Molecularly imprinted polymer nanoparticles for olanzapine recognition: application for solid phase extraction and sustained release

Abstract

Recently, scientists have drawn more attention to polymeric nanoparticles as potential drug carriers. In this study, we synthesized high selective imprinted polymer nanoparticles using olanzapine as the template. The aim of this study was to prepare efficient imprinted polymer nanoparticles from an olanzapine template for the controlled release of olanzapine as a therapeutic drug for central nervous system (CNS) diseases at different pH values, and solid-phase extraction (SPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The morphology of the nanoparticles was determined using scanning electron microscopy (SEM) images. Drug release, binding properties and dynamic light scattering (DLS) of the molecularly imprinted polymers (MIPs) were studied in this investigation. The adsorption isotherm was fitted with Langmuir and Freundlich models. The performance of the MIPs for the controlled release of olanzapine was assessed in two different media (SDS 1% and PBS). Results revealed that the MIPs have potential application in controlled drug release. Moreover, cytotoxicity of the MIP nanoparticles was measured on a NIH/3T3 cell line using a MTT method. Furthermore, the MIPs were applied to extract olanzapine from human blood plasma samples. The limit of detection (LOD) and limit of quantification (LOQ) were evaluated and were 0.18 μg L⁻¹ and 0.39 μg L⁻¹, respectively. These results collectively illustrate that MIP nanoparticles can be employed as an efficient technique for the extraction of the olanzapine from human plasma.

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1. Introduction

Over the past few decades, pharmaceutical and medicinal scientists have focused on designing and developing effective techniques to establish promising novel drug delivery systems. The aims of such efforts were to find a novel system capable of controlling the rate of drug release, the period of drug delivery, and eventually delivering the drug to its target site.¹ Besides, a controlled drug delivery system improves the bioavailability of the medicine via the prevention of premature degradation of the drug. Moreover, also enhancing uptake whilst maintaining drug concentration within the therapeutic window through controlling the drug release rate can desirably reduce side effects upon systemic therapy. Of further relevance, targeting drug delivery can also improve treatment procedures by minimizing side effects owing to lower doses of the corresponding medicine.²

Nano-carriers are colloidal systems ranging from 1 to 300 nm which are able to carry a given therapeutic agent. They can be fabricated from a variety of different materials, including polymers (micelles, dendrimers and polymeric nanoparticles), lipids (liposomes), viruses (viral particles) and organometallic compounds (nanotubes).³ Compared with other compositions, polymeric materials, especially polymer nanoparticles, have the best features and capabilities. Polymeric structures with desirable physical and chemical properties can provide appropriate drug delivery systems and allow high loading of medicines.⁴ Moreover, they provide control over drug release kinetics which can be readily modified by functionalization to display a variety of surface-attached ligands for various purposes. Additionally, it should be stated that many polymers have a long history of safe use in humans.⁵

Recently, one of these fascinating techniques, namely the imprinted polymer, has drawn more attention by scientists for medical and analytical approaches.^6–8 Molecular imprinting is known to be an efficient technique for tailor-made binding sites in polymer networks with a memory of the shape, size and functional group of the template molecules.^9,10 Molecularly imprinted polymers (MIPs) have great potential in a number of applications in the life sciences which highlights them as desirable drug carriers.¹¹ Promising applications of MIPs include tailor-made separation materials,¹² molecular recognition materials for biosensors,¹³ catalysis material,¹⁴ antibody mimics, molecular diagnostic tools,^15,16 and drug delivery systems,¹⁷ such as MIP membranes,¹⁸ MIP gels,¹⁹ and MIP micro and nanoparticles.²⁰ Among all these applications, most of the attention has been focused on MIP particles as a drug delivery system and MIP recognition materials for analytical purposes due to their recognition, selective rebinding ability, flexibility and multi-usage in practical applications.²¹ However, these two applications probably have more challenges compared to other MIP applications.²² In this regard, inherent limitation in size, diameter and morphology of the imprinted polymers associated with conventional methods of molecular imprinting synthesis simply means that further investigations need to be performed.²³ Although traditional molecularly imprinted polymers are synthesized by bulky polymerization due to its simplicity, post-treatment like grinding and sieving is required. Irregularly shaped milled particles have a broad particle size distribution. On the other hand, sieved particles cause a useful yield decrease of almost 50% less than the initial decline.²⁴ Additionally, irregularly shaped particles may cause some limitation in the removal and rebinding of templates.^25,26 This is especially important for imprinting macromolecules, such as protein and oligosaccharides.^17,27

Several approaches have been used to obtain molecularly imprinted particles with a controlled size and shape distribution. As early as 1973, suspension polymerization was used as a technique to produce smaller droplet sizes by Ugelstad et al.²⁸ who scaled down the droplet size to several hundred nanometers by shearing the suspension.²⁹ In order to obtain a stable emulsion with a homogeneous size, a co-stabilizer was added to the dispersed phase to suppress the diffusion processes in the continuous phase, so nanodroplets were stabilized by a hydrophobic agent against Ostwald ripening during the miniemulsion polymerization. Miniemulsion polymerization is particularly suited for the one-step preparation of molecularly imprinted nanoparticles.^30–32 Considering two major advantages of the miniemulsion, a polymerization method that provides the possibility of synthesizing the nanoparticles during a single-stage process without using organic solvent, we employed such a technique as the primary imprinting polymerization system for preparing surface imprinted polymeric beads. Miniemulsion polymerization can routinely produce mono-dispersed particles of a size between 50–500 nm. We also hypothesized that the imprinted nanoparticles would provide an extended surface area for template molecular uptake. Given the excellent heat transfer property, miniemulsion polymerization is extremely suitable for industrial applications.

Another critical factor for choosing the proper carrier for a drug delivery system (DDS) is the controlled release of the drug at the target site in the right dose and for the required time. A controlled drug delivery system offers the possibility to maximize the drug efficacy and safety which provides a reasonable rate of dose delivered in its target site in the body, resulting in a prolonged administration frequency along with increasing patient compliance.³³

Nowadays, the central nervous system (CNS) poses a unique challenge for drug delivery. The blood-brain barrier (BBB) significantly hinders the passage of systemically delivered therapeutics and the brain’s extracellular matrix limits the distribution and longevity of the locally delivered agent.⁵ Polymeric nanoparticles as drug carriers are considered as a promising solution for this problem, and has resulted in a vast array of scientists working on the design and synthesis of polymeric nano-carriers for drug delivery, particularly molecularly imprinted nanopolymers for CNS drug delivery.

Olanzapine (OLZ), 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine, is an approved atypical antipsychotic drug for treatment of schizophrenia and bipolar disorders. OLZ belongs to the class of thienobenzodiazepine compounds and is available as a tablet, an orally-disintegrating “wafer”, which rapidly dissolves in saliva (Zyprexa). It is also available in 10 milligram vials for intramuscular injection. These dosage forms exhibit low bioavailability due to an extensive first pass metabolism and non-targeted delivery results in numerous side effects.^34,35 Since the target site of the olanzapine is in the brain, designing a desirable strategy with the goal of improving the bioavailability of this medication through preventing its first pass metabolism seems reasonable. Moreover providing an approach in terms of bypassing the BBB and targeting the receptor site to achieve the desired OLZ concentration at its site of action appears beneficial.

For a study of the extraction in the solid phase by MIPs, it should be stated that a few types of extraction methods are routinely used in analytical toxicology, such as liquid-liquid extraction (LLE),³⁶ liquid-liquid micro-extraction (LLME),^37,38 solid phase extraction (SPE),³⁹ solid-phase micro-extraction (SPME),⁴⁰ and molecularly imprinted solid phase extraction (MISPE).^41–43 Although SPE is the most convenient method for extraction due to its simplicity in terms of labor intensity and solvent consumption, MISPE is currently a growing field in clean up techniques for the analysis of biological samples. SPE is a very common type of sample extraction and clean up technique for biological fluids in analytical chemistry.^44–48

Herein, we focused on the preparation of non-covalent molecularly imprinted particles consisting of ethylene glycol dimethacrylate (EGDMA) as a crosslinker and methacrylic acid (MAA) as functional monomer using the miniemulsion polymerization process. Sodium dodecyl sulfate (SDS) was used as the surfactant and hexadecane was employed as the hydrophobic agent to prevent Ostwald ripening of the miniemulsion. To characterize the MIP nanoparticles synthesized via miniemulsion polymerization, scanning electron microscopy (SEM), thermal gravimetric analysis and dynamic light scattering were employed. The efficiency of the molecular imprinting effect was examined by binding experiments and quantified by UV absorption.

2. Results and discussion

2.1. Miniemulsion polymerization

Emulsions are defined as micro heterogeneous systems, comprising at least one immiscible liquid dispersed in another in the form of droplets (oil-in-water or water-in-oil dispersion), with a diameter ranging between 50 and 5000 nm. Emulsions are divided into three types: macroemulsions, miniemulsions and microemulsions. The major difference between these three classes is based on the droplet size (dispersed phase) in these systems, and their relative stability. These systems feature a minimum stability which can be enhanced by adding suitable substances, like surfactants or finely ground solids.

In order to discuss miniemulsion polymerization, it will be necessary to review the mechanism of macroemulsion polymerization. Macroemulsion polymerization is a complex process. Nucleation mechanisms are generally divided into three types: micellar, homogeneous, and droplet. Theoretically, all three types of mechanisms can occur simultaneously in every reaction. However, it is the superiority of one mechanism that causes authors to consider only one in their studies. In macroemulsion polymerization, micellar and homogenous nucleation are dominant. This is because the large size of the monomer droplets, and their consequently low interfacial area, make them ineffective in competing for water-borne free radicals.

Miniemulsions are produced by the combination of a high shear to break up the emulsion into submicron monomer droplets, and a surfactant/co-stabilizer system to retard monomer diffusion from the submicron monomer droplets. It is evident that destabilization of emulsions or miniemulsions can occur by collision of the droplets resulting in coalescence (a bimolecular process). The handling of this problem is a standard question in colloid science and is usually solved by addition of appropriate surfactants which provide the necessary colloidal stability to the droplets against coalescence by collision, controlled by the type and amount of the employed surfactant. The monomer droplets change quite rapidly in term of the size throughout sonication. High shear is provided by a sonicator or a mechanical homogenizer. With increasing the time of the ultrasonication, the droplet size decreases and therefore the entire oil/water interface increases. At the beginning of homogenization, the polydispersity of the droplets is quite high, but by constant fission and fusion processes, the polydispersity decreases and the miniemulsion reaches a steady state, where a dynamic equilibrium of droplet fission and fusion rates will be achieved. This also means that miniemulsions come to the minimal particle sizes under the applied conditions, while they make use of the surfactant in the most effective way possible. The surfactant is necessary to retard droplet coalescence caused by Brownian motion, settling or Stokes law creaming or settling. The co-stabilizer (also referred to in earlier works as a co-surfactant) prevents Ostwald ripening. When a liquid emulsion is subjected to a high shear, small droplets will result.

2.2. Thermal analysis

Differential scanning calorimetry (DSC) is one of the thermal decomposition analyses which has many applications in chemistry such as detecting changes in enthalpy and the specific heat capacity. To investigate the thermal properties of the nanoparticles, DSC analysis was conducted. The DSC thermograms of the pure OLZ, non-imprinted nanoparticles, and leached and non-leached imprinted nanoparticles are shown in Fig. 1. Pure OLZ exhibited an endothermic peak for the melting temperature at about 195 °C. A recent study on DSC results for all imprinted and non-imprinted nanoparticles showed that these particles have a thermal stability up to approximately 300 °C, due to a high level of crosslinking of the synthesized polymers that makes it promising for use in different applications.⁴⁹ All polymers showed similar thermal behavior, but in non-leached imprinted polymers, a transition was observed around 195 °C, for the melting the OLZ, due to the non-leached nanoparticles.

Fig. 1 DSC thermograms for OLZ (a), non-leached MIP₂ (b), leached MIP₂ (c) and NMIP (d).

2.3. Study of morphology and porosity of nanoparticles

The morphology of the MIP and NMIP (non-molecularly imprinted polymer) particles was determined from scanning electron microscopy (SEM) images, as depicted in Fig. 2. The morphology of the polymer particles is spherical and uniform. As shown in Fig. 2, both MIP and NMIP particles were nano sized, 66.4 nm and 89.8 nm, respectively. Unlike the MIP particles, NMIP particles were larger in size which might be due to the patternless polymerization for NMIP in which the polymerization process occurs without a template. Particle size distribution was measured with dynamic light scattering (DLS) analysis. Fig. 3 illustrates a narrow particle size distribution for the nanoparticles. The polydispersity index (PdI) and z-average for the MIPs were 0.163 and 145, and for the NMIPs they were 0.215 and 189, respectively. The difference between the SEM and DLS particle diameters was due to polymer swelling which occurred in the DLS test conditions.

Fig. 2 Scanning electron micrographs, MIP₂ (a), NMIP (b).

Fig. 3 Particle size distribution of MIP₂ (a) and NMIP (b) measured by dynamic light scattering.

2.4. Characterization

2.4.1. FT-IR. On the basis of theoretical calculations, undoubtedly, MAA monomers strongly interact with the template which was identified and used for synthesis of the MIP nanoparticles. One surprising observation was that FT-IR spectra of the NMIP and leached and non-leached MIP particles displayed a similar characteristic peak, indicating a similarity in the backbone structure of the different polymers. The IR spectra of the non-leached imprinted polymer (MAA co-EGDMA) are shown in Fig. 4. The hydrogen binding with the –COOH group of MAA, the C=O stretching, the OH stretching, and the bending vibrations at 1716, 3426.95, and 1391.9 cm⁻¹ in the non-leached MIP₂ particles were shifted to 1736.56, 3544.15, and 1390.5 cm⁻¹ in the corresponding leached MIP₂, respectively. Other absorption peaks match with those of the MIP₂ particles, as well as the NMIP particles: 1258, 1154 cm⁻¹ (symmetric and asymmetric ester C–O stretch bonds), 1636 cm⁻¹ (stretching vibration of residual vinylic C=C bonds), 2955.2, 2961.5 cm⁻¹ (CH₂ asymmetric stretching vibration) and 954 cm⁻¹ (out-of-plane bending vibration of vinylic C–H bond).

Fig. 4 Infrared spectra of the leached (a) and non-leached (b) MIP particles.

2.4.2. Thermo-gravimetric analysis (TGA). TGA was performed at the maximum heating rate of 20 °C min⁻¹ in an oxygen atmosphere. As plotted in Fig. 5, TGA revealed two decomposition states regarding the non-leached MIP particles as follows; one mass loss between 110 to 180 °C (up to 5% weight loss), ascribed to the free monomer and cross-linker decomposition and solution evaporation. Another initiated at 195 °C, related to the OLZ decomposition, since the melting point of OLZ is 195 °C. All the materials were completely decomposed around 460 °C. These observations indicate that the particle arrangement in non-leached and leached MIPs is more comparable with the NMIP particles, as the former exhibited decomposition above 300 °C, and; the latter starts to decompose at ∼260 °C onwards. Besides, the non-leached and leached MIP particles have similar degradation patterns above 400 °C. The complete decomposition of the polymeric matrix occurs for both at temperatures above 450 °C.

Fig. 5 TGA plots for leached MIP, non-leached MIP and NMIP.

2.5. Effect of pH on drug loading

The effect of pH on the sorption of the OLZ was examined by varying the pH of OLZ solutions from 3 to 12. Several batch experiments were performed by equilibrating 50 mg of the imprinted particles with 5 mL of the solution containing 0.05 mM of OLZ under the desired levels of pH. The results for different polymers demonstrated that pH has a great effect on drug loading (Fig. 6). The percentage of OLZ retention increased up to a pH of 6.8 and then decreased with a further increase of pH. A difference of about 90% between MIP and NMIP was observed at the pH of 6.8. A similar effect was observed to a lesser extent at lower and higher pH values which may be attributed to the protonation of the functional group of OLZ and to the deprotonation of the carboxyl groups of the polymer, respectively.⁵⁰

Fig. 6 Effect of pH on the rebinding efficiency of olanzapine. 50 mg of the imprinted polymers; sample volume: 5 mL; olanzapine concentration: 0.05 mM; room temperature.

2.6. Adsorption capacity of polymers

Another important factor studied in this investigation was the adsorption capacity of a sorbent to quantitatively remove a specific amount of template from the solution. The carboxylic acid in the cavities of the MIP has a very high hydrophilic affinity with respect to OLZ. In addition, the amines of the OLZ undergo hydrogen bonding with the carboxylic acid group of the MIP particles. In summary, the hydrogen bonding and the hydrophilic forces are the major interactions between OLZ and MIP particles. Once the system has come to equilibrium, the amount of free template in the solution was measured to determine the amount of the adsorbed template. The remaining OLZ in the supernatant was measured using HPLC-UV. The adsorption isotherm (Q) of the OLZ for MIP and NMIP particles is depicted in Fig. 7A. According to these results, the maximum amount of OLZ that can be adsorbed by MIP is 95 mg g⁻¹at a pH of 6.8. As all the accessible specific cavities of the MIP particles are saturated, the retention of the analyte is mainly due to non-specific interactions, which can be identical for MIP and NMIP particles. The results show that the imprinted particles possess a recognition ability for OLZ which can be attributed to the complementary cavities created by OLZ templates. Results of equilibrium studies were analyzed using the Langmuir and Freundlich isotherms to find the best fitted model for describing the adsorption process. The original forms of the Langmuir and Freundlich isotherms, are given by eqn (1) and (2), respectively;

q_e = K_FC_e^1/n (2)

where q_m is the maximum amount of adsorption (mg g⁻¹), K_L is the affinity constant (L mg⁻¹), K_F is a constant, which is a measure of adsorption capacity and n is a measure of adsorption intensity. Corresponding linear forms of the Langmuir and Freundlich isotherms can be expressed as eqn (3) and (4), respectively:

log q_e = 1/n log C_e + log K_F. (4)

Fig. 7 (A) Adsorption isotherms of olanzapine onto MIP and NIP nanoparticles. (B) The adsorption isotherms fitted with the Langmuir model. (C) The adsorption isotherms fitted with the Freundlich model.

As plotted in Fig. 7B and C, the two isotherms can be fitted by the Langmuir and Freundlich models. It was observed that the equilibrium data were best matched by the Langmuir isotherm with good correlation coefficient values; the detailed values are reported in Table 1.

Table 1 Isotherm parameters for OLZ adsorption by MIP and NIP nanoparticles

	Langmuir			Freundlich
	qm (mg g^−1)	KL (L mg^−1)	R^2	KF (L mg^−1)	1/n	R^2
MIPs	114.9425	0.0104	0.9977	9.1960	0.3906	0.9425
NIPs	34.1296	0.0033	0.9838	0.4815	0.6195	0.9881

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2.7. In vitro sustained release studies

The ability of the MIP nanoparticles to deliver OLZ, especially via sustained release, was evaluated using drug release rate analysis and compared with drug release from NMIP particles in two different media, sodium dodecyl sulfate (SDS 1% pH 6.8)⁵¹ and phosphate buffer saline (PBS pH 7.4), to simulate the intestinal environment and physiological environment, respectively. The result of the release profile of OLZ from the nanoparticles is shown in Fig. 8. The initial quick release of OLZ might be due to weakly adsorbed OLZ molecules at the surface of the nanoparticles. It should be noted that NMIP nanoparticles released a considerable amount of OLZ within 45 hours (about 95% of OLZ in SDS and 84.5% in PBS) while MIP released the same amount of drug within 136–140 hours in SDS and PBS. Subsequently, the release of OLZ from MIP nanoparticles was slower and more delayed (up to 300 hours release of 100% drug in SDS and 340 hours in PBS). There was a comparable difference in the release kinetics between the different nanoparticles under these conditions. The release profiles of the imprinted nanoparticles were slower than that of the non-imprinted nanoparticles. This notable difference in the release of the imprinted nanoparticles and NMIP particles can be attributed to the existence of a specific site in the imprinted nanoparticles that had a strong interaction with the OLZ molecules.

Fig. 8 In vitro drug release profile of 60 mg OLZ imprinted nanoparticles in SDS as an intestine simulated environment (pH = 6.8) and PBS as a physiological simulated environment (pH = 7.4) at 37 °C (mean ± S.D. n = 3).

2.8. In vitro cytotoxicity

The relative cytotoxicity of the MIP nanoparticles based on poly (methacrylic acid) (PMAA) was measured on a NIH/3T3 cell-line using a MTT viability assay. The MTT method is based on the ability of a mitochondrial dehydrogenation enzyme in viable cells to cleave the tetrazolium ring of the pale-yellow MTT and form formazan crystals with a dark-blue color. The dependent relationship between the number of surviving cells and the absorbance intensity of the formed formazan determines the cytotoxicity of MIP nanoparticles based on PMAA in aqueous solution. As demonstrated in Fig. 9, MIP nanoparticles (50 mg mL⁻¹) barely exerted any cytotoxic effect on NIH/3T3 cells. The MTT results indicated that cell viability percentage did not significantly alter upon MIP particle exposure during a 7-day incubation period (less than 13%), which is in accordance with the live and dead assay images.

Fig. 9 MTT assay: effect of MIP nanoparticles on the viability of 3T3 cells. 3T3/NIH cells were incubated with MIP nanoparticles (1, 3, 5 and 7 days), MTT reduction was determined.

2.9. Real sample analysis

To demonstrate the potential of MIP nanoparticles for the selective sample clean up, the MIP nanoparticles were employed in the purification of spiked OLZ in human plasma. Common solid phase extraction materials showed a lack of selectivity, except the immunosorbents (ISs) which are highly selective, although their development is too expensive and time consuming. Hence, such materials are not suitable for most real sample analysis.⁵² These drawbacks and the necessity of a rapid and selective clean-up method have collectively urged the development of a SPE procedure involving moleculary imprinted polymers, MISPE.^53,54 Accordingly, the simple preparation and high porosity of MIP nanoparticles can provide fast, simple, and selective extraction with higher column efficiencies. Compared with the ISs, MIPs have a lower cost, a higher load capacity, and resistance to elevated temperature and pressure.⁵⁵ Blood samples were drawn from the Iranian blood transfusion service (Tehran, Iran) and five schizophrenic patients of the Psychiatric Clinic of Tehran Medical University subjected to treatment with olanzapine tablets at a constant daily dose for at least 4 weeks. Aqueous media were applied for the loading of the solution, and the wash procedure was carried out using acetone in order to obtain the maximum recovery of the analytes. The chromatograms obtained for the plasma samples are illustrated in Fig. 10. The amount of OLZ bound by the MIP and NMIP particles was 92 ± 1.2% and 13 ± 1.4%, respectively. Results revealed the existence of the specific sites in MIP particles and their good performance in the binding of OLZ. This efficient method allowed us to obtain cleaner extracts and suppress interfering peaks arising from complex biological matrices. Results from the HPLC analysis are reported in Table 2, which indicate that the MIP extraction of olanzapine for the plasma sample had good precision (4.5% for 50.0 μg L⁻¹) and recovery (between 83–94). Typical chromatograms (represented in Fig. 10) revealed that the MIP particles can be used for sample clean up. A broad peak in the chromatogram was omitted when the MIP particle sorbent was used. As shown in Table 3, the limit of detection (LOD) and limit of quantification (LOQ) for olanzapine in plasma samples were 0.18 and 0.39 μg L⁻¹, respectively. The number of repeats for the experiment was three.

Fig. 10 HPLC chromatograms obtained after percolation of 2 mL human serum spiked with 50.0 μg L⁻¹ of OLZ with a cleanup step comprising the (A) NMIP particles and (B) MIP particles monitored at 251 nm; conditions: column ACE 5 μm, C18 4.6 mm × 250 mm at +40 °C, eluent 50 mM phosphate buffer salt (pH 7.4) : acetonitrile : methanol 67 : 22 : 11 (v/v) at flow rate of 1.0 mL min⁻¹.

Table 2 Assay of olanzapine in human plasma by a SPE-HPLC procedure

Sample	Spiked value (μg L^−1)	Recovery% ± SD^a
		MIP	NIP
a Average of three determinations.
Human serum	5	83 ± 1.1	10 ± 1.2
	10	92 ± 1.2	13 ± 1.4
	25	86 ± 1.5	12 ± 1.0
	50	94 ± 1.6	10 ± 1.7

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Table 3 Relative comparison of the developed method with existing methods for the determination of olanzapine

Method	LOD	LOQ	Samples	Reference
HPLC	3 × 10^3 μg L^−1	8 × 10^3 μg L^−1	Pharmaceuticals	56
LC-MS-MS	—	0.25 μg L^−1	Human urine	57
Stability-indicating HPLC	34.5 μg L^−1	1.1 × 10^2 μg L^−1	Bulk and formulations	58
HPLC-ED	1 μg L^−1	3 μg L^−1	Human plasma	59
Stability-indicating UPLC	0.14 μg L^−1	0.44 μg L^−1	API and pharmaceutical dosage forms	60
Spectrophotometric determination	6.6 × 10^3 μg L^−1	20 × 10^3 μg L^−1	Pharmaceuticals	61
GC-EI-MS	0.5 μg L^−1	0.5 μg L^−1	Human plasma	62
Kinetic method	4 × 10^3 μg L^−1	0.1 × 10^3 μg L^−1	Pharmaceuticals, serum	63
Flow-injection chemiluminescence	0.1 μg L^−1	—	Pharmaceuticals, serum, urine	64
Proposed method	0.18 μg L^−1	0.39 μg L^−1	Human plasma	This work

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3. Experimental

3.1. Materials

Methacrylic acid (MAA) was obtained from Merck (Darmstadt, Germany), which was distilled under vacuum prior to usage in order to remove the stabilizers. Ethylene glycol dimethacrylate (EGDMA) and 2,2-azobis isobutyronitrile (AIBN) were purchased from Sigma-Aldrich (Steinheim, Germany). AIBN was recrystallized from methanol before usage. Sodium dodecyl sulfate (SDS) and hexadecane (Merck, Hohenbrunn, Germany) were used without further purification. Olanzapine (OLZ) was obtained from Temad Co. (Iran). Dialysis tubes (Sigma dialyses tube Mw cutoff 12 kDa) were heated in an aqueous solution of 2%wt sodium hydrogen carbonate and 0.05%wt ethylene diamine tetra acetic acid (EDTA) and then kept under refrigeration in an aqueous solution of 0.5%wt sodium azide until use. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was obtained from Sigma. Dimethyl sulfoxide (DMSO) was used after purification by distillation under vacuum and dried with calcium hydride (CaH₂). NIH/3T3 cell lines were obtained from the National Cell Bank of Iran. Other reagents and solvents were of analytical grade.

3.2. Instruments

The instruments used for the general procedures were an ultrasonicator (SONO SWISS SW 12H), high-speed homogenizer (IKA, T25, Ultra-Turrax, USA), centrifuge (Sigma 3K30, Germany), oven (Memmert UNB 400, Germany), and a thermo-gravimetric analyzer (Perkin Elmer TGS-2). The FT-IR spectra of the ground polymers were recorded on a Bruker model EQUINOX 55. The high performance liquid chromatography (HPLC) system consisted of a Water 515 pump, a 486 water UV/vis detector, and a model 7725i Rehodyne injector with a 20 μL sample loop. Chromatographic separation was achieved on an ACE 5 μm, C18 column of 4.6 mm × 150 mm. A Windaus two-channel peristaltic pump model D-38678 was used to pump solvents during the MISPE experiments. Scanning electron microscopy (SEM) was performed for morphology analysis on a Philips XL30 microscope (Philips/FEI, Inc. Netherland). pH measurements were conducted with a model 780 Metrohm Switzerland combined glass electrode.

3.3. MIP and NMIP preparation with miniemulsion polymerization

The schematic representation of imprinting and removal of OLZ from the imprinted polymer is shown in Scheme 1. The molecularly imprinted polymer for OLZ was prepared via miniemulsion polymerization from a reagent mixture obtained by mixing OLZ, MAA, EGDMA, and hexadecane as represented in Table 4. In brief, the cross linker (EGDMA), functional monomer (MAA), and hydrophobic agent (hexadecane, 0.2 ml) were mixed. Afterwards, the template (OLZ) and initiator (AIBN, 0.3 mmol) were added to the mixture. The mixture obtained was sonicated in an ultrasonic bath for 18 minutes to ensure that all the materials were dissolved reaching a clear mixture. This procedure also facilitated the formation of the interaction between the drug and the monomers. The solution was gently agitated afterwards for 30 minutes until a hydrogen interaction between MAA and OLZ occurred and a complex was formed. This organic phase solution was slowly added into 30 ml of deionized water containing 15 mg dissolved SDS through a syringe equipped with a 0-G angiocatheter using a high-speed homogenizer at 24 000 rpm for 4 minutes. In order to de-gas the sample, the prepared emulsion was exposed to ultrasonic irradiation for 2 minutes and deoxygenated with dry nitrogen for 5 minutes. According to the standard miniemulsion polymerization procedure, for preparing MIP and NMIP nanoparticles, polymerization was carried out in a three-necked glass reactor equipped with a mechanical stirrer and a nitrogen gas inlet to maintain a nitrogen atmosphere. To maintain the desired reaction temperature, the reactor was immersed in a water bath with thermostatic control at a temperature of 70 ± 1 °C for 16 hours. At the end of polymerization, the obtained nanoparticles were collected by centrifuging at 17 000 rpm for 30 minutes. In order to remove the excess surfactant and the remaining monomer and impurities, the polymer particles were dialyzed against 1 L deionized water (four times for 3 h). Subsequently, the produced nanoparticles were centrifuged and dried in an oven at 50 °C overnight. For removing the drug from non-leached imprinted polymers, batch-mode solvent extraction with 40 ml of methanol containing 10% acetic acid (v/v) was used and the non-leached polymer was washed five times for 1 hour, until no template was detected from the washing solvent by UV spectrophotometry at a wavelength of 251 nm. The nanoparticles were finally washed with 40 ml deionized water and 40 ml acetone, and the resulting leached imprinted nanoparticles were dried at 50 °C overnight. Likewise, non-molecularly imprinted polymers (NMIPs) were synthesized similarly to the MIPs, except they were formed without the template. As depicted in Table 4, a different ratio of monomer, MAA, to template was used in the experiment. To find the optimum proportion of the functional monomers to the template (OLZ) upon miniemulsion polymerization, 30 mg of the leached polymer particles and NMIPs with various ratios of template to monomer molecules (1 : 2, 1 : 4, 1 : 6 and 1 : 8) were dispersed in 8 ml of the OLZ solution at a concentration of 0.05 mM in mixture solution of water/acetonitrile (v/v 4 : 1) via 2 minutes sonication followed by 30 minutes gentle agitation. Particles were collected by centrifugation, the concentration of the supernatant was measured using UV spectrophotometry, and the amount of drug absorbed was calculated. As represented in Table 4, the optimal ratio of the monomers to template was 4 : 1, which had both the best specific affinity and the highest retention of 86% while such a value was 9% for the corresponding NMIP. Excess of the functional monomer with respect to the template yielded a higher non-specific affinity. Therefore, the typical 1 : 4 : 16 (template : monomer : cross linker) ratio was used for further studies.

Scheme 1 The schematic representation of the MIP synthesis for olanzapine.

Table 4 Template : monomer ratios and the percentage of olanzapine recovery by each polymer

Polymer	Template : monomer	OLZ (mmol)	MAA (mmol)	SDS (ml)	Hexadecane (ml)	EGDMA (mmol)	AIBN (mmol)	Recovery^a (%)
a Average of three determinations.
MIP1	1 : 2	2	4	30	0.2	16	0.3	42 (±1.2)
MIP2	1 : 4	1	4	30	0.2	16	0.3	86 (±1.0)
MIP3	1 : 6	0.5	4	30	0.2	16	0.3	64 (±1.6)
MIP4	1 : 8	0.25	4	30	0.2	16	0.3	61 (±1.3)
NMIP	—	—	4	30	0.2	16	0.3	9 (±1.2)

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3.4. Packing of OLZ-MIP and operation of the on-line SPE-HPLC system

OLZ-MIP and NMIP columns were prepared by packing 100 mg of the optimized polymers into an empty polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. Then the cartridge was conditioned sequentially with 1 mL of methanol, 1 mL of ultra-pure water and 1 mL ammonium phosphate (25 mmol L⁻¹) at pH 3.0. Extraction experiments included loading the column with 5 mL of a sample containing 50 μg L⁻¹ OLZ at a flow rate of 1.0 mL min⁻¹. Afterwards, the cartridge was rinsed with 1 mL acetone : acetonitrile (3 : 1), 1 mL methanol : acetic acid (9 : 1) and 1 mL ultra-pure water, a full vacuum was applied through the cartridge for 20 minutes to remove residual moisture, then 1 mL dichloromethane and a vacuum were applied for 2 minutes to remove residual solvent and dry the cartridge. Finally, the elution phase was performed by passing 3 × 1 mL of phosphate buffer salt : acetonitrile : methanol 67 : 22 : 11 (v/v) through the cartridge. At the end, each eluted sample was injected into the analytical column and analyzed by HPLC.

3.5. Binding experiments

For measuring the bonding of the template to the polymer matrix, 50 mg of MIP nanoparticles was added to 10 ml of OLZ solution at appropriate conditions (pH = 6.8) in various concentrations of the drug. The mixtures were placed in a thermostated environment at 25 °C for 24 hours under uninterrupted stirring and then filtered via filter paper. The residual concentration of OLZ in the solution was measured using HPLC-UV. The quantity (Q) of OLZ bound to the MIP and NMIP particles was calculated by the following equation:⁶⁵

Q = [(C₀ − C_t) × V]/W

where C₀ (mg L⁻¹) is the initial concentration and C_t is the residual concentration of OLZ; V (L) is the initial volume of the solution and W (g) is the weight of MIPs or NMIPs.

3.6. Extraction procedure for human plasma samples

The blood samples were drawn from the Iranian blood transfusion service (Tehran, Iran) and five schizophrenic patients of the Psychiatric Clinic of the Tehran Medical University that they had subjected to treatment with olanzapine tablets at a constant daily dose for at least 4 weeks. All samples were stored at −20 °C, except plasma samples in which we analyzed olanzapine levels (kept at – 80 °C), until use. Before extraction with MIP nanoparticles, some treatments were necessary due to the possibility of protein bonding of the olanzapine or other disruptive factors. Hence, the plasma samples were diluted with 25 mM ammonium acetate in alkaline pH and then centrifuged at 6000 rpm for 30 minutes to remove excess proteins. After filtering the supernatant via a cellulose acetate filter (0.2 μm pore size), the filtrate was collected in a glass vial and stored at −20 °C until the next step of the analysis performance. 2 mL of the filtered sample was directly percolated through the MIP or NMIP particles.

3.7. Adsorption capacity experiments

In the measurement of adsorption capacity of MIP and NMIP absorbents, 50 mg samples of the absorbents were added to 100 mL OLZ solution at concentrations of 10–500 μg ml⁻¹. The suspensions were mechanically shaken at room temperature, followed by centrifuging and removal of the absorbents.

3.8. Cytotoxicity study (MTT assay)

Cell viability is considered a significant parameter to be evaluated in order to determine the toxic effects of prepared polymeric vehicles for drug delivery. In order to determine the cytotoxicity of the MIP nanoparticles, an MTT assay was carried out on NIH/3T3 cell lines. Cells were plated at a density of 13 × 10³ cells per cm² (4 × 10³ cells per well) onto a 96-well culture plate in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) for 24 hours (5% CO₂, 95% humidity at 37 °C). Afterwards, MIP nanoparticles were dispersed in DMEM and added to the full-growth media of confluent NIH/3T3 cells to yield a final concentration of 50 mg mL⁻¹ and incubated for 1, 3, 5 and 7 days. Cell viability was determined using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA) assay containing calceinacetoxymethyl ester (calcein AM) and ethidium homodimer-1 (EthD-1). An MTT [3-(4, 5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide] assay was performed at several time points (1, 3, 5 and 7 days) in order to measure cell viability. Briefly, phosphate buffered saline (PBS) containing 5 mg mL⁻¹ MTT was added to each well. The cells were then incubated for 4 hours at 37 °C resulting in the formation of formazan crystals due to the action of succinate-tetrazolium reductase belonging to the mitochondrial respiratory chain on MTT. The crystals were solubilized afterwards using 60 μL of DMSO (Sigma, Germany). Absorbance at 540 nm was measured using a plate reader. Using the standard curve plotted for the known number of cells, the number of the viable cells in each plate was determined and was reported as percent control. Results were obtained upon three discrete experiments.

3.9. In vitro drug release in various media

In vitro release of OLZ from the MIP nanoparticles was determined under sink conditions (the volume of the release medium used was enough to dissolve at least five times the quantity of drug present in the nanoparticles). To this end, 60 mg of MIP or NMIP nanoparticles was added to a certain amount of the OLZ solution at a concentration of 0.05 mM in a mixture solution of water/acetonitrile (v/v 4 : 1). For better dispersion, the suspension was exposed to ultrasonic irradiation for 2 min and was mechanically shaken at room temperature for 1 h, followed by centrifuging and removal of the absorbents. The remaining OLZ in the supernatant was measured by HPLC-UV. Then, 5 mg of OLZ-loaded nanoparticles were dispersed in 5 ml of 1% wt. sodium dodecyl sulfate (SDS, pH 6.8) aqueous solution and placed into a dialysis bag with a molecular weight cutoff of 12 000 Da (Sigma, Germany). The dialysis bag was immersed in a flask containing 45 ml of release medium (1%wt SDS). The whole assembly was shaken at 100 rpm at 37 °C. At predetermined time intervals, 3 ml of the release medium was removed and replaced with fresh medium. To make the 45 ml volume, the outer dialysis bag remained constant. The content of the OLZ in the sample was measured by HPLC-UV and the cumulative release percentage of the OLZ was calculated. In another experiment, 5 mg of the OLZ-loaded nanoparticles was dispersed in 5 ml of phosphate buffer saline (PBS, pH 7.4) and placed into a dialysis bag. In this case, the dialysis bag was placed in flask containing 45 ml of PBS (pH 7.4) containing 30% (v/v) alcohol (sink conditions). The whole assembly was shaken at 100 rpm at 37 °C and the procedure was carried out based on the aforementioned protocol. The content of OLZ in the sample released in PBS was measured using HPLC-UV and a cumulative release percentage of OLZ was calculated. In both media the release of OLZ from NMIP particles was calculated and compared with the release from MIP particles.

4. Conclusion

The aim of the present study was to prepare polymeric imprinted nanoparticles with a high loading capacity as a drug carrier to execute a sustained release of olanzapine in CNS diseases. Furthermore, we sought to determine the effect of this polymeric drug delivery system on solid phase extraction (SPE) for analytical purposes. MIP nanoparticles possess high chemical, mechanical, and thermal resistance and provide an appropriate capacity for drug transportation and extraction from human blood plasma. In this study, we developed uniformly sized MIPs via miniemulsion polymerization through a non-covalent mechanism. The relationship between the morphology and porosity of the particles, and the binding and release properties were perused which collectively provided useful guidelines for controlling the MIP particle properties for a desired application, including analytical fields, and drug delivery systems. The key factors controlling recognition and release in the imprinted polymer matrices included mole ratios of the monomer to olanzapine and the effect of the environment pH on drug loading, which were discussed during our investigation. The results also stated that the best ratio of the monomer to olanzapine was 4 : 1 and the best medium for efficient drug loading was at a pH of 6.8. In addition, in vitro release experiments were performed. For this purpose, intestine media was simulated by SDS solution (1% wt) and physiological media was simulated by PBS, which were used to provide release studies from olanzapine loaded nanoparticles. The results point towards the ability of MIP particles to control olanzapine release, supporting a release mechanism, in which the release rate of the drug from the matrices depends on the selective interaction between the drug and the imprinted cavities, the pH level, and temperature of the dissolution medium. By exploring the drug release profile from MIP and NMIP, under the same conditions, molecular specificity and specific sites of the template in MIP particles were claimed. The higher drug loading and slower drug release from MIP particles confirmed that claim. Cytotoxicity of the MIP nanoparticles on NIH/3T3 cell lines was studied and results revealed that synthetic MIP particles have low cytotoxicity in these cells. Moreover, the ability of the MIP particles in analytical application like SPE was studied. The recoveries for the spiked human plasma samples were between 83–94% in 5–50 μg L⁻¹ respectively. Taken together, it can be concluded that the technique has great potential in developing selective extraction methods for other compounds. For olanzapine adsorbed onto MIP nanoparticles, isotherm studies showed that the experimental equilibrium data could be well fitted by the Langmuir adsorption model. The analytical ability of OLZ-MIP for SPE purposes was compared with other previously reported methods, regarding some analytical characteristics. The MIP particles show a low LOD and LOQ. According to the different studies conducted in this investigation, it can be suggested that we can use this technique for other drugs and provide drug delivery carriers and polymeric sorbent for analytical purposes via a convenient and simple method with lower cost.

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