparticles was found to 186  21.45 nm, which was found to decrease up to 179  12.37 nm at 6000 rpm; beyond that, at speeds of 7000 and at 8000 rpm (F7– F8), drastic increment in particle size, to 245  19.14 and 451  15.17 nm was observed, probably due to aggregation. Homogenization time is also considered to be important, and it was studied over the range of 7 to 12 min. With homogenization for 7 min, the particle size was found to be 359.4 nm, which was found to decrease at 8 min, at the same speed as earlier. The particle size was found to be in a decreasing order, such that at 9, 10, and 11 min (F10 to F12), the sizes were 260  23.54, 260  23.54, and 216  15.61 nm, respectively (Table II). In the final optimized TCN formulation (F16), the particle size distribution, EE, and zeta potential of the modified CNPs were found to be 215  34.71 nm, 70  2.71% and  17.06 mV, respectively. The zeta potential values of unmodified nanoparticles were found to be higher ( 34.33 mV) than that of the TCNs, which indicates the greater positive charge, which is why unmodified CNPs have a better electrostatic interaction with the negatively charged machine. Better mucoadhesion for up to 15 h indicates the formation of disulfide bond between TCS and the CS-rich subdomains of mucus glycoproteins. Thus, modified nanoparticles are good candidates for the delivery of CNS by the nasal route.

In vitro drug release of modified and unmodified CNPs In a comparative evaluation of the release pattern of both modified and unmodified TCNs, if we consider the initial phase of the first two and a half hours, the release pattern of unmodified CNPs was observed to be faster in comparison with that of the modified nanoparticles. The probable reason is the presence of drug on the outer surface of nanoparticles, while in the modified nanoparticles, due to the nature of in situ gel formation, a high density gel network complex may be produced, which restricts the water penetration where drug diffusion takes place. After 3 h, the release of drug from the modified nanoparticles was found to be uniform, and this pattern was observed for a longer period of time. If we compare the release pattern for 13 h, modified CNPs were found to have 80% cumulative release, while the unmodified nanoparticles had a cumulative release of 68% (Figure 3).

Figure 3. In vitro drug release profile of the TCs and the non-modified CNPs.

all the animals, but when applying the forced swim induced a depressive condition, drastic reduction was observed. Further, coadministration of different formulations of selegiline hydrochloride yielded different results. Between the two formulations and the plain drug, TCNs at 10 mg/kg were found to successfully restore the sucrose consumption as compared to other formulations (Figure 5).

Locomotor activity The locomotor activity is also associated with the depressive condition, in which locomotor activity was found to be highly impaired. The observed results show that on the first day, the highest locomotor activity was observed, which was found to be decreased on introducing the harsh depressive condition (forced swim, leading to a drastic reduction in locomotor activity). If we compare the first day to the seventh day, there was a significant reduction in locomotor activity observed, which was found to be restored on treatment. Selegiline-loaded TCNs at a dose of 10 mg/kg were observed to be highly effective compared to plain drug and unmodified nanoparticles (Figure 6).

Biochemical estimation It is well-established that in the pathophysiology of depression, the level of radicals is found be significantly higher

Behavioral assessment Immobility stress Immobility, referred to as behavioral despair in animals, is claimed to reproduce a condition similar to human depression (Porsolt et al. 1977a). The immobility score in the disease condition was found to be the highest, which was significantly shortened on administration of TCNs. Plain selegiline does not produce a significant alteration in immobility time. The treatment with unmodified CNPs was found to produce responses, but not at the desired range (Figure 4).

Sucrose preference test The sucrose preference test is a valuable and valid behavioral indicator of chronic stress in rats. In sucrose preference testing, subsequent to day-zero, sucrose was consumed by

Figure 4. Effect of TCNs on the immobility time in depression-induced rats. Values were expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs; ep  0.05 as compared to CNPs.

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8  D. Singh et al.

Figure 5. Effect of TCNs on the sucrose uptake in depression-induced rats. Values are expressed as mean  SEM. **p  0.05 as compared to 7th day SPT; ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low); dp  0.05 as compared to TCNs (high).

than that in the normal physiological condition. In our present study, the nitrite level was found higher in the disease condition and significantly reduced on nasal administration of the modified nanoparticle formulation developed, while the plain drug solution was unable to reach the requirement of therapy. There have been discussions about oxidative stress contributing to the pathophysiology of mood disorders (Forlenza and Miller 2006, Sarandol et al. 2007, Ozcan et al. 2004). The nitrite level after the completion of the forced swim test was found to be the highest, at about 445 mg/mL. Plain selegiline HCl was not found to be significantly important upon nasal route of administration, while the TCNs at a dose of 10 mg/kg were found to significantly attenuate the level

of nitrite; here we also studied the CNPs, but they did not produce the desired response (Figure 7). Under the normal physiological conditions, multiple defense mechanisms exist to protect against these free radicals, including the restriction of their production through the maintenance of a high oxygen gradient between the ambient and cellular environments, and their removal by non-enzymatic and enzymatic antioxidants (Davies 2000). Oxidative stress occurs when redox homeostasis is tipped towards an overbalance of free radicals, due to either their overproduction or deficiencies in antioxidant defense (Sies 1997). Oxidative stress mechanisms have been implicated in the pathogenesis of psychiatric disorder. The brain is considered to be highly sensitive to oxidative damage for

Figure 6. Effect of TCNs on the locomotor activity in depression-induced rats. Values are expressed as mean  SEM. *p  0.05 as compared to 7th day locomotor activity; ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline.

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Selegiline hydrochloride-loaded TCNs  9 several reasons, which include the high oxygen utilization and hence the generation of free radical by-products, and the reducing potential of certain neurotransmitters (Valko et al. 2007). There is a significant association between depression and polymorphisms in oxidative and nitrosative (O & NS) genes, like manganese superoxide dismutase (SOD), catalase, and myeloperoxidase. Animal models of depression very consistently show lowered antioxidant defense and activated O & NS pathways in the peripheral blood and the brain. In animal models of depression, anti-depressants consistently increase lowered antioxidant levels and normalize the damage caused by O&NS processes (Maes et al. 2011). The natural antioxidant enzymes are highly impaired in the disease condition due to their excessive consumption or low level of production. The levels of catalase and GSH were extremely reduced in disease condition, while upon treatment they were found to be restored to normal levels. In the pathophysiology, decreased antioxidant status by induction of oxidative and nitrosative (IO&NS) pathways is well-established, and major depression has been characterized by a significant reduction in numbers of key antioxidant enzymes, which is the hallmark of depression (Figure 8). The lowered antioxidant capacity may impair protection against reactive oxygen species (ROS), causing damage to fatty acids, proteins, and DNA by O & NS stress. Increased ROS in depression is demonstrated by increased levels of plasma peroxides and xanthine oxidase. Damage caused by O&NS is shown by increased levels of malondialdehyde (MDA), a by-product of polyunsaturated fatty acid peroxidation and arachidonic acid, and increased 8-hydroxy-2-deoxyguanosine, indicating oxidative DNA damage (Maes et  al. 2011). The malondialdehyde in the forced swim- and the tail suspension-induced depression was found to be at peak level, which was highly attenuated on treatment with selegiline HCl in different dosage forms, which produces response at different extents. Among these, the results of administration of TCNs have indicated the lowest level of malondialdehyde, and are comparable with CNPs, while the results of administration of TCNs at a lower dose of 5 mg/kg were not found to be comparable with those at a high dose of 10 mg/kg (Figure 9). A number of studies have suggested that oxidative stress, characterized by the imbalance between production of free radicals and the antioxidant capacity of an organism, may contribute to the neuropathology of neurological and psychiatric diseases, including major depression (Ng et  al. 2008). Glutathione is known as the body’s master antioxidant, usually found in every cell of body, and is the leading component of the body’s defense system. It directly quenches reactive hydroxyl free radicals, other oxygen-centered free radicals, and radical centers on DNA and other bio molecules. In our study, the GSH level in the disease condition was found to be at the lowest level, and on treatment with TCNs at 10 mg/kg, it was found to return to normal range, while a low response was found on administering the same formulation at a dose of 5 mg/kg. Catalase is a natural endogenous antioxidant enzyme, and its concentration was found to be highly impaired in disease

Figure 7. Effect of TCNs on nitrite level in depression-induced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low).

Figure 8. Effect of TCNs on GSH in depression-induced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low); dp  0.05 as compared to TCNs (high); ep  0.05 as compared to CNPs.

Figure 9. The effect of TCNs on LPO in depression-induced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low); dp  0.05 as compared to TCNs (high).

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10  D. Singh et al. conditions, which was found to be restored on treatment. The over-production of free radicals and the disturbances in their scavenging by catalase and associated antioxidant enzymes leads to oxidative stress. It has been reported that selegiline has neuroprotective and antioxidant properties, since it increases SOD and CAT activities (Ebadi et al. 2002, Magyar and Haberle 1998). Our study supports the antioxidant potential of the TCNs at a dose of 10 mg/kg (Figure 10). In different phases of the whole study with the forced swim test and the tail suspension test, the SOD level was found to be highly impaired in the disease condition, and was restored on treatment, at different levels. Among these, the responses to TCNs at a higher dose were found to be desirable as compared to those with plain selegiline and TCNs at a low dose. The free radical- neutralizing potential of selegiline is supported by the results demonstrated by Cohen and coworkers, and it is also supported by the another study on the underlying mechanism of the beneficial effects of selegiline associated with enhanced activity of free radical scavenging enzymes such as superoxide dismutase (SOD) and catalase (Figure 11) (Magyar and Haberle 1998).

Effect of TCNs on mitochondrial enzyme complex activity Mitochondrial oxidative damage is based on the fact that the mitochondrial respiratory chain is the major source of superoxide anion generation (Rinwa et  al. 2013). Energy production in the mitochondria is catalyzed by the protein complexes, namely NADH-ubiquinol oxidoreductase (complex I), succinate-ubiquinol oxidoreductase (complex II), ubiquinol cytochrome c oxidoreductase (complex III), and cytochrome C oxidase (complex IV) (Jezek and Hlavata 2005). There was a significant alteration in the activities of mitochondrial enzyme complexes I &II, and a decreased number of viable cells (complex III). The level of cytochrome C oxidase enzyme (complex IV) in forced swim-induced depression was also observed (Figures 12–15). Selegiline treatment in dosage forms of modified nanoparticles significantly restored the activities of mitochondrial enzyme

Figure 10. The effect of TCNs on catalase in forced swim-induced depression-induced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low).

Figure 11. The effect of TCNs on SOD in depression-induced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to selegiline; cp  0.05 as compared to TCNs (low); dp  0.05 as compared to TCNs (high).

complexes I and II, and improved the number of viable cells and the level of cytochrome C oxidase enzyme, as compared to the control group. However, the plain drug did not produce any significant effect on mitochondrial complex activities, and further coadministration of modified nanoparticles of selegiline significantly potentiated its protective effects. Gardner and Boles (2011) have demonstrated the alteration of the mitochondrial complex in depression. Besides, mitochondrial impairment may generate an excess of nitric oxide (Liu et al. 1997b), which further leads to oxidative damage (Clementi et al. 1998). Moreover, evidence suggests that the mitochondria progressively get damaged and lose their functional integrity due to reactive oxygen species (ROS), which thereby enhances the oxidative damage (Turrens 2003). This suggests that mitochondrial dysfunction might be the key factor in the production of ROS, which further leads to oxidative damage in depression.

Measurement of drug concentration in the brain Currently, the use of polymeric nanoparticles is one of the most promising approaches for CNS drug delivery (GarciaGarcia et al. 2005). In the measurement of drug concentration in the brain, the maximum amount of drug in the brain was found to be 8.2 mg/mL. The concentration of plain selegiline in the brain was found to be lower, while the highest concentration measured was that of TCNs. In the case of unmodified CS, the highest concentration in the brain was found to be 4 mg/mL (Figure 16). The surface properties of the nanoparticles represent a key factor in the drug delivery system for CNS targeting, as they protect the embedded drugs against chemical or enzymatic degradation. Increasing the chances of the active molecule reaching the target site, TCNs were found to absorbed by the adsorption-mediated transcytosis due to the reaction of the positively-charged TCNs with the negatively-charged endothelial cell at the outer layer of the BBB (Tosi et al. 2008), which is an advantage TCNs have over anionic nanocarriers. Selegiline hydrochloride is highly metabolized by the oral route due to this advantage, and drug concentration in the brain is found to be increased, while the amount of plain

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Selegiline hydrochloride-loaded TCNs  11

Figure 12. Effect of TCNs on the level of complex I in depressioninduced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to TCNs (low); cp  0.05 as compared to TCNs (high).

Figure 14. The effect of TCNs on the level of complex III in depressioninduced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to TCNs (low); cp  0.05 as compared to TCNs (high).

selegiline was found to be negligible in comparison with the amount of TCNs.

Conclusion The strategy of thiolation of CS (neuroprotective activity already well-known) and its novel application in the preparation of nanoparticles, making use of their advantages of strong mucoadhesion, excellent permeation enhancement, in situ gelling nature, and nose-to-brain delivery for effective treatment of depression, was found to be a big success by itself. In a comparative evaluation from all aspects, the TCNs were found to be more significant than the unmodified nanoparticles. In an evaluation of drug on animals, the results were found to be of good significance, and showed that the TCNs successfully attenuate the oxidative stress and restore the activity of the mitochondrial complex. In an evaluation of behavioral parameters, a drastic reduction in the dura-

Figure 13. Effect of TCNs on the level of complex II in depressioninduced rats. Values are expressed as mean  SEM. ap  0.05 as compared to disease control; bp  0.05 as compared to TCNs (low); cp  0.05 as compared to TCNs (high).

Figure 15. The effect of TCNs on the level of complex IV in depressioninduced rats. Values are expressed as mean  SEM ap  0.05 as compared to disease control; bp  0.05 as compared to TCNs (low); cp  0.05 as compared to TCNs (high); dp  0.05 as compared to CNPs.

Figure 16. The amount of various formulations found in the brain, following intranasal administration. The values of drug concentration in the brain are expressed as mean  SEM.

12  D. Singh et al. tion of immobility was seen, showing that TCNs successfully restore the impaired locomotor activity, and normal sucrose consumption was found on treatment. The aforementioned results of the nanoparticulate formulation developed were found to offer a promising approach for emerging diseases like depression, especially when the efficacy of the model drug by the oral route is not of much significance.­

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Acknowledgement The authors are thankful to Shree Praveen Garg, Chairman, ISF College of Pharmacy, Moga, Punjab, INDIA for his continuous support and encouragement.

Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.­

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