Self-Assembled, Chitosan Grafted PLGA Nanoparticles for Intranasal Delivery: Design, Development and Ex Vivo Characterization

July 3, 2017 | Autor: C. Pardeshi | Categoria: Chitosan, Surface modification, Mucoadhesive Drug Delivery System, PLGA Nanoparticles
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Self-Assembled, Chitosan Grafted PLGA Nanoparticles for Intranasal Delivery: Design, Development and Ex Vivo Characterization a



Shailesh S. Chalikwar , Bhushan S. Mene , Chandrakant V. Pardeshi , Veena S. Belgamwar a

& Sanjay J. Surana



Department of Pharmaceutics and Quality Assurance, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India Accepted author version posted online: 25 Feb 2013.

To cite this article: Shailesh S. Chalikwar , Bhushan S. Mene , Chandrakant V. Pardeshi , Veena S. Belgamwar & Sanjay J. Surana (2013): Self-Assembled, Chitosan Grafted PLGA Nanoparticles for Intranasal Delivery: Design, Development and Ex Vivo Characterization, Polymer-Plastics Technology and Engineering, 52:4, 368-380 To link to this article:

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Polymer-Plastics Technology and Engineering, 52: 368–380, 2013 Copyright # Taylor & Francis Group, LLC ISSN: 0360-2559 print=1525-6111 online DOI: 10.1080/03602559.2012.751999

Self-Assembled, Chitosan Grafted PLGA Nanoparticles for Intranasal Delivery: Design, Development and Ex Vivo Characterization Shailesh S. Chalikwar, Bhushan S. Mene, Chandrakant V. Pardeshi, Veena S. Belgamwar, and Sanjay J. Surana

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Department of Pharmaceutics and Quality Assurance, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India

The objective of present study was to modify the surface of Poly(D,L-lactide-co-glycolide acid) (PLGA) nanoparticles (NPs) with chitosan to enhance the mucoadhesive potential of carrier system. Grafting of chitosan on PLGA surface was carried out via amide bond formation mediated by carbodiimide and confirmed by FTIR spectroscopy. Self-assembled PLGA NPs containing chlorpromazine hydrochloride were fabricated by 23 factorial design. The improved mucoadhesive potential was confirmed by several tests including in vitro mucoadhesion study. Ex vivo permeation was satisfactory. Histopathological study on sheep nasal mucosa revealed safe mucoadhesion. They were also found to be robust on accelerated stability study. Keywords Chitosan; Complexation; Mucin; Mucoadhesion; PLGA; Polymer grafting; Surface modification

INTRODUCTION Polymeric drug delivery systems have attracted increasing attention during the last two to three decades. Poly(D,L-lactide co-glycolide acid) (PLGA) has been used for decades to deliver variety of drug molecules including small molecular weight compounds, peptides and proteins[1,2,3]. PLGA is an attractive polymer for delivery of biopharmaceuticals, approved by FDA, several PLGA based formulations have received worldwide marketing approval, non toxic, synthetic, biodegradable, biocompatible, outstanding controlled release characteristics and most widely used biomaterial because of its safety and enhanced drug stability, including drug carriers for hydrophobic as well as hydrophilic drugs[4,5,6]. Nevertheless, due to short residence time on the nasal mucosa, absorption of the PLGA incorporated drug is strongly limited. By improving the nanoparticulate delivery systems by modification of the Address correspondence to S. S. Chalikwar, Department of Pharmaceutics & Quality Assurance, R. C. Patel Institute of Pharmaceutical Education & Research, Karwand Naka, Shirpur, 425405, Dist: Dhule, Maharashtra, India. E-mail: [email protected]

surface with mucoadhesive agents, a prolonged residence time on mucosal surfaces can be achieved with PLGA. Chitosan is most widely selected as a conjugating material for PLGA nanoparticles (NPs) because of its recognized mucoadhesivity, biodegradability, biocompatibility and ability to enhance the penetration of large molecules across mucosal surfaces. Hence, in present study surface modification was under taken to improve the interaction of the hydrophobic PLGA NPs with the nasal and intestinal absorbing epithelia[7]. The interaction between cationic amino groups on chitosan and anionic moieties such as sialic and sulfonic acids on the mucus layer is responsible for its mucoadhesiveness[8]. Thus far, many lipophilic drugs have been encapsulated easily into PLGA NPs. However, encapsulation of water soluble ionic drugs, such as chlorpromazine hydrochloride (CPZ HCl), may be a great challenge for formulation of PLGA NPs. In order to enhance the encapsulation efficiency (EE) and there by oral bioavailability of CPZ HCl, in our study, Dextran sulphate (DS) was used as counter ion polymer which form complex with cationic CPZ HCl for neutralization of charge and resulted into hydrophobic complex which would allow the instantaneous entrapment of the complex into PLGA. Nanotechnology is an emerging interdisciplinary technology and widely used as a drug carrier system which is designed in such way that it can achieve adequate stability, improved absorption, controlled release, quantitative transfer and, therefore, the expected pharmacodynamic activity. The main objective of the nanotherapy is to guide drug molecules specifically and directly to diseased tissues. The aim of site-specific delivery is to minimize side effects on host encountered with conventional therapy[9,10]. Schizophrenia is a psychotic disorder characterized by a disintegration of thought processes and emotional responsiveness. It also commonly manifests as auditory hallucinations, paranoid or bizarre delusions, or disorganized speech and thinking. The onset of symptoms mostly


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occurs in young adulthood and global lifetime prevalence observes about 0.3–0.7%[11]. Chemically CPZ HCl is 2-chloro-10-(3-dimethylaminopropyl) phenothiazine hydrochloride, potent antipsychotic agent. It was first used to treat mental illness in Paris during 1951. CPZ HCl is thought to elicit their antipsychotic action via interference with central dopaminergic pathways in the mesolimbic areas of the brain. Some major side effects seen with it include sedation, amnesia, hypotension, dry mouth, constipation, urinary retention and lowering of seizure threshold. FDA has approved it in 1954 and is available in as injectable, oral and rectal dosage forms[12]. When rapid action is required to control acute severe symptomatology, intramuscular route is used primarily while for the long term treatment of psychiatric illness oral sustained release capsules are used. CPZ HCl can be useful for long term treatment of psychiatric illness via nasal drug delivery, as a strategy to enhance therapeutic efficacy through avoidance of hepatic first pass metabolism which is extensive in case of selected drug candidate[13]. CPZ HCl belongs to Biopharmaceutical Classification System (BCS) ‘‘Class I’’ having plasma half life of 30 h. It has low oral bioavailability of 30–50% because of its extensive first pass metabolism in liver[14]. Therefore, oral dosage forms required to be frequently administered with large doses. An alternative parenteral route of administration could have provided greater bioavailability; however, it causes the patient incompliance such that patient needs to be hospitalized and its safety problems. In addition parenteral administration is very irritating and is not advised; its use is limited to severe hiccups, surgery and tetanus. Therefore, alternative routes of administration are needed to improve its therapeutic efficacy. The blood–brain barrier (BBB) prevents most substances from freely diffusing and penetrating into the central nervous system (CNS) from the bloodstream in order to maintain brain homeostasis. As this barrier is also the primary obstacle for delivery of drugs to the brain, various methods of circumventing the BBB have been attempted. In the last decade, intranasal (IN) administration has attracted considerable interest, since it provides a noninvasive method for bypassing the BBB and delivering therapeutic drugs directly to the CNS. Nasal mucosa is relatively permeable and highly vascularized, providing rapid absorption of the applied drug. Nasal delivery also avoids degradation of drugs in the gastrointestinal tract and first pass metabolism in liver. Rapid absorption into the systemic circulation after nasal administration has been demonstrated with several drugs active in the central nervous system[15]. Drugs administered to the nasal cavity can travel along the olfactory and trigeminal nerves to reach many regions within the CNS. However, it has few limitations like short residence time of formulation within a nasal cavity due to the mucocilliary


clearance etc. The main limitation of nasal drug delivery is low membrane permeability of high molecular weight polar molecules such as proteins and peptides[16]. But, now a day, such problems overcome by the use of mucoadhesive polymers, some of which would even demonstrate additional permeation enhancing capabilities[17]. It is hypothesized that PLGA NP based nasal delivery system of CPZ HCl would provide brain targeting and sustained release of drug within the brain. This benefit would help to improve its clinical utility, decrease the dose and frequency of dosing, reduce side effects and improve therapeutic efficacy. EXPERIMENTAL Materials CPZ HCl was a kind gift from Torrent Pharmaceuticals Pvt. Ltd., Ahmadabad (India). PLGA and chitosan low molecular weight (viscosity 20–200 cps, 75–85% deacetylation grade) were purchased from Sigma Aldrich, USA. Dextran sulphate (sodium salt), mucin, polyvinyl alcohol (PVA) and dialysis membrane were purchased from HIMEDIA, Mumbai, India. N, N – dicyclohexyl carbodiimide (DCC) was purchased from S. D. Fine Chem Ltd., Mumbai, India. Water used in all the studies was distilled and filtered through 0.22-mm nylon filter paper before use. All other reagents and solvents used were of analytical grade. Experimental Design Present day use of design of is an attractive approach in polymer technology[18]. Thus a 23 simple factorial design was employed using Design Expert software (version 8.0.1; Stat-Ease, Inc., Minneapolis, MN, USA) for preparation of CPZ HCl loaded PNs (Table 1) and to study the effect of different independent variables on its properties. Three factors were evaluated each at two levels and experimental trials were performed at all possible eight combinations. Drug to dextran sulphate ratio (X1), sonication time (X2) and surfactant concentration (X3) were selected as independent variables. Two responses, particle size (Y1), EE (Y2) were measured for each trial and taken as dependent variables (Table 1). For optimization of the desirable batch, particle size was kept at minimum level, while EE was kept at maximum level. Preparation of Chitosan Grafted PLGA Nanoparticles Loaded with CPZ HCl Depolymerization of Chitosan Chitosan was depolymerized as previously described method by Chattopadhyay and Inamdar[19]. Low molecular weight chitosan resulted from depolymerization upon hydrolysis by nitrous acid as a depolymerising agent. A 2% solution of chitosan in acetic acid was prepared. Predissolved



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TABLE 1 Independent variables along with their formulation code, levels and respective average particle size, PDI, zeta potential, EE (%) and DL(%) of different batches of PNs Formulation code

Drug: DS ratio (X1)

F1 F2 F3 F4 F5 F6 F7 F8

1:1 (þ1) 1:1 (þ1) 1:1 (þ1) 1:1 (þ1) 1:0.5 (1) 1:0.5 (1) 1:0.5 (1) 1:0.5 (1)

Sonication Surfactant Avg. particle time (min) concentration size (nm)a (X2) (%) (X3) (Y1) 1 2 1 2 1 2 1 2

(1) (þ1) (1) (þ1) (1) (þ1) (1) (þ1)

1 2 2 1 1 2 2 1

(1) (þ1) (þ1) (1) (1) (þ1) (þ1) (1)

700.0  27 390.2  19 527.3  23 463.9  12 617.6  24 306.1  17 503.0  21 453.2  20

PDIa 0.402  0.04 0.221  0.05 0.312  0.08 0.187  0.08 0.296  0.07 0.173  0.09 0.462  0.11 0.324  0.09

Zeta potentiala (mV) EE %a(Y2) 24.5  2.8 8.03  1.3 12.6  1.7 21.0  2.0 24.7  2.3 5.67  0.7 11.3  1.2 21.7  2.1

30.94  2.78 32.45  2.91 27.92  2.34 36.72  3.72 21.38  2.15 23.89  2.26 18.61  1.73 25.66  2.27

DL%a 4.05  0.07 3.86  0.08 3.49  0.06 4.59  0.09 2.98  0.04 2.67  0.05 2.32  0.03 3.20  0.05

[(þ1) indicates high level and (1) low level)];. a These results are mean  standard deviation. (n ¼ 3).

dilute solution of sodium nitrite was then added gradually to chitosan solution and stirred vigorously for 2 h at room temperature to get desired viscosity level. The depolymerized chitosan was then precipitated out by 5 M sodium hydroxide. The precipitates of chitosan was then washed 3 to 4 times with methanol and dried at 60 C. Activation of Carboxylic Group of PLGA PLGA (100 mg) dissolved in dichloromethane. About 6 mg of DCC added in the preceding solution and stirred for 30 min at room temperature to activate the carboxyl group of PLGA. The resultant solution was filtered and precipitated by dropping into ice-cold diethyl ether and the activated PLGA was completely air dried at room temperature[20,21]. Preparation of CPZ HCl PLGA NPs PLGA NPs prepared by a combined self-assembled and nanoprecipitation technique as a previously described method[22] with slight modifications. PVA was used as surfactant for the preparation of PNs. Aqueous solutions containing 12.5 mg of CPZ HCl (i.e., with weight ratio of 12.5% to PLGA) and DS (Table 1) were prepared and added dropwise in previously prepared PVA aqueous solution (Table 1) under magnetic stirring (Remi Motors, Mumbai, India). Then, activated PLGA polymer (100 mg) was transferred to acetonitrile to form organic phase. This organic solution was injected dropwise in to the aqueous solution. The resultant emulsified solution sonicated for 1 or 2 min using Probe Sonicator (PCI Analytics Ltd., Mumbai, India) and gently stirred at room temperature by using a magnetic stirrer at a rate of 120 rpm overnight to evaporate the organic solvent. Nanoparticles were collected by centrifugation at 8000 rpm using cooling centrifuge (Remi Instruments. Ltd., Mumbai, India).

Grafting of Chitosan on the PLGA NP Surface Centrifuged above PLGA NPs were dissolved in 10 ml of 0.1 M phosphate buffer, pH 4.5 to form suspension. Earlier depolymerized chitosan (60 mg) dissolved in 6 ml of 0.5% acetic acid, pH 3.5, and added to the above suspension. The mixture was magnetically stirred (vigorously) at room temperature for 12 h. Chitosan conjugated NPs were centrifuged and washed with 0.5% acetic acid in order to remove free chitosan. Finally washed and freeze-dried using freeze dryer (Vir Tis, Benchtop 4.0 K XL, USA)[23]. Confirmation of Grafting of Chitosan on PLGA NPs Fourier Transformed Infrared (FTIR) Study FTIR spectra of chitosan grafted PLGA NPs (optimized batch F4), pure PLGA, pure chitosan were recorded with FTIR spectrometer (8400 S, Shimadzu) using the KBr disk method (2 mg sample in 200 mg of KBr). The scanning range was 250  3500 cm1 and the resolution was 1 cm1[24]. Particle Size and Zeta Potential Measurement Particle size and zeta potential was measured before and after surface modification of PNs (optimized batch F4) using a Zetasizer (Nano ZS 90 Malvern Instruments, UK) as per procedure described in characterization of CPZ HCl PNs. The study was conducted in triplicates. Characterization of CPZ HCl PNs Particle Size, Polydispersity Index and Zeta Potential Photon correlation spectroscopy (PCS) was used to measure size of all formulation samples, which analyzed the fluctuations in light scattering resulting from the Brownian motion of the particles, using a Zetasizer Nano ZS 90 (Malvern Instruments, UK). Nanoparticle formulations were diluted with double distilled water to get


optimum 50–200 kilo counts per second (kcps) for measurements. Polydispersity index was also studied[25,26]. The surface charge of the NPs was determined by measuring the zeta potential using the same equipment[26,27]. Zeta potential measurements were run at 25 C with electric field strength of 23 V=m [28,29].

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Entrapment Efficiency (EE %) and Drug Loading (DL %) For the determination of EE, 10 mg of lyophilized NPs were digested in 2 ml of dichloromethane. The formed solution was centrifuged at 14,000 rpm for 10 min and the supernatant was suitably diluted with dichloromethane and analyzed spectrophotometrically at kmax 254 nm. Values of EE % and DL % were calculated using Equations (1) and (2), respectively [26,30]. EE% ¼

DL% ¼

Amount of drug recovered in nanoparticles  100 Total amount of drug added ð1Þ Amount of drug recovered in nanoparticles  100 Total mass of polymer ð2Þ

Mucus Adsorption Study Mucus Glycoprotein Assay To access mucoadhesive potential of prepared CPZ HCl PNs mucus glycoprotein assay was carried out. It was performed according to a method previously reported by Patil et al.[31]. Mucus glycoprotein assay determine the amount of mucin adsorbed onto the surface of NPs by knowing the concentration of free mucin. Adsorption of Mucin on PNs Mucin aqueous solutions with different concentrations (0.1, 0.2, 0.4, 0.8, 2 mg=ml) were prepared. A weighed quantity of NPs were dispersed in the above mucin solutions (2 ml), vortexes, and shaken at room temperature for 1 h. Then, the dispersions were centrifuged at 4000 rpm for 4 min, and the supernatant was used for the measurement of the mucin content by mucus glycoprotein assay. From standard calibration curve of mucin, concentration was calculated and then from the difference between the total amounts of mucin added and the free mucin in the supernatant, amount of mucin adsorbed onto the nanoparticles surface was calculated. Using Freundlich and Langmuir equations, data obtained were interpreted. In Vitro Mucoadhesion Study The mucoadhesive property of prepared NPs was determined by falling liquid film technique. A freshly cut piece of sheep nasal mucosa obtained from local abattoir


(Shirpur, India) of 2 cm2 area within 1 h of sacrificing the animal which was cleaned by washing with isotonic saline solution. Accurately weighed 100 mg of freeze dried NPs were placed on mucosal surface, which was attached over a polyethylene plate. Simulated nasal electrolyte solution (SNES) of 100 ml of volume was placed on NPs. To allow the polymer to interact with the membrane, this plate was incubated for 15 min in desiccator at 90% relative humidity and finally fixed at an angle of 45 relative to the horizontal plane. Phosphate-buffer saline (PBS) solution (pH 6.6) warmed at 37 C was peristaltically pumped at a rate of 5 ml=min over the tissue. One hour after administration of NPs, the amount of drug in collected perfusate was determined spectrophotometrically. The amount of NPs corresponding to the amount of drug in the perfusate was determined. The amount of adhered NPs was estimated as the difference between the amount of applied NPs and the amount of flowed NPs. Percent mucoadhesion was computed using the following Equation (3)[32]. Invitro mucoadhesion ð%Þ

Amount of adhered PNs  100 Amount of applied PNs ð3Þ

In Vitro Drug Release Study In vitro drug release from CPZ HCl NPs was performed in PBS solution (pH 6.6), using dialysis bag method as per method previously reported by Seju et al. (2011)[33] with slight modification. Dialysis membrane (cut off 12,000 Da) was soaked overnight in PBS solution prior usage. Aqueous CPZ HCl NPs dispersion equivalent to 2 mg of drug was placed in dialysis bag and sealed at both ends. The dialysis bag was immersed in a beaker containing 200 ml of PBS solution at 37  2 C and magnetically stirred at 100 rpm. Samples were withdrawn at predetermined time intervals and the sink condition was maintained replacing with fresh pre-warmed PBS solution at same temperature. The withdrawal samples were diluted if necessary and analyzed for estimation of CPZ HCl by UV spectrophotometer (1700, Shimadzu, Tokyo, Japan) at kmax 254 nm. The study was performed in triplicates. Drug Release Kinetics To study the drug release kinetics, the data obtained from in vitro drug release study was fitted to various kinetic models such as zero order [Equation (4)] as cumulative amount of drug released versus time; first-order [Equation (5)] as log cumulative percentage of drug remaining versus time and Higuchi’s model [Equation (6)] as cumulative percentage of drug released versus square root of time. Zero-order C ¼ K0 t




where K0 is the zero-order rate constant expressed in units of concentration=time and t is the time in min. A graph of concentration versus time would yield a straight line with a slope equal to K0 and intercept the origin of the axes. First-order

0.45 mm nylon filter, diluted if necessary and analyzed using a UV Visible spectrophotometer at kmax 254 nm. Each removed sample was replaced by an equal volume of fresh pre warmed diffusion medium having same temperature [33]. The study was performed in triplicates.

Log C ¼ Log C0  Kt=2:303

Histopathological Study Histopathological studies were carried out using isolated sheep nasal mucosa obtained from a local abattoir within 2 h of sacrificing the animal. It was cleaned by washing with isotonic saline solution and sectioned into two pieces. One piece was used as control (untreated nasal mucosa), and another piece was treated with NPs suspension in PBS solution and used as test (treated nasal mucosa). After 2 h, they were fixed in 10% neutral carbonate buffered formalin solution, routinely processed and embedded in paraffin. For the viability of the tissues, the experiment was carried out in a cell culture incubator (Sanyo Incubator, Model MCO-5AC, Japan) to assure optimal conditions. Paraffin sections were cut on glass slides and stained with hematoxylin and eosin (HE). Sections were examined under a microscope (Motic, Prompt Plus DMB 1, 2.0 version, Italy), to detect any damage to the tissue during nasal administration[33,36].


where C0 is the initial concentration of drug, K is the firstorder constant and t is the time. Higuchi’s model Qt ¼ Kt1=2


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where Qt is the amount of drug released in time t, K is the kinetic constant and t is the time in min. Mechanism of Drug Release To evaluate the drug release mechanism from the CPZ HCl PNs, data obtained from the in vitro drug release studies was subjected to Korsmeyer–Peppas model [Equation (7)] as log cumulative percentage of drug released versus log time. The release exponent n and K values were calculated from the slope of straight line. Mt=M1 ¼ Ktn


Where Mt represents the amount of the released drug at time t, M1 the total amount of drug released after an infinite time, K the diffusional constant of the drug polymer system and n is an exponent that characterizes the mechanism of drug release. Value of n indicates drug release mechanism from the delivery system [34,35]. Ex Vivo Permeation Study An ex vivo drug permeation study was performed using Franz diffusion cell across sheep nasal mucosa as permeation barrier, obtained from the local slaughter house within 1 h of sacrificing the animal. Freshly excized sheep nasal mucosa was dipped immediately in PBS solution (pH 6.6). Cartilages were removed properly; the mucosal membrane was isolated and washed with PBS solution. Mucosal membrane mounted in the diffusion cell with mucosal and serosal surfaces facing the donor and receiver compartment, respectively. Receiver compartment was filled with 25 ml of fresh PBS solution. Ex vivo diffusion was conducted by reconstituting the lyophilized CPZ HCL PNs (equivalent to 2 mg of drug) with 1 ml of PBS solution and placed onto the stabilized sheep nasal mucosal membrane in the donor compartment and stirred slowly on a magnetic stirrer. The temperature was maintained at 37  0.5 C with the help of a circulating water bath. Samples from receiver compartment were withdrawn at predetermined time intervals, filtered through

Scanning Electron Microscopy (SEM) Morphology of NPs was studied by using scanning electron microscope[6]. The optimized freeze dried formulation was kept onto metal plate and dried under vacuum to form a dry film that was then observed under the scanning electron microscope (LEO 440i; Leo Electron Microscopy, Ltd., Cambridge, UK) at the required magnification and at voltage of 20 kV. Differential Scanning Calorimetry (DSC) The thermal behavior of pure drug; polymers such as pure chitosan, pure PLGA, pure dextran sulphate; physical mixture of drug and above polymers in 1:1 ratio, and freeze-dried optimized NP formulation was studied using a differential scanning calorimeter (DSC 1 stare system, Mettler-Toledo, Switzerland) at a heating rate of 10 C= min in the range of 35 C to 350 C, under nitrogen environment at 40 ml=min. X-Ray Diffraction (XRD) The crystallinities of drug, placebo lyophilized PN formulation, drug loaded lyophilized PN formulation and physical mixture of drug and placebo lyophilized PN formulation (in 1:1 ratio) were analyzed by using X-ray diffractometer (Bruker AXS D8 Advance, Germany). The samples were mounted on a sample holder and XRD patterns were recorded in the range of 3–80 at a chart speed of 5 per min[20].



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Accelerated Stability Study Accelerated stability study was carried out according to International Conference on Harmonization (ICH) Q1A (R2) guidelines and as previously reported by Chalikwar et al., 2012[37]. It was performed at 25  2 C=60  5% RH with respect to particle size, zeta potential and EE. Freshly prepared freeze dried powder of optimized batch was filled in 3 different amber coloured glass vials, sealed and placed in stability chamber (CHM-10S, Remi Instruments Ltd., Mumbai, India) and maintained at 25  2 C=60  5% RH for a period of total 3 months. The dried powder samples subjected for stability test were re-dispersed in distilled water and analyzed with a sampling interval of 1 month for particle size, zeta potential and EE. RESULTS AND DISCUSSION Preparation of Chitosan-Grafted PLGA Nanoparticles Loaded with CPZ HCl CPZ HCl, being a hydrophilic drug, cannot be directly targeted to the brain. Self assembled and nanoprecipitation technique was employed to encapsulate hydrophilic moiety in the core of hydrophobic PLGA polymer. Dextran sulphate (DS) is a anionic counterion polymer that form the complex with cationic drug, resulted into neutralization of anionic and cationic charge on DS and CPZ HCl, respectively, and that supported to the high degree of hydrophobicity[22] and CPZ-DS complex was successfully embed in PLGA core. Depolymerization of chitosan causes the reduction in molecular mass that favours the access of the free amino group for further coupling with carboxylic group of PLGA. Carboxylic group of PLGA NPs were activated by DCC forming o-acylurea as intermediate that reacts with primary amino groups of chitosan and resulted into the formation of amide bonds[23]. The scheme for the surface modification of CPZ HCl PLGA NPs with chitosan is represented in Figure 1.

FIG. 1. Scheme forsurface modification of CPZ HCl PLGA NPs with chitosan.

Confirmation of Grafting of Chitosan on PLGA NPs Fourier Transform Infrared Study Confirmation of conjugation between PLGA NPs and chitosan was done by FTIR study (Figure 2). Intensity of peak at 1756 cm1 for PLGA ester C¼O stretching was found to be less intense for chitosan grafted PLGA NPs (Figure 2a) compared to pure PLGA (Figure 2b) and peak at 1626 cm1 is a characteristic frequency for the C¼O stretching of amide bonds, which was observed in chitosan-grafted PLGA NPs (Figure 2a)[38].

FIG. 2. FTIR spectra of a) chitosan-grafted PLGA NPs, b) pure PLGA, and c) pure chitosan.

Particle Size and Zeta Potential Measurement Confirmation of conjugation between PLGA NPs and chitosan was also done by measuring the particle size and zeta potential before and after modification on PLGA

PNs[23]. Particle size of chitosan grafted PLGA NPs before modification was found to be 339  07 nm, while after modification it was found to be 463.9  12 nm, while zeta



potential changes from negative to positive, i.e., from 18.2  1.7 mV to þ 21.0  2.0 mV. These results also supports to earlier FTIR study.

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Characterization of CPZ HCl NPs Particle Size, Polydispersity Index and Zeta Potential Average particle size, polydispersity index, zeta potential of all batches of PNs is reported in Table 1. The (polydispersity index PDI) was studied to determine the narrowness of the particle size distribution. All CPZ HCl PNs showed zeta potential values towards positive indicating an immobilization of cationic chitosan at the surface of NPs and under suppression of original negative charge. Effect of Surfactant Concentration on Particle Size As the concentration of PVA increased from lower to higher concentration (i.e., from 1% to 2%) resulted into the reduction in particle size of CPZ-PLGA NPs (Figure 3a). At lower surfactant concentration, NPs were aggregated to produce larger size. It is easy to understand that an insufficient amount of emulsifier would fail in stabilizing all the NPs and thus some of them would tend to aggregate. As a result, NPs with larger size would be produced[39,40].

Effect of Sonication Time on Particle Size Prolonged time of sonication resulted into decrease in particle size (Figure 3b). Because longer duration of sonication causes release of high energy and their input into the system produce rapid dispersion of organic solution as nanodroplets of small size. It also prevents the agglomeration of particles and rapidly emulsified into nanosized droplets[30,39]. Entrapment Efficiency (EE %) and Drug Loading (DL %) The EE % and DL % of all designed batches of CPZ-PLGA NPs are reported in Table 1. By considering the results in terms of particle size and EE, batch F4 was screened as optimized batch. Effect of Surfactant Concentration on EE Surfactant is mainly used in NP preparations to prevent their aggregation and formation of larger globules. But, experimentally it was observed that, as the concentration of surfactant increased from 1% to 2% then EE was decreased (Figure 3c), in agreement with earlier report[40]. Effect of Sonication Time on EE Sonication time showed linear relationship with EE. Longer duration of sonication causes proportionate increase in EE (Figure 3 d). Because high sonication time releases more energy that help in preventing drug leaching out from NPs and ultimately contributes to increase in EE[30]. Effect of Drug to DS Ratio on EE A 1:1 drug-to-dextran ratio showed higher EE as compared with 1:0.5 ratio (Figure 3e). From the obtained results it was revealed that concentration of DS play major role in entrapment of drug into PLGA NPs[22]. Mucus Adsorption Study Mucus Glycoprotein Assay As the concentration of mucin increased, the amount of mucin adsorbed on surface of PNs was also increased. Thus, there was existence of stronger interaction between mucin and chitosan. The linearity range was found to be in the range of 0.05–0.25 mg=ml with the linear regression equation, y ¼ 0.003x þ0.029.

FIG. 3. Effect of (a) surfactant concentration on particle size, (b) sonication time on particle size, (c) surfactant concentration on EE, (d) sonication time on EE, and (e) drug to dextran sulphate ratio on EE. (Color figure available online.)

Adsorption Isotherm The data obtained from adsorption studies were fitted to Freundlich and Langmuir equations. Constant values of Freundlich isotherm (n and K) and Langmuir isotherm (a and b) were obtained from straight lines and reported in Table 2. It was found that the values of R2 were significantly higher (p < 0.05) for the Langmuir equation as compared to the Freundlich equation. This indicates



TABLE 2 Isotherm constants for Langmuir and Freundlich equations of batch F4 Freundlich isotherma n 1.052

K 0.177

R2 0.986

Langmuir isothermb a 1.444

b 0.082

R2 0.994


x=m ¼ K  Ce1=n;. 1=(x=m) ¼ aþb  1=Ce. x=mis mucin adsorbed, K and n are Freundlich isotherm constants, a and b are Langmuir isotherm constants, and Ce is the free mucin.

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a more specific adsorption process where electrostatic interactions are involved. The adsorption of mucin to chitosan is expected to be dominated by the electrostatic attraction between the positively charged chitosan and negatively charged mucin[31]. This study was only performed on optimized batch (F4). In Vitro Mucoadhesion Study Mucoadhesion study was carried out to ensure the adhesion of NPs (optimized batch F4) to nasal mucosa for prolong period of time at the site of absorption. NPs have shown mucoadhesion of 87  8.9% and could satisfactorily adhere on sheep nasal mucosa. The functional groups present on surface of mucoadhesive polymers are hydroxyl, carboxyl, amine and amide. A linear polysaccharide, chitosan contains the amine functional groups contents hence showed higher percentage of mucoadhesion on nasal mucosa. The interaction between cationic amino groups on chitosan and anionic moieties such as sialic and sulfonic acids on the mucus layer is responsible for its mucoadhesiveness[8,41]. In Vitro Drug Release Study This study was performed on all prepared batches but results were reported only for optimized batch. The dissolution profile showed biphasic behavior consisting of initial burst release followed by a slow release phase for the period of 48 h (Figure 4). The initial burst release can be attributed to the presence of swelling of polymer, i.e., chitosan was grafted over the surface of PLGA NPs; a slight improvement of initial burst could be achieved. As the PLGA is hydrophobic polymer, the initial burst of the drug was not continued; instead, sustained release of drug was attributed from PLGA NPs. Drug Release Kinetics The data obtained from in vitro drug release studies of F4 batch was fitted to the different models like zero-order, first-order and Higuchi’s model. The regression coefficient

FIG. 4. In vitro drug release profile of optimized batch F4 of CPZ HCl PNs. (Color figure available online.)

(R2) for zero-order, first-order and Higuchi’s model was found to be 0.862, 0.903 and 0.979, respectively. Thus, the best fitted model was found to be Higuchi’s model, as it showed the highest linearity. Mechanism of Drug Release The corresponding plot of log cumulative percentage drug release versus log time of the Korsmeyer–Peppas equation indicated regression coefficient (R2 ¼ 0.879), n and the K values were found to be 0.647 and 0.634, respectively. The n value indicated that the optimized formulation followed the non-Fickian or anomalous diffusion mechanism of drug release[34]. Ex Vivo Permeation Study The optimized formulation was subjected to ex vivo permeation studies using sheep nasal mucosa. Percent drug permeation across the sheep nasal mucosa form NPs was found to be 9.01  0.602 at the end of 4 h, as depicted in Figure 5. However, unlike in vitro drug release studies, no burst or biphasic release behavior could be observed. It would be logical to attribute this to the barrier properties of both PLGA matrix and the nasal mucosa, both of which act as rate limiting membranes for permeation of drug from the PLGA NPs. The results are in consistent with previous study[33]. Histopathological Study It was necessary to examine histological changes in NPs treated (test) mucosa and its comparison with untreated (control) mucosa. The photomicrograph of both was depicted in Figure 6. No change in all components like goblet cell, mucus layer was appeared in both Figures 6a and 6b. Both the membranes were normal and no severe signs such of epithelial necrosis were detected. Thus, these

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FIG. 5. Ex vivo permeation study of optimized batch F4 of CPZ HCl PNs. (Color figure available online.)

results indicate that the NPs seem to be safe for nasal administration. Scanning Electron Microscopy (SEM) The shape and surface morphology of CPZ HCl NPs was examined by scanning electron microscope and depicted in Figure 7. The SEM photomicrograph of optimized NPs confirmed their non-spherical shape. They possessed smooth surface without any rupture. This morphology would help in slow clearance and longer retention in nasal cavity.

FIG. 7.

SEM photograph of CPZ HCl PNs of optimized batch F4.

for chitosan at 62.25 C (B), for PLGA at 49.00 C (C), and for dextran sulphate at 60.6 C (D). No considerable shift in the position of endothermic peaks was observed in the DSC thermogram of physical mixture of drug and used polymers (E). Thus, DSC study concluded the absence of any chemical incompatibility between drug and polymers. Absence of endothermic peak of CPZ-HCl in freeze dried NPs (F) revealed that molecular inclusion of drug in to the polymers and conversion of crystalline state of drug to amorphous state. Peak at

Differential Scanning Calorimetry (DSC) DSC is a highly useful means of detecting drug-excipient incompatibility in the formulation. The DSC thermograms of pure drug (A), pure chitosan (B), pure PLGA (C), pure dextran sulphate (D), physical mixture of drug and above polymers in 1:1 ratio (E), and freeze dried optimized PNs (F) are depicted in Figure 8. The DSC thermogram showed a sharp endothermic peak for CPZ-HCl at 197.18 C (A),

FIG. 6. Light photomicrograph of the nasal mucosa (a) untreated sheep nasal mucosa (control) (b) PNs-treated sheep nasal mucosa (test). (Color figure available online.)

FIG. 8. DSC thermogram of A) pure drug, B) pure chitosan, C) pure PLGA, D) pure dextran sulphate, E) physical mixture of drug and used polymers, and F) freeze-dried CPZ HCl PNs of optimized batch F4.



TABLE 4 Summary of results of regression analysis for responses Y1 and Y2 Models


Response 0.9743 (Y1) Linear model Response 0.9935 (Y2) Linear model

Adjusted Predicted R2 R2











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Y1: Particle Size, Y2: EE, R2: Regression Coefficient, SD: Standard Deviation, CV: Coefficient of Variance.

FIG. 9. XRD pattern of A) pure drug, B) placebo lyophilized PN formulation, C) drug-loaded lyophilized PN formulation, and D) physical mixture of drug and placebo lyophilized PN formulation.

163.58 C in freeze-dried optimized PN (F) formulation was due to the presence of mannitol, used as cryoprotectant during lyophilization. X- Ray Diffraction (XRD) X-ray diffraction patterns of pure crystalline drug (A), placebo lyophilized PN formulation (B), drug-loaded lyophilized PN formulation (C), and physical mixture of drug and placebo lyophilized PN formulation (in 1:1 ratio) (D) are shown in Figure 9. The typical crystalline nature of drug was defined by observing sharp principal peaks of CPZ HCl between 5 to 30 on 2h scale (Figure 9A). The broad peak of placebo freeze dried PN formulation was recorded (Figure 9B) and distinctive sharp peaks of drug were disappeared in drug loaded freeze dried PN formulation (Figure 9C). Although both broad peak and sharp peak were obtained in physical mixture of drug and placebo PN formulation (Figure 9D). These results were in good support with DSC results, indicating the CPZ

HCl encapsulated in core of surface modified NPs and conversion of drug from crystalline to amorphous state. Accelerated Stability Study Accelerated stability studies of freeze-dried PNs of optimized batch was conducted using particle size, zeta potential, and EE as prime parameters. The results are reported in Table 3. It was observed from these results that there was slight but linear increase in average particle size, where as slight but linear decrease in zeta potential and EE was observed on three-month storage. So, it can be concluded that, there were no significant alteration in average particle size, zeta potential and EE. Hence, they were found to be stable at 25  2 C=60  5% RH for a total period of 3 months. Optimization Data Analysis Fitting of Data to the Model The three factors with lower and upper design points in coded and uncoded values are shown in Table 1. All the responses observed for eight formulations prepared were fitted to various models using Design-Expert1 software. It was found that the best-fitted model was linear for both particle size and EE. The values of R2, adjusted R2,

TABLE 3 Accelerated stability studies of CPZ HCl PNs of optimized batch Test period Stability parameter Particle sizea (nm) Zeta potentiala (mV) EEa (%) a

0 month 463.9  12 21.0  2.0 36.72  3.72

1 month

2 month

3 month

479.8  24 19.72  1.8 33.06  2.86

490.6  17 18.91  2.2 31.18  2.11

496.1  20 17.21  1.6 29.94  1.91

These results are mean  standard deviation. (n ¼ 3).



TABLE 5 Summary of results of analysis of variance for measured response Parameters DF

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Particle size Model





104782.26 34927.42 50.60

Residual Total EE Model

4 7

2761.27 690.32 107543.53 35617.74



84.01 202.89

Residual Total

4 7

1.66 253.69

0.41 84.42

– –

– –

Significance p

0.0012 significant – –
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