Preparation and study of tramadol imprinted micro-and nanoparticles by precipitation polymerization: microwave irradiation and conventional heating method.

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IJP 14056 1–8 International Journal of Pharmaceutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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Preparation and study of tramadol imprinted micro-and nanoparticles by precipitation polymerization: Microwave irradiation and conventional heating method

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Mahmoud Seifi a,b , Maryam Hassanpour Moghadam c, Farzin Hadizadeh a , Safa Ali-asgari b , Jafar Aboli b , Seyed Ahmad Mohajeri c, * a b c

Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Department of Chemistry, Shahrood Branch, Islamic Azad University, Shahrood, Iran Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 March 2014 Received in revised form 29 April 2014 Accepted 30 April 2014 Available online xxx

In the present work a series of tramadole imprinted micro- and nanoparticles were prepared and study their recognition properties. Methacrylic acid (MAA), as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker and different solvents (chloroform, toluene and acetonitrile (ACN)) were used for the preparation of molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Several factors such as template/monomer molar ratio, volume of polymerization solvent, total monomers/solvent volume ratio, polymerization condition (heating or microwave irradiation) were also investigated. Particle size of the polymers, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), rebinding, selectivity tests and release study were applied for evaluation of the polymers. The optimized polymers with smaller particle size and superior binding properties were obtained in acetonitrile under heating method. MIPA4 with a size of 42.6 nm and a binding factor (BF) of 6.79 was selected for selectivity and release tests. The polymerization was not successful in acetonitrile and toluene under microwave irradiation. The MIPA4 could selectively adsorb tramadol, compared to imipramine, naltrexone and gabapentin. The data showed that tramadol release from MIPA4 was significantly slower than that of its non-imprinted polymer. Therefore, MIP nanoparticles with high selectivity, binding capacity and ability to control tramadol release could be obtained in precipitation polymerization with optimized condition. ã 2014 Published by Elsevier B.V.

Keywords: Binding affinity Molecularly imprinted nanoparticles Microwave Precipitation polymerization Tramadol

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1. Introduction

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Molecular imprinting technique is one of the most attractive and promising method to create template-shaped cavities in polymeric network with memory of the template molecule to be used in molecular recognition. The active binding site in a molecularly imprinted polymer (MIP) has a unique geometric structure which is particularly selective for a template molecule (Beltran et al., 2007; Haginaka, 2009; Kan et al., 2009; Mohajeri and Ebrahimi, 2008; Mohajeri et al., 2011). Several factors (e.g. template–monomer interactions, the stoichiometry and

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* Corresponding author at: Mashhad University of Medical Sciences, Pharmacodynamics and Toxicology, BuAli sq., Ferdowssi sq. Mashhad, Khorasan Razavi, Iran. Tel.: +98 511 7112611/9125145695/9196773117; fax: +98 511 7112470. E-mail addresses: [email protected], [email protected] (S.A. Mohajeri).

concentration of the template and monomers, the kind and polarity of the porogen and the temperature of polymerization) influence the selectivity performance of MIPs (Batra and Shea, 2003; Mijangos et al., 2006). Many efforts have been done over the years to develop MIPs for different applications (Malaekeh-Nikouei et al., 2012; Mohajeri and Ebrahimi, 2008; Mohajeri et al., 2010, 2012; Pérez-Moral and Mayes, 2004). The MIPs have been evaluated recently as the new drug delivery systems to increase the drug loading capacity and sustain the drug release in aqueous media (Cirillo et al., 2004; Mohajeri et al., 2012). Typically, MIPs were prepared by bulk polymerization as brittle monoliths which are then ground and sieved to create a large surface area and appropriate size of particles (Esfandyari-Manesh et al., 2011; ski et al., 2012). Unfortunately, this procedure is timeLulin consuming, causes loss of materials, the cavities of the MIPs may be destroyed, reduced the efficiency of binding assay due to difficult access to the depth of the polymer matrix (Yoshimatsu et al., 2007) and also the irregular shape of such MIP particles generally give

http://dx.doi.org/10.1016/j.ijpharm.2014.04.071 0378-5173/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Seifi, M., et al., Preparation and study of tramadol imprinted micro-and nanoparticles by precipitation polymerization: Microwave irradiation and conventional heating method, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j. ijpharm.2014.04.071

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M. Seifi et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx

less binding recognition in adsorption procedure (Abouzarzadeh et al., 2012; Esfandyari-Manesh et al., 2011; Poma et al., 2010). To simplify and optimize the synthesis procedure and to enhance the performance of MIP particles, alternative synthetic tactics have been applied to avoid the need for grinding the polymer monolith, sieving and separating the imprinted particles (Abouzarzadeh et al., 2012; Mohajeri et al., 2011). Several methods such as suspension polymerization (Zhang et al., 2009), core–shell emulsion polymerization (Gao et al., 2011) and mini-emulsion polymerization (Curcio et al., 2009) have been reported for preparation of polymeric imprinted micro- and nanoparticles. Although, these methods have clear value, optimization of dependable experimental protocols can be lengthful, the general enforceability is suspicious in some cases, and the residual emulsifier or stabilizer, remain in polymerization media, potentially affect the selectivity of the final imprinted polymer. The precipitation polymerization, as an alternative to above mentioned methods, is a marvelous and simple approach which could be applied as a general method for producing high-quality spherical imprinted particles. Precipitation polymerization is a surfactantfree method that involves polymerization of monomers in an excess of solvent (Wang et al., 2007). This method has been also used for preparation of MIP nanoparticles. Due to the higher surface area-to-volume ratios in nanoparticles, the imprinted cavities are more easily accessible for the templates, and the binding kinetics are improved (Gao et al., 2011; Poma et al., 2010). MIPs are stable under different conditions such as heating, organic solvents and different pH values. Their stability and other beneficial properties, make them attractive for numerous applications such as chemosensor (Guan et al., 2008), surface plasmon resonance (SPR) sensor (Matsui et al., 2005), water treatment (Caro et al., 2005), artificial antibodies (Ramstrom et al., 1996a), separation of enantiomers from a chiral compound (Ramstrom et al., 1996b; Schweitz et al., 1997a, 1998), based stationary phases for capillary electro chromatography (Schweitz et al., 1997a,b), solid-phase extraction (Andersson, 2000) and high performance liquid chromatography (HPLC) stationary phase (Lai et al., 2007). Previous studies indicated that the MIP nanoparticles have a good potential for the controlled delivery of drugs and can increase the time of drug release (Ciardelli et al., 2004; Kryscio and Peppas, 2009; Puoci et al., 2004). Also, MIP nanoparticles can enhanced the capacity of template loading versus the monolith polymers that synthesized by bulk polymerization (Cacho et al., 2004). The present research was focused on the synthesis of tramadol

{(1R, 2R)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol} imprinted micro- and nanoparticles with high loading capacity and selectivity. Tramadol is a centrally acting synthetic analgesic compound that is not derived from natural sources (Sindrup et al., 1999). Tramadol overdose and poisoning has been increased recently especially in young adults with a history of drug abuse and mental disorders (Shadnia et al., 2008). Nausea and vomiting, depression, tachycardia, seizures and prolonged hypoglycemia are the most common symptoms of tramadol poisoning (Marquardt et al., 2005; Mugunthan and Davoren, 2012; Taghaddosinejad et al., 2011). Also, this toxic molecule is a potent water pollutant chemical agent which should be detected and removed from waste water in a water treatment process (Rúa-Gómez and Püttmann, 2012). Therefore, preparation of molecularly imprinted polymeric beads is valuable for application in HPLC column and chemical sensor design for analysis of tramadol in water and biological fluid. Also, the molecularly imprinted polymeric beads could be evaluated as a drug delivery system for sustaining the tramadol release and thereby decreasing its toxicity and adverse effects following oral administration. In this work, a series of tramadol imprinted polymers were prepared using MAA and EGDMA as monomers and ACN, toluene and chloroform as polymerization solvent. The polymerization was initiated and continued under microwave irradiation or conventional heating method and the performance of the final MIP particles was studied in different experiments.

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2. Materials and methods

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2.1. Chemicals

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Methacrylic acid (MAA, purity: 98%) and ethylene glycol dimethacrylate (EGDMA, purity: 98%) were purchased from Sigma–Aldrich (Steinheim, Germany); chloroform, toluene, acetonitrile (ACN), methanol and acetic acid were of high purity or HPLC grade and obtained from Merck (Darmstadt, Germany). 2,20 -Azobisisobutyronitrile (AIBN, purity: 98%) was obtained from ACROS (Geel, Belgium). All other chemicals and reagents were of the highest available purity and used as purchased. Tramadol hydrochloride and gabapentin were provided by Daru Pakhsh Company (Tehran, Iran), imipramine hydrochloride by Sobhan Darou Company (Rasht, Iran) and naltrexone hydrochloride by Iran Daru Company (Tehran, Iran) (Fig. 1). Tramadol base (as the template for imprinting process) was prepared by alkalinization of

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Fig. 1. Structures of chemicals used in this study.


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tramadol hydrochloride solution (100 mg mL1) with sodium hydroxide (1 M). The precipitate was washed several times with deionized water to remove the water-soluble impurities and dried at room temperature in the darkness.

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2.2. Apparatus

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Chromatographic determination of tramadol was carried out on a Younglin (Anyang, South Korea) Acme 9000 HPLC system equipped with a SP930D solvent delivery module, SDV50A solvent mixing vacuum degasser, column oven CTS30, UV730 dualwavelength UV/vis detector and ODSA C18 (4.6 mm 150 mm, 5 mm) column. The injection volume was 20 mL and the column temperature was fixed at 40 C. The data analysis was performed by Autochro 3000 software. Acidity of the solutions was adjusted using a pH meter model 3510 digital, Jenway (Essex, UK) equipped with a combined glass-calomel electrode. FT-IR spectra of nanosized particles were recorded on a PerkinElmer Spectrum-Two (Waltham, USA) using KBr discs in the range of 450–4000 cm1. The size measurement and distribution of the particles were carried out by Zetasizer model Nano-ZS (Malvern Zetasizer ZS, Malvern, UK). An ultracentrifuge, model MPW-350R (Warsaw, Poland) was applied for separation of nanoparticles from solution. Microwave polymerization method was carried out using Milestone Microwave Apparatus (Shelton, USA).

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2.3. Polymer synthesis

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2.3.1. Conventional heating method Molecularly imprinted particles were prepared using precipitation polymerization under the heating conditions (Table 1). Tramadol base as the template and MAA as the functional monomer were dissolved in solvent (in a 50 mL round bottom balloon) and stored at room temperature in the darkness for 12 h to form effective template –monomer complexes before polymerization. Then, EGDMA as the cross-linker and AIBN as the initiator were added to the solution. The mixture was sonicated in a bath sonicator at 60 C for 15 min, sparged with oxygen-free nitrogen for 5 min and sealed while nitrogen gas was blown. After polymerization, the particles were washed with acetone (2 mL 10 mL) and then with methanol-acetic acid (90/10, v/v). The suspension was centrifuged at 15,000 rpm (18227 g) for 25 min to remove the supernatant. Washing step was continued until no tramadol or other compound could be detected by

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HPLC–UV in supernatant (bleeding test). Finally, the particles were washed with HPLC grade methanol to remove residual acetic acid and dried at room temperature for 24 h. A control non-imprinted polymer (NIP) was prepared in the absence of the tramadol, following the same procedure described above.

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2.3.2. Microwave irradiation method Prepolymerization steps were done as mentioned above to prepare monomer compositions (Table 1). The polymerization was carried out in a 250 mL single-necked balloon, equipped with a condenser coil, nitrogen duct and vigorous magnetic stirring. The balloon was placed in the microwave synthesizer. Then the solutions were adequately mixed by vigorous agitation and bubbled with a nitrogen gas stream for 15 min. Microwave radiation was accomplished for 120 min at 70 C. The temperature was reached to 70 C after 3 min. NIP particles were prepared in the absence of the template as described above.

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2.4. Adsorption experiments

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Dry polymer particles (7.5 mg) were incubated, in 1.5 mL aqueous solution, with drug (20 mg mL1) and were shaken for 24 h at room temperature. The mixture was centrifuged (15,000 rpm for 25 min) and the concentration was determined in supernatant by HPLC–UV. The amount of bound drug was calculated from the difference between initial and final concentrations in solution. (Mohajeri et al., 2011) The amount of drug binding was calculated by Eq. (1): (Mohajeri et al., 2010)

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Binding ¼

areastd areaequ concentrationstd V sample areastd

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(1) 187

binding factor (BF) value was calculated according to Eq. (2): (Mohajeri et al., 2011) BF ¼

K MIP K NIP

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(2) 189

where K was the partition coefficient for each polymer and calculated by Eq. (3): (Sahebnasagh et al., 2013) K¼

bounddrug=gpolymer ðfreeÞ

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(3) 191

where, (free) was the free unbound drug in solution after equilibrium whereas, bound drug was the amount of drug bound

Table 1 Compositions and polymerization conditions for preparation of tramadol-imprinted polymers.

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Polymers

Template (mmoL)

MAA (mmol)

EGDMA (mmol)

AIBN (mmol)

Solvent (mL)

Synthesis method

Temperature ( C)

RPM

Time (h)

Z-average (nm)

PDI

MIPC1 MIPC2 MIPC3 MIPC4 NIPc MIPT1 MIPT2 MIPT3 NIPT MIPA1 MIPA2 MIPA3 NIPA MIPA4 NIPA4 MIPm1 MIPm2 MIPm3 MIPm4 NIPm

0.3 0.4 0.6 1.2 – 0.3 0.4 1.2 – 0.367 0.735 1.47 – 0.36 – 0.3 0.4 0.6 1.2 –

2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.94 2.94 2.94 2.94 1.44 1.44 2.4 2.4 2.4 2.4 2.4

12.6 12.6 12.6 12.6 12.6 2.37 2.37 2.37 2.37 2.91 2.91 2.91 2.91 3.556 3.556 12.6 12.6 12.6 12.6 126

40 40 40 40 40 33 33 33 33 40 40 40 40 40 40 40 40 40 40 40

Chloroform (50) Chloroform (50) Chloroform (50) Chloroform (50) Chloroform (50) Toluene (33) Toluene (33) Toluene (33) Toluene (33) Acetonitril (40) Acetonitril (40) Acetonitril (40) Acetonitril (40) Acetonitril (40) Acetonitril (40) Chloroform (100) Chloroform (100) Chloroform (100) Chloroform (100) Chloroform (100)

heating heating heating heating heating heating heating heating heating heating heating heating heating heating heating microwave microwave microwave microwave microwave

50 50 50 50 50 60 60 60 60 60 60 60 60 60 60 70 70 70 70 70

150 150 150 150 150 50 50 50 50 50 50 50 50 50 50 600 600 600 600 600

24 24 24 24 24 24 24 24 24 48 48 48 48 48 48 2 2 2 2 2

>5 mm >5 mm >5 mm >5 mm >5 mm 445.3 1179 1786 1196 197.8 393.8 1444 2021 42.6 1836 4838 5867 2033 5711 4946

>0.5 >0.5 >0.5 >0.5 >0.5 0.188 0.177 0.305 0.29 0.06 0.1 0.11 0.87 0.02 0.168 1 0.536 0.48 0.454 0.296


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Fig. 2. Particle size distribution of MIPA4 measured by photon correlation spectroscopy using a dynamic light scattering (DLS).

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sonicated for 30 min, subsequently dropped onto copper grids coated with amorphous carbon film and dried thoroughly in an electronic drying cabinet at a temperature of 25 C and a relative humidity of 45%.

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2.9. Release study

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Dry MIP and NIP nanoparticles (60 mg) were incubated in 20 mL tramadol hydrochloride (50 mg mL1) at room temperature and shaken for 24 h. The mixture was centrifuged (15,000 rpm for 25 min) and supernatant was filtered and analyzed by HPLC. After drying, the drug-loaded nanoparticles (6 mg 10 mg) were suspended, in 6 different tubes, in 2 mL normal saline and the drug released was determined at different incubation times (5, 10, 20, 30, 45 and 60 min).

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3. Results and discussion

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to per gram dry polymer. Obviously, the BF values calculated for tramadole (as the template) binding to MIPs were reported as the imprinting factor (IF) of MIPs.

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2.5. Analysis of chemicals

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An isocratic method was applied for analysis of compounds. The mobile-phase composition was potassium dihydrogen phosphate (0.01 M, pH 5.89)-methanol acetonitrile (70/20/10, v/v/v), and the flow rate (1.2 mL min1) for tramadol, acetic acid (2%)-acetonitrilewater (2/40/58, v/v/v) and flow rate (1.2 mL min1) for gabapentin and imipramin hydrochloride and ammonium dihydrogen phosphate (0.005 M, pH 2.7)- acetonitrile (60/40, v/v) and flow rate (1.2 mL min1) for naltrexone hydrochloride. The UV–vis detector was set to 218 nm for tramadol hydrochloride whereas; it was set to 275 nm, 240 nm and 211 nm for gabapentin, imipramin hydrochloride and naltrexone hydrochloride, respectively.

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2.6. Preparation of samples for FT-IR spectrum

3.1. Synthesis of tramadol imprinted nanoparticles

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2.7. Size distribution of particles

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The synthesized polymers were analyzed by photon correlation spectroscopy using a dynamic light scattering (DLS). The MIPs and NIPs were re-suspended in deionized water by bath sonication, and characterized by Zetasizer Nano ZS (Malvern Inc., UK) at the 90 detection angle (Jacobsen et al., 2009; Yoshimatsu et al., 2007). To evaluate the size distribution of the polymer, 100 mL of the suspension (5 mg mL1) were added to 1 mL deionized water, in a test tube. The samples were sonicated (30 min) in the bath sonicator at 60 C. Then the particle size average and distribution were reported.

In a study by Javanbakht et al. a tramadol imprinted polymer monolith was prepared using MAA as a functional monomer and EGDMA as a cross-linker by bulk polymerization (Javanbakht et al., 2010). In the present work, tramadol imprinted micro- and nanoparticles were synthesized by precipitation polymerization under microwave irradiation or in a conventional heating method. Different factors such as template concentration, template/functional monomer molar ratio, functional

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Fourier-transform infrared spectra (4000–400 cm1) of washed MIPA4 (without tramadol) and unwashed (containing tramadol) MIPA4 in KBr (20/80, w/w) were recorded by PerkinElmer spectrometer (model Spectrum-Two; PerkinElmer, USA).

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2.8. Transmission electron microscopy (TEM)

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High-resolution electron microscopy, selected-area electron diffraction patterns and energy-dispersive X-ray spectroscopy were performed using high-resolution transmission electron microscopy (HR-TEM; JEOL-2100) equipped with an energydispersive X-ray spectrometer (EDS, INCA) operated at 200 kV with a Gatan Orius SC600 CCD camera. The sample for TEM was prepared as follows: the aqueous suspended of the polymers was

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Fig. 3. TEM of MIPA4 was performed using high-resolution transmission electron microscopy (HR-TEM; JEOL-2100).


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Fig. 5. The amount of tramadol binding (mg) to 7.5 mg of MIP and NIP polymer. Each data represents mean SEM (n = 3).

Fig. 4. FT-IR spectra of the washed and unwashed MIPA4 nanoparticles.

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monomer/cross-linker molar ratio (Yilmaz et al., 1999), polarity of the solvent (Esfandyari-Manesh et al., 2011), temperature, appropriate agitation during the polymerization (Li and Stöver, 1993) and reaction time influence the final characteristics of the obtained MIPs in terms of particle size, capacity, affinity and selectivity for the target analyte (Guan et al., 2008). The polymers were synthesized using chloroform, toluene, and acetonitrile (ACN) as porogenic solvents. Also, different template/MAA/EGDMA molar ratios were applied in polymerization (Table 1). In microwave irradiation method, the monomers were not polymerized in toluene and ACN. Thus, microwave irradiation method was performed in chloroform. The particle size of the MIP and NIP polymers were between 2 mm and 5.8 mm in this method. Total monomers/porogen volume ratio plays an important role in the size of MIP particles. Applying less monomer/volume ratios decreases the particle size of the final MIP (Esfandyari-Manesh et al., 2011; Yoshimatsu et al., 2007; Zhu et al., 2007). The monomers/porogen volume ratio in microwave irradiation method was 0.15 mmol mL1 whereas; the value for MIPC1 to MIPC4 was 0.3 mmol mL1 (Table 1). The particle size of MIPC1–C4 was more than 5 mm. The monomers/porogen volume ratios in MIPT1–T3 (prepared in toluene) and MIPA1–A3 (prepared in ACN) were 0.145 and 0.146, respectively. In spite of their similar monomer/solvent ratios, the size of the MIPs significantly decreased in MIPA1–A3 series. This finding indicated the effect of solvent in particle size during polymerization. According to the previous works the polymers synthesized in ACN usually have less particle size compared to the polymers prepared in other solvents (Abouzarzadeh et al., 2012; Esfandyari-Manesh et al., 2011; Yoshimatsu et al., 2007; Zhu et al., 2007). Therefore, we focused on ACN to achieve imprinted nanoparticles. Functional molar ratio is another important factor in preparation of MIP nanoparticles. Thus, changing the functional monomer/crosslinker monomer ratio from 1.01 (in MIPA1–A3) to 0.4 (in MIPA4) decreased the particle size from 197 nm (MIPA1) to 42.6 nm (MIPA4). Therefore, applying the suitable solvent (ACN), monomer/solvent ratio of 0.125 and monomer/cross-linker ratio of 0.4, we reached to the particle size of 42.6 nm with a polydispersity index (PDI) of 0.02 in MIPA4 (Figs. 2 and 3). According to the

results using different concentrations of template affect the size of the final polymer particles. In conventional heating method, the tramadole/total monomers molar ratios were between 0.02 and 0.25. The data showed that increasing the tramadole/total monomers molar ratios (in each MIP series) increased the size of MIP polymer particles. Also, a significant difference was found between particle size of MIPs and their NIP. Previous study indicated that the particle size differences between MIPs and NIPs were may be due to molecular interaction between MAA and template. In the absence of template, functional monomer (MAA) can form hydrogen-bonded dimmers in the non-imprinting system, and the pre-polymerization solution contains both functional monomer dimmers and free functional monomers. In the imprinting system, there are additional molecular interactions between functional monomer and template, which might somehow affect the growth of cross-linked polymer nuclei (Abouzarzadeh et al., 2012). As a fact, the monomer concentration applied in polymerization is the same in both NIP and MIP systems. But, due to the presence of more functional monomer dimmers in pre-polymerization solution in the non-imprinting system, the number of polymerization nuclei is usually less than that in imprinting system whereas; the growing size of the final cross-linked non-imprinted nuclei is significantly higher than that of MIP particles. The data showed that the polymer particles in optimized MIP are usually smaller than that in their NIP particles (Table 1).

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3.2. FT-IR characterization

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Due to the similar backbone structure and monomer composition of the polymers, the FT-IR spectra of MIP and NIP polymers are usually the same (Azodi-Deilamia et al., 2010). Also, washed and unwashed MIPA4 polymers had similar characteristic peaks (Fig. 4). Due to presence of hydrogen bond between the functional groups in tramadol molecule and COOH group in MAA, the bending vibrations at 1385, 1725 and 3443 cm1 in the unwashed MIPA4 were shifted to 1390, 1730 and 3567 cm1 in the corresponding washed MIPA4. According to this result washing procedure was

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Table 2 Binding factors of MIPs. polymers

MIPC1

MIPC2

MIPC3

MIPC4

MIPT1

MIPT2

MIPT3

MIPA1

MIPA2

MIPA3

MIPA4

MIPm1

MIPm2

MIPm3

MIPm4

BF

1.08

1.11

1.01

0.76

1.14

1.08

0.98

1.49

3.68

1.49

6.79

1.52

1.49

1.52

1.35


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Fig. 6. Drug binding (mmol) to 7.5 mg polymer in a 20 mg/mL aqueous solution after 24 h incubation in room temperature. Each data represents mean SEM (n = 3).

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performed completely and no bleeding would be seen during rebinding tests.

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3.3. Adsorption experiments

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Tramadole binding to MIPs was usually more than that to their related NIPs. It shows that the imprinting was successfully happened in all MIPs (except MIPC4) (Fig. 5). According to the results, the maximum BF values (3.68 and 6.79) were obtained from the MIPs prepared in ACN (MIPA2 and MIPA4) (Table 2). It can be concluded that MAA-tramadole interactions are more effective in ACN compared to other solvents. Therefore, the imprinted cavities and MAA monomers in the binding sites work more specific in MIPA series in comparison to other MIPs. Also the MIPA4 had the maximum tramadole binding and BF between MIPs. This data revealed that decreasing the size of the imprinted particle, significantly increases the binding capacity and specificity of the MIP particles. Other researchers have shown that the specific binding sites and imprinted cavities are usually more accessible for the template in micro and nano MIPs (Abouzarzadeh et al., 2012; Esfandyari-Manesh et al., 2012; Guan et al., 2008).

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3.4. Selectivity of MIPA4

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In the next step the selectivity of the MIPA4, as the optimized nano-sized MIP, was evaluated using different water soluble drugs such as imipramine hydrochloride, naltrexone hydrochloride and gabapentin, compared to tramadol hydrochloride. Thus, the drug binding (mmol) to 7.5 mg MIPA4 and NIPA4 was determined after 24 h incubation in an aqueous solution (20 mg mL1). The data showed that MIPA4 had significantly higher affinity for binding tramadol than NIPA4 (Fig. 6). However, the average amount of drug binding to imprinted polymer was more than that of NIP, the difference between MIPA4 and NIPA4 was not statistically significant for other drugs (P-value > 0.05). The drug binding to the NIP can be explained by nonspecific binding and random action and reaction of the template molecules with free functional groups in the polymer matrix (Esfandyari-Manesh et al., 2011); whereas the template binding to MIP could be explained by specific binding (in imprinted cavities) and nonspecific binding in the other sites of polymer. Imipramine hydrochloride, naltrexone hydrochloride and gabapentin have different chemical structures, compared to tramadol. Therefore, the tramadol imprinted cavities had less affinity to adsorb these drugs. The results indicated the selectivity

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Fig. 7. Tramadol released from 10 mg polymer in normal saline. Each data represents mean SEM (n = 3).

of MIPA4 for tramadol binding. In our previous work, the results had also demonstrated the selectivity of clozapine submicron MIP compared to its NIP in a rebinding test (Mohajeri et al., 2011).

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3.5. Drug release studies

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In the next step tramadol release from MIPA4 and NIPA4 was evaluated in aqueous media. The aim of the release test was to evaluate the nano-sized imprinted and non-imprinted polymers in controlling the drug release in normal saline (Fig. 7). Fig. 7 indicated that 100% of drug was released after 5 min from NIPA4 matrix; whereas 100% drug release was occurred after 45 min from MIPA4. This finding obviously demonstrated the ability of MIPA4 and its imprinted binding sites in sustaining and controlling tramadol release in aqueous media. Our previous works have indicated higher affinity of MIPs to their template or their similar analogues and their ability in slowing the release in water, compared to the NIPs (Malaekeh-Nikouei et al., 2012, 2013; Mohajeri et al., 2012; Tabassi et al., 2013).

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4. Conclusion

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Molecularly imprinted nanoparticles for tramadol as template have been successfully synthesized by precipitation polymerization method using MAA as a functional monomer and EGDMA as a cross-linker in acetonitrile under heating method. Different factors such as template and monomers concentrations, template/ monomer and monomer/cross-linker molar ratios, porogen type, polymerization condition and time were considered in preparation and optimization of MIP particles. The optimized uniform MIP nanoparticles (MIPA4) with tramadol/MAA/EGDMA molar ratio of 1/4/10 showed the highest binding factor (6.97) and the smallest particle size (42.6 nm) according to DLS and TEM. The binding properties, selectivity, and ability to control the drug release were evaluated for MIPA4. The selectivity test showed that MIPA4 nanoparticles had higher binding affinity to tramadol, in comparison with naltrexone, imipramin and gabapentin which may be prescribed with tramadol and detected simultaneously in different tissues. MIPA4 nanoparticles could also sustain the tramadol release in normal saline media, compared to non-imprinted particles. This study demonstrated that different factors influence the size and binding properties of the imprinted micro- and nanoparticles. In the present work, the MIPA4 nanoparticles with

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the smallest size were selected as the optimized MIP with superior binding and release properties and selectivity for tramadol in aqueous media.

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Kaszuba et al. (2008).

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Acknowledgment

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We gratefully acknowledge the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support through the grant number 910259. Authors declare that there is no conflict of interests in this study. The results described in this paper were part of a MSC student (Mahmoud Seifi) thesis.

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