OPTIMIZED METHOD FOR OBTAINING CHITOSAN NANOPARTICLES LOADED WITH XANTHINE DERIVATIVE

Transcription

1 Rev. Med. Chir. Soc. Med. Nat., Iaşi 2015 vol. 119, no. 3 PHARMACY ORIGINAL PAPERS OPTIMIZED METHOD FOR OBTAINING CHITOSAN NANOPARTICLES LOADED WITH XANTHINE DERIVATIVE Florentina Lupascu 1, Oana Dragostin 1, Luminita Confederat 1, P. Dubruel 2, Mamoni Dash 2, S. Keshari 2, Andreea Pânzariu 1, Ioana Vasincu 1, Lenuta Profire 1 * University of Medicine and Pharmacy Grigore T. Popa - Iasi Faculty of Pharmacy 1.Department of Pharmaceutical Chemistry Ghent University - Ghent, Belgium 2.Department of Polymer Chemistry & Biomaterials Research Group *Corresponding author. lenuta.profire@umfiasi.ro OPTIMIZED METHOD FOR OBTAINING CHITOSAN NANOPARTICLES LOADED WITH XANTHINE DERIVATIVE (Abstract): The use of nanotechnology is the current trend for drug delivery approaches. Aim: This research was focused on the development of an optimized method for preparation and characterization of chitosan nanoparticles (CSNPs) and xanthine derivative loaded CSNPs. Materials and methods: In order to develop appropriate method different parameters such as the concentration of chitosan and tripolypho s- phate as well as the stirring speed were varied. The aggregation of the nanoparticles (NPs) was prevented by using Tween 80 as a surfactant. Results: Following the CSNPs optimization process nanoparticles in the range of nm were obtained. The optimized method was used for the loading of xanthine derivative in the CSNPs. The particle size of CSNPs loaded with xanthine derivative was 267 nm and the loading efficiency was ~ 86%. In presence of the surfactant, the NPs size decreases slightly from 267 nm to 193 nm with increasing the concentration of Tween 80. The CSNPs loaded with xanthine derivative remai n stable after 72 h of storage. Conclusions: The CSNPs and xanthine derivative loaded CSNPs obtained after the optimization method by modifying several independent variables can be characterized as having small size and a good stability over time. Given th ese important features CSNPs are promising materials for targeted drug delivery system. Keywords: NANO- PARTICLES, IONIC GELATION, CHITOSAN, TRIPOLYPHOSPHATE. Drug carrier systems constitute an important research field due to some characteristics such as the capacity to encapsulate bioactive compounds, cross biological barriers and to protect macromolecules such as proteins, peptides from degradation in biological media and to deliver drugs to a target site with following controlled release (1). Nanogels (nanometer sized hydrogel particles) have attracted growing interest over the last several years in terms of their potential for applications in pharmaceutical fields, like drug delivery systems for improve the pharmacokinetic profile of drugs such as poor gastro-intestinal absorption, low drug solubility, and rapid metabolism. In the last years have been important developments of several synthetic as well as natural polymers and has researched the possibility of their use for pharmaceutical applications (2). 925

2 Florentina Lupascu et al. Natural polymers can be generally divided into four different categories, including polysaccharides, proteins, nucleic acids, and lipids. One polysaccharides large used is chitosan (CS) and it is defined as polymeric carbohydrate structures composed of repeating monosaccharide units linked by glyosidic bonds (3). Recently CS, the second most abundant natural polymer after cellulose, has attracted great attention in pharmaceutical and biomedical fields because of its advantageous biological properties such as: widely found in nature, biocompatibility, nontoxicity and biodegradability (4, 5). The manufacturing process of CS particles as carriers for drug delivery system, especially in terms of nanoparticles (NPs), has widely been studied (6). Several different techniques are usually employed to obtain NPs from CS including ionic gelation emulsion, synthesis with carboxymethyl cellulose, formulation using glutaraldehyde, synthesis with alginate and polymerization with poly (hydroxyethyl methacrylate). Among them the ionic gelation technique (IGT) has attracted considerable attention because it uses nontoxic cross-linker, organic solvent free and has convenient and controllable benefits (7, 8). IGT using syringe dropping is usually employed to prepare chitosan nanoparticles (CSNPs), in which sodium tripolyphosphate (TPP) solution (ph 7-9) was dropped into the acidic solution (ph 4-6) of CS. Despite of the easy procedures, it is uneasy to control the process for reproduction (9). The obtaining of CSNPs by IGT involve electrostatic interactions between the positively charged primary amino groups of chitosan and negatively charged groups of polyanions employed as crosslinkers to determined ph values, behaving as a metastable thermodynamic system which is highly sensitive to variations in ionic strength. For this reason appropriate choice of CS amount, the time spent in the reaction, the rate of addition and concentration of cross-linker agent TPP, the stirring speed applied during the reaction, ph of the solution are factors that affect the properties of CSNPs: particle size, long-term stability during storage, drug loading and release efficacy. It can be concluded that preparation process of CSNPs could be manipulated by varying the preparation conditions. TPP is the most frequently non-toxic polyanion used as cross-linker agent for preparation CSNPs (2). It is know that NPs have a high tendency to aggregate after a storage period as a result of increasing the surface area in NPs formulations (10). During the preparation process of CSNPs the mechanical energy associated with the reaction stirring speed may exceed the electrostatic repulsion energy between nanoparticle positive surface charges, thus eventually triggering aggregation tendency. This represents a recurrent problem in the formation of single nanoparticles. Tween 80 decreases the surface tension at the interface between materials so this agent can be used mainly to limit aggregation of NPs and therefore acts in the reducing of their sizes (11). Therefore, developing CSNPS with small particle size is the key to improve its encapsulation efficacy and other functionalities. This paper aims to describe the protocol for obtaining CSNPs in order to be used as drug delivery systems for xanthine derivatives. MATERIAL AND METHODS Materials. Chitosan low molecular weight (CSLMW), TPP and Tween 80 were purchased from Sigma Aldrich. Chitosan 926

3 Optimized method for obtaining chitosan nanoparticles loaded with xanthine derivative had a molecular weight (M w ) of 190, ,000), viscosity of 200 cps and degree of deacetylation (DD) of 75-85% as indicated by the supplier. All other reagents and solvents were of high grade purity and were obtained from commercial suppliers. Methods Nanoparticles formulation CSNPs were prepared via IGT proposed by Fabregas et al. (10). In order to optimize the method different concentrations of CSLMW 0.05%, %, 0.25% w/v were chosen. Briefly, CSLMW was dispersed in acetic acid 2% (v/v) and the mixture was left under stirring overnight at room temperature to obtain clear CS gel. Then 2 ml of TPP aqueous solution (% and 0.5%) were added drop wise to 0.25 ml CSLMW solution (0.05%, % and 0.25%) with a micropipette under 700 rpm and stirring speed of 1000 rpm. The conversion of the solution from clear to opaque appearance indicated nanoparticles formation. The final suspension was stirred for 2 h to obtain an efficient reticulation and then centrifuged. The NPs (sediment) could be observed from the bottom of centrifuge tube. The supernatant was removed and CSNPs were dispersed in distilled water for further characterization. The size and distribution of the CSNPs were monitored using a dynamic light scattering set-up equipped with a Brookhaven BI-9000 AT digital correlator (Brookhaven Instrum. Corp, N.Y.) at the temperature of 25⁰C. For improving the CSNPs diameter and for avoiding the aggregation, the Tween 80 (10 μl and 20 μl) was added to a sample of 0.25 ml CSLMW gel (0.05%, % and 0.25% w/v) under stirring for 30 min. The particle size distribution was reported as polydispersity index (PDI) from the DLS analyzer, in which PDI <1 represented the good polydispersion (12). Particle size and polydispersity measurements were run in triplicate. Chitosan nanoparticles loaded with xanthine derivative The xanthine derivative (2-{2-[2-(1,3- dimethylxanthine-7-yl)acethyl]hydrazono}- 5-benzylidene-3-(4-brom-phenyl)- thiazolidine-4-one) (13) (2 mg) was dissolved in 5 ml of glacial acetic acid and the solution was mixed with 20 μl Tween 80. Then, 0.25 ml CSLMW gel (0.05% w/v) was added and the mixture was stirred for one hour. Afterwards, 2 ml of TPP solution (0.5% w/v) were added dropwise slowly using syringe dropping method and the suspension was stirred for another 2 h. The conversion of the solution from clear to opaque appearance indicated nanoparticles formation. The particles formed were separated by centrifugation. The supernatant was removed and spectrophotometrically analyzed for loading efficiency evaluation, and xanthine derivative-loaded CSNPs were dispersed in distilled water for further characterization. Particle size and polydispersity measurements were run in triplicate. Loading efficiency (LE) The amount of xanthine derivative in the CSNPs was evaluated using UV spectrophotometric method (UVIKNO XL, BIOTECH Instruments). After centrifugation of the xanthine loaded CSNPs suspension, the supernatant was spectrophotometrically analyzed at 280 nm for evaluating the content of xanthine derivative in the TPP solution. The loading efficiency (%) was calculated using the following formula: LE (%) = C 1 /C 0 x 100 where: C 0 = initial concentration of the xanthine derivative (mg/ml); C 1 = concen- 927

4 Florentina Lupascu et al. tration of the xanthine derivative in the TPP solution (mg/ml) (14). RESULTS AND DISSCUSION Chitosan nanoparticles formulations Chitosan (CS) undergoes nucleophilic interaction with the cross-linking agent (TPP) resulting in CSNPs formation. Chitosan with a pka of 6.3 is polycationic and presents NH 3 + sites when dissolved in acidic medium (acetic acid). When sodium tripolyphosphate (Na 5 P 3 O 10 ) is dissolved in water it dissociates to give both hydroxyl and phosphoric ions. In the present study, the ph of TPP was selected to be 9.0 and at this ph both hydroxyl and phosphoric ions are and may compete with each other to interact with the NH 3 + of chitosan (13). The hydroxyl ions get linked to the amino groups by deprotonation. The chemical structure of chitosan (a), tripolyphosphate (b) and xanthine derivative (c) are presented in figure 1. Fig.1 The chemical structure of chitosan (a), tripolyphosphate (b), xanthine derivative (c) The size of the particles according to different concentration of CSLMW, TPP and stirring speed during ionic gelation are shown in tab. I. By varying these parameters NPs in the range of nm were obtained. The increase in CS concentration was associated with an increase in particle size due to the large number of amino groups that are able to interact as reported in the literature. As can be seen in table I, the average diameter of NPs was about 200 nm when CS concentration was 0.05% w/v while the size increased more than four times when the concentration of CS was 0.25% w/v. At the same time it was noticed that at the higher concentration of TPP the particle size decreased with increasing cross-linking degree of the CSNPs which adopt a more compact and stable structure. The stirring speed during NPs formation also influences their size; thus by increasing the speed from 700 rpm to 1000 rpm there was a decrease in the NP size. NPs have a high surface area and this may lead to very high aggregation after long periods 928

5 Optimized method for obtaining chitosan nanoparticles loaded with xanthine derivative of storage. After 72 h of storage, the aggregation tendency was more pronounced, the NPs size being higher comparative with the initial size. The PDI changed from 79 to for NPs stored less than 48 h, indicating good dispersity for all CSNPs. Conc. of CSLMW solution (%) TABLE I The size of CSNPs obtained by ionic cross-linking method Conc. of Stirring Particle average Particle average Particle average TPP solution (%) (rpm) (nm) (nm) speed size after 48 h size after 72 h size initial (nm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 35 CSNPs-xanthine derivative and loading efficiency Because particle size is the most important characteristic of NPs systems in terms of drug release, for drug loaded CSNPs the following formulation was chosen: CSLMW 0.05%, TPP 0.5% under 1000 rpm stirring speed during ionic gelation. The size of CSNPs loaded with xanthine derivative obtained without Tween 80 is slightly increased (267 nm), but the size decreases in the presence of Tween 80 as shown in Table II. Also seen was a similar LE% after variation in surfactant concentration which means that the entrapment efficiency was not affected. TABLE II The size of CSNPs-xanthine derivative and the loading efficiency Xanthine Initial average Average Tween 80 derivative particle size particle size (μl) (mg) (nm) after 72 h (nm) PDI LE (%) ± ± ± ± ± ± 20 7 ± ± ± ± ± ± 2.4 CONCLUSIONS The aim of this study was to develop and optimize a method for preparation of CSNPs in order to be used as delivery system for xanthine derivative. Some parameters such as concentration of CS, TPP, Tween 80 and the stirring speed during ionic gelation are very important in 929

6 Florentina Lupascu et al. NP formation process. The method was optimized by modifying these independent variables in order to obtain small size NPs, and the optimized method was used for loading the xanthine derivative. The optimized formulation led to xanthine loaded CSNPs with the size in the range nm depending on surfactant concentration, without agglomeration effects and with an adequate percentage of loading efficiency. ACKNOWLEDGEMENTS This paper was published under the frame of European Social Found, Human Resources Development Operational Programme , project no. POSDRU/159/1.5/S/ REFERENCES 1. Elmizadeh H, Khanmohammadi M, Ghasemi K, Hassanzadeh G, Nassiri M, G, Preparation and optimization of chitosan nanoparticles and magnetic chitosan nanoparticles as delivery systems using Box-Behnken statistical design. J Pharm Biomed Anal 2013; 80: Lopez-Leon T, Carvalho ELS, Seijo B, Ortega JL, Bastos-Gonzalez D, Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior. J Colloid Interf Sci 2005; 283: Yang J, Han S, Zheng H, Dong H, Liu J, Preparation and application of micro/nanoparticles based on natural polysaccharides. Carbohydrate polymers 2015; 123: Shen JM et al. Chitosan based luminescent/magnetic hybrid nanogels for insulin delivery, cell imaging, and antidiabetic research of dietary supplements. Int J Pharm 2012; 427: Cornelia V, Pieptu D, Dumitriu RP, Panzariu A, Profire L, Chitosan/Hyaluronic acid polyelectrolyte complex hydrogels in the management of burn wounds. Med Surg J 2013; 117(2): Pasanphan W, Rimdusit P, Choofong S, Piroonpan T, Nilsuwankosit S, Systematic fabrication of chitosan nanoparticle by gamma irradiation. Radiat Phys Chem 2010; 79: Toole MG et al. Curcumin encapsulation in submicrometer spray dried chitosan/tween 20 particles. Biomacromol 2012; 13: Basu SK, Kavitha K, Rupeshkumar M, Evaluation of Ionotropic Cross-Linked Chitosan/Gelatin B Microspheres of Tramadol Hydrochloride. AAPS Pharm. Sci. Tech. 2011: 12(1): Jafarinejad S, Gilani K, Moazeni E, Ghazi-Khansari M, Najafabadi AR., Mohajel N, Development of chitosan-based nanoparticles for pulmonary delivery of itraconazole as dry powder formulation. Powder Technol 2012; 222: Fabregas A et al. Impact of physical parameters on particles size and reaction yield when using the ionic gelation method to obtain cationic polymeric chitosan-tripolyphosphate nanoparticles. Int J Pharm 2013; 446: Kheradmandnia S, Vasheghani-Farahani E, Nosrati M, Atyabi F, Preparation and characterization of ketoprofen-loaded solid lipid nanoparticles made from beeswax and carnauba wax. Nanomed- Nanotechnol 2010; 6: Zhang H, Zhao Y, Preparation, characterization and evaluation of tea polyphenole Zn complex loaded b-chitosan nanoparticles. Food Hydrocolloids, 48 (2015) Lupascu F et al. Synthesis and evaluation of antioxidant activity of some new benzylidene- thiazolidine-xanthine derivatives. Med Surg J 2013; 117: Pawar H, Douroumis D, Boateng JS, Preparation and optimization of PMAA-chitosan-PEG nanoparticles for oral drug delivery. Colloid Surface B 2012; 90: