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Nov 19, 2013 - piroxicam (PX), a hydrophobic anticancer drug. High drug entrapment efficiencies (>40 %) of the PEG-PMMI-CholC6 micelles were observed ...
J Polym Res (2013) 20:295 DOI 10.1007/s10965-013-0295-1

ORIGINAL PAPER

pH-responsive micelles composed of poly(ethylene glycol) and cholesterol-modified poly(monomethyl itaconate) as a nanocarrier for controlled and targeted release of piroxicam Zhaleh Pourmoazzen & Massoumeh Bagheri & Ali Akbar Entezami & Kazem Nejati Koshki Received: 22 August 2013 / Accepted: 2 October 2013 / Published online: 19 November 2013 # Springer Science+Business Media Dordrecht 2013

Abstract A novel monomethyl itaconate-based copolymer (PEG-PMMI-CholC6) bearing cholesteryl (CholC6) and poly(ethylene glycol) (PEG) side chains with specific degrees of side-chain substitution (DSChol =4.85 and DSPEG =16.41) was synthesized by performing a reaction involving cholesterol-containing poly(monomethyl itaconate) (PMMICholC6) and polyethylene glycol monomethyl ether (PEG, M W∼2000). In aqueous solution, reversible pH-responsive micelle-like aggregates of PMMI-CholC6 and PEG-PMMICholC6 amphiphilic copolymers formed, with their phase transitions occurring around pH 3.8 and 5.12, respectively. The presence of the PEG groups improved the hydrophilicity of the copolymer and suppressed excessive micelle aggregation. The critical micelle concentration (CMC) of the PEG-PMMI-CholC6 copolymeric micelles at pH5.12 was about 1 mg/L. DLS and TEM studies revealed that the spherical micelles had mean diameters of 40 %) of the PEG-PMMI-CholC6 micelles were observed due to the enhanced surface Z. Pourmoazzen : M. Bagheri (*) Chemistry Department, Faculty of Science, Azarbaijan Shahid Madani University, P.O. Box: 53714-161, 5375171379 Tabriz, Iran e-mail: [email protected] A. A. Entezami Polymer Laboratory, Organic Chemistry Department, Faculty of Chemistry, Tabriz University, Tabriz, Iran K. N. Koshki Faculty of Advanced Biomedical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

hydrophilicity of these micelles. In vitro release studies performed in buffer solutions at pH 1.2, 4.5, and 7.4 indicated that the PEG-PMMI-CholC6 delivery system can act as a stable nanocarrier allowing controlled drug release at target sites in the pH range 4.5–7.4. Interestingly, MTT assays indicated that the PEG-PMMI-CholC6 micelles did not inhibit HeLa cells regrowth, even at high micellar concentrations. These results suggest that PEG-PMMI-CholC6 micelles have great potential to be safely used in tumor-targeting chemotherapy. Keywords Poly(ethylene glycol) . Self-assembly . Monomethyl itaconate . pH-responsive . Amphiphilic polymers . Targeted drug delivery

Introduction Drug delivery systems have interested researchers for the past three decades. The ideal drug delivery system should be biocompatible and nontoxic, have a high loading capacity, be of a suitable size, and be sufficiently stable to prevent uptake by the reticuloendothelial system and excretion from the body. It is also desirable for such a system to reduce the side effects of drugs on healthy cells and tissues [1]. To achieve effective targeted delivery and satisfactory therapeutic applications, various synthetic polymers have been used to deliver therapeutic agents such as drugs and genes [2]. Drug delivery systems that are capable of releasing their payloads in response to stimuli have received much attention in recent years, as such systems can be used to target tissues, to reach specific intracellular locations, or to promote drug release [3, 4]. Polymeric systems that respond to various stimuli, such as light, temperature, pH, redox potential, interactions with guest molecules, and interactions with biological receptors, are becoming more prevalent in applications relating to biology,

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drug delivery, and nanotechnology [5, 6]. Among various polymeric systems with stimuli-responsive elements, amphiphilic polymers have attracted a great deal of interest for two reasons: (i) they can self-assemble into various supramolecular structures and can thus noncovalently encapsulate guest molecules; (ii) the release of guest molecules can be triggered by external stimuli [7]. The self-assembly of amphiphilic molecules in aqueous media is of fundamental interest for applications in biotechnology and medicine because most drug molecules are hydrophobic, so self-assembling amphiphilic systems can be utilized for drug delivery [8]. Polymeric micelles are nanoscopic constructs that possess a core/shell architecture. They are obtained from the self-assembly of amphiphilic polymers in aqueous solutions above the critical micelle concentration (CMC). The core, the hydrophobic domain, acts as a reservoir and protects the drug payload, whereas the hydrophilic shell mainly confers aqueous solubility and steric stability to the ensemble [9, 10]. The design and preparation of pH-sensitive micelles is an exciting research field that seeks to exploit the attractive properties of polymeric micelles to improve the selective delivery of therapeutic molecules using physiological triggers [11]. Different organs, tissues, and cellular compartments have different pH values, which makes pH a suitable stimulus for the release of the therapeutic molecules from the micelles. In addition, it is proposed that micelles are taken up by cells via an endocytic process [12]. At the start of the endocytic pathway, the pH is near to the physiological pH (7.4), but further along the pathway it drops to a lower pH (5.5–6.0) in endosomes and approaches pH 5.0 in lysosomes [13]. Therefore, polymeric micelles that are responsive to pH gradients can be designed to release their payloads selectively in tumor tissue or within tumor cells. Additionally, the oral route is preferred for the delivery of drugs because it is simple to implement and improves patient compliance and quality of life. Alternatively, drugs administered by the oral route experience pH gradients as they transit from the stomach (pH 1–2) to the duodenum (pH 6), and along the jejunum and ileum (pH 6–7.5) [14]. Poly(monomethyl itaconate) (PMMI) is a hydrophilic polymer with pH-sensitive properties and excellent biocompatibility [15, 16]. In our previous work, we synthesized a series of amphiphilic polymers based on cholesteryl-modified PMMI (PMMI-CholC6) [17]. Because of their biocompatibility, their potential for interactions with cholesteryl receptors on the cell surface, and their ability to drive self-assembly, cholesteryl groups were selected as hydrophobic segments to functionalize the PMMI chains. We recently reported the micellar self-assembly and the pHsensitive behavior of PMMI-CholC6 polymers that were synthesized as novel candidates for pH-responsive nanocarriers employed in controlled drug release (Bagheri et al., unpublished). Furthermore, novel double hydrophilic

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poly(monomethylitaconate)-co-poly(N,N-dimethylaminoethyl methacrylate) (PMMI-co -PDMAEMA) and cholesterolconjugated PMMI-co-PDMAEMA micelles were studied in detail as potential pH-responsive nanocarriers for improved cancer therapy [18]. The presence of polyethylene glycol (PEG) chains on the surface of a polymeric nanoparticle can reduce its adsorption of serum proteins, through a mechanism known as the steric repulsion effect. Moreover, the addition of pendant PEG groups to a polymer leads to some useful properties, such as biocompatibility and suppression of the uptake of nanoparticles by the reticuloendothelial system. This increases the blood circulation time of the nanoparticles [19]. Given these advantages of modification with PEG molecules, the work described in the present paper focused on the synthesis and self-assembly of PEG-PMMI-CholC 6 from the amphiphilic polymer PMMI-CholC 6 using appropriate amounts of PEG and CholC6 pendant groups such that PEGPMMI-CholC6 aggregates in aqueous solution present enhanced stability, respond to external stimuli, and show biological functions. We were able to test the sensitivity of this polymer to acidic and basic stimuli. 4-Hydroxy-2-methyl-N -(py ridin-2-yl)-2 H -1,2benzothiazine-3-carboxamide-1,1-dioxide (piroxicam, PX) is a hydrophobic nonsteroidal anti-inflammatory drug (NSAID) that is used to cure and prevent inflammation and has antitumor properties. This NSAID has shown the ability to inhibit the viability of human colon cancer cells in vitro [20].

Chemical structure of Piroxicam (PX)

Therefore, we developed new PEG-PMMI-CholC6-based micelles that encapsulate PX and evaluated the pH dependence of the release profile of PX from the micelles as well as the cytotoxicity of the micelles towards HeLa cells. The micelles showed pH-responsive release, making them a potential candidate for targeted cancer drug delivery.

Experimental Materials Cholesteryl-functionalized polymers (PMMI-CholC6) with different degrees of cholesteryl side-chain substitution

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(DSchol) were synthesized in our laboratory previously, using PMMI and 6-cholesteryl-1-hexanol (CholC6) [17]. The PMMI-CholC6 with DSchol =4.85 was selected for use in this study. The chemical structure of PMMI-CholC6 is shown in Scheme 1. Polyethylene glycol monomethyl ether (PEG-OH, M W =2000) and a dialysis membrane (MWCO=10,000) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), pyrene, PX, potassium dihydrogen phosphate, sodium hydrogen phosphate, and sodium chloride were purchased from Merck (Darmstadt, Germany). N ,N Dimethylformamide (DMF) and tetrahydrofuran (THF) were dried before use, and all other chemicals were used as received. Measurements Spectroscopic characterization was performed using various instruments. FT-IR spectra were recorded on a Bruker (Ettlingen, Germany) PS-15 spectrometer. 1H NMR spectra were taken on a 400-MHz Bruker (Rheinstetten, Germany) SP-400 Avance spectrometer using chloroform, dimethyl sulfoxide (DMSO), and deuterium oxide as solvents and tetramethylsilane as the internal standard. A Mettler–Toledo (Columbus, OH, USA) 822 DSC (differential scanning calorimeter) was used to determine phase-transition temperatures at heating and cooling rates of 10 °C/min. The instrument was calibrated for temperature and enthalpy using an indium standard. The pH measurements were carried out with a Metrohm (Herisau, Switzerland) 774 pH-meter equipped with glass and Ag/AgCl reference electrodes and calibrated with standard HCl and acetate buffer at an ionic strength of 0.10 M (NaCl). Fluorescence spectra were recorded on a PerkinElmer (Waltham, MA, USA) LS50B luminescence spectrometer at room temperature. The morphology of the self-assembling micelles was observed by transmission electron microscopy (TEM) using a Philips (Amsterdam, Netherlands) SM10 TEM operated at an accelerating voltage of 150 keV linked to an Epson (Long Beach, CA, USA) HP8300 photo flatbed scanner. Dynamic light scattering (DLS) measurements were performed on a Malvern Instruments (Malvern, UK) ZEN3600 zetasizer with a He-Ne laser beam at 511 nm and 25 °C. Samples were filtered with a 0.2-μm filter of mixed cellulose acetate to remove any interfering dust particles. The polydispersity and the number-average and weight-average molecular weights were determined using a Shimadzu (Kyoto, Japan) 6A gel permeation chromatograph (GPC) with a Waters (Milford, MA, USA) Ultrastyragel 250 (7.8 × 300 mm) column (DMF was used as eluent at 40 °C; the flow rate was 1 ml/min) and a differential refractive index detector; calibration was performed with polystyrene standards.

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Synthesis of PEG-modified PMMI-CholC6 (PEG-PMMI-CholC6) The synthetic pathway to PEG-modified PMMI-CholC6 (PEGPMMI-CholC6) is shown in Scheme 1. A mixture of PMMICholC6 (3.42 mmol of the COOH group, 0.5 g) and DCC (3.42 mmol, 0.72 g) in 5 mL of dry DMF was placed in a reaction flask and stirred for 4 h at room temperature. Then a solution of DMAP (0.937 mmol, 0.1 g) and PEG-OH (M W = 2000; 0.684 mmol, 1.84 g) in 5 mL of dry DMF was added dropwise for almost 2 h to the reaction flask. The reaction mixture was stirred for at least 48 h. Precipitated dicyclohexylurea was removed by filtration and the filtrate was poured into 20 mL of diethyl ether to give a white precipitate of PEG-PMMI-CholC6. FT-IR (KBr): 3421 (OH, carboxylic acid), 2887 (aliphatic CH2), 1737 (ester C=O), 1670 (carboxylic acid C=O), 1464 (CH2, bending), 1360 (CH3, bending), 1113 (O-CH2) cm−1. 1H NMR (DMSO-d6): 0.62 (s, CH3 from cholesterol), 0.85 (d, CH3 from cholesterol), 0.89 (d, CH3 from cholesterol), 0.93 (d, CH3 from cholesterol), 1.13 (s, CH3 from cholesterol), 2.50 (s, –CH2– in polymer backbone), 2.87 (s, H, CH 2COO), 3.05 (s, CH3 from (–CH2CH2O)n CH3), 3.14 (s, CH2 from –CH2COOCH3), 3.30 (s, CH3 from –COOCH3), 3.48 and 3.67 (s, CH2 from –(CH2CH2O)n –CH3), 3.40 (t, CH2 from CH2OCO), 4.14 (t, CH2 from –CH2OCOO–), 4.48 (m, CH from cholesterol), 5.41 (m, =CH from cholesterol), 7.92 (1H, –COOH) ppm. Determination of phase-transition pH The pH values at which phase transitions occurred for PMMICholC 6 and PEG-PMMI-CholC 6 were determined by monitoring the change in transmittance of 0.5 wt% polymer in solution when the pH was varied from 2.00 to 9.00 at a wavelength of 500 nm. At the phase-transition pH, the optical transmittance was 50 %. Fluorescence measurements The phase transition and CMC at pH 5.00 (the phase-transition pH) of the PEG-PMMI-CholC6 polymer were determined by a fluorescence method using pyrene as a hydrophobic fluorescence probe in all determinations. Aliquots of pyrene solution (1.54×10−5 M in acetone, 100 ml) were added to 50ml containers and the acetone was allowed to evaporate. Different concentrations (0.10–5.00 g/L) of the polymer solution in water at different pH values (adjusted by adding HCl or NaOH) were prepared at room temperature. Sample solutions (10 mL) at the desired concentrations were added to the above containers containing the pyrene residue. It should be noted that all of the aqueous buffer sample solutions contained excess pyrene at the same concentration of 6.17×10−7 M. These sample solutions were then stirred at room temperature

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Scheme 1 Synthetic route to PEG-PMMI-CholC6

for 24 h to dissolve and equilibrate the pyrene. Excitation was carried out at 306 nm, and emission spectra were recorded from 345 to 650 nm. Both the excitation and the emission bandwidth was 5 nm. The CMC value was obtained from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations.

correlation coefficient (R 2) was at least 0.998 and 0.999, respectively. The drug-loading efficiency and drug-loading capacity were calculated as follows:

Micelle preparation

where A is the total mass of PX used, B is the mass of unloaded PX in the precipitate after centrifugation, and C is the mass of the polymeric sample.

PEG-PMMI-CholC6 was dissolved in 0.65 mL of 1,4-dioxane at an initial polymer concentration of 0.95 % (w/w) (6.2 mg/mL). The sample was then stirred for 1 h to ensure complete dissolution of the polymer. To induce self-assembly into micelles, a buffer solution at pH 5.12 was added dropwise at a rate of 0.2 % (w/w)/min. Water was added until a final water concentration of 50 % (w/w) was obtained [21] (the water concentration determines the resulting morphology [22]). The solution was stirred overnight to remove the organic solvent. The micelle solutions remained colloidally stable after dioxane removal. The PMMI-CholC6 micelles were passed through a dialysis membrane (MWCO=10,000) because of the insolubility of the polymer in volatile organic solvents. 6.2 mg/mL of the polymer in DMF were dialyzed against 200 mL of buffer solution at pH 3.34. The micelles had formed in the dialysis bag after 24 h.

Loading capacityð%Þ ¼ ðA−BÞ=C  100

ð1Þ

Loading efficiencyð%Þ ¼ ðA−BÞ=A  100;

ð2Þ

In vitro drug release test The in vitro release behavior of PX-loaded micelle solutions was investigated using the dialysis membrane diffusion technique in buffer solutions at pH 1.2, 4.5, and 7.4 at a temperature of 37 °C. At predetermined intervals, 3-mL aliquots of the test solution, which was immersed in 25 mL of the release medium (buffer solution at pH 1.2, 4.5, or 7.4), were withdrawn and replaced with an equal volume of fresh release medium. The drug concentration in the extract was determined by measuring its absorbance at 348 nm in a UVvis spectrophotometer (note that all experiments were carried out in triplicate). MTT assay and cell treatment

Drug loading The PX-loaded polymeric micelles were prepared by a dialysis method as follows. Briefly, 30 mg of polymer and 6 mg of PX were dissolved in 5 mL of DMF. Then the solution was dialyzed against phosphate-buffered saline (PBS) at pH 3.34 and 5.12 for 24 h using a dialysis bag (MWCO 10,000). The unloaded drug concentration (C , mg/mL) was measured by UV-vis spectrophotometry of the dialyzate at 348 nm. Standard solutions at pH 7.4 and 1.5 were prepared at concentrations ranging from 0.003 to 0.1 mg/mL. The

The cytotoxic effects of PX, the PMMI-CholC6 polymeric micelle solution, and the PX-loaded polymeric micelle solution on T47D cells were studied by performing MTT assays for 48 h. Briefly, 2500 cells/well were cultivated in a 96-well culture plate. After 24 h of incubation at 37 °C, the cells were treated with different concentrations of PX, PMMICholC6 polymeric micelles, and PX-loaded polymeric micelles (0–800 ppm) for 48 h (experiments were performed in quadruplicate). Then the medium in each well was carefully removed and 50 μl of 2 mg/ml MTT were added to each well.

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This was incubated in the dark for 4.5 h, before 200 μl of DMSO were added. Thereafter, Sorensen’s glycine buffer was added and the absorbance of each well was measured at 570 nm for 15–30 min. The mean OD of each well was calculated for data analysis. Then the cell viability was calculated according to: cell viability (%) = mean OD of the test wells/mean OD of control wells × 100. Finally, graphs were plotted using SPSS 16.0 that allowed us to determine the IC50 values of PX, PMMI-CholC6 polymeric micelle solution, and PX-loaded polymeric micelle solution towards T47D cells.

Results and discussion Synthesis and characterization of PEG-PMMI-CholC6 In this study, our aim was to develop PEG-PMMI-CholC6 micelles for targeted PX delivery. PEG-PMMI-CholC6 was designed to respond to changes in pH, thus leading to superior micellar stability, preventing premature drug release, prolonging the circulation time, increasing drug accumulation at tumor tissues, and boosting its uptake by tumor cells. In our previous work, PMMI-CholC6 samples were synthesized with different DSchol [17, 18]. The DSchol of each sample was determined by conductivity measurements and 1H NMR analysis. In the present work, we synthesized PEG-PMMICholC 6 copolymers with different molar ratios of PEG:CholC6 (20:5, 30: 5, 40:5, 20:10, 30:10, 40:10, and 40:25) by reacting PMMI-CholC6 with PEG in the presence of DCC and DMAP (Scheme 1). Among these copolymers, only the sample with 20:5 mol% PEG:CholC6 had pHresponsive properties. Therefore, the PMMI-CholC6 with 20 mol% of pendant carboxylic acid groups (DSchol =4.85) was used to prepare the PEG-PMMI-CholC6 copolymer with 20:5 PEG:CholC6. 1 H NMR and FT-IR spectroscopy were used to confirm the structure of the synthesized PEG-PMMI-CholC6. FT-IR spectra of the unmodified and modified PMMI-CholC6 (with DSchol = 4.85) were analyzed in order to elucidate their structures (Fig. 1). This analysis identified the peaks

Fig. 1 FT-IR spectra of PMMI-CholC6 (a) and PEG-PMMI-CholC6 (b)

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associated with PMMI-CholC6 and the PEG side chains. Both the modified and the unmodified PMMI-CholC6 contained carboxylic acid groups that produced a broad peak from 3200 to 3500 cm−1 due to O–H stretching vibrations (Fig. 1a and b). Comparison of the IR spectrum of the PEG-PMMI-CholC6 with that of PMMI-CholC6 highlighted a new absorption feature at 1106 cm−1 in the PEG-PMMI-CholC6 spectrum, which was assigned to the asymmetric stretching vibrations of ether groups [23]. Another difference caused by the PEG moieties in the PEG-PMMI-CholC6 was an increase in the C–H stretching resonances from the PEG group, which were found at 2887 and 2960 cm−1 (Fig. 1b). These results clearly show that PEG-PMMI-CholC6 was obtained. The 1H NMR spectra of PMMI-CholC6 and PEG-PMMICholC6 (with DSchol =4.85) are displayed in Fig. 2. In the 1H NMR spectrum of PEG-PMMI-CholC6, contributions from both PMMI-CholC6 and the PEG side chains are present. The

Fig. 2a–b 1H NMR spectra of a PMMI-CholC6 and b PEG-PMMICholC6

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singlet peak at 3.05 ppm denoted h in the 1H NMR spectrum (Fig. 2b) originates from methoxy protons at the ends of the PEG side chains. In addition, broad peaks relating to the main chain methylene units within the PEG groups are clearly visible at δ =3.48 and 3.67 ppm (Fig. 2b). The peaks from the protons in the cholesteryl side chains (albeit at low intensities) and those on the polymer backbone were still present after the reaction. The degree of PEG substitution on the polymer backbone (DSPEG) was determined based on the ratio of the integral of the signals from the methyl protons in the cholesteryl groups at around 0.85 ppm (d, Fig. 2b) to the integral of the signals from the methoxy protons of the PEG group at around 3.05 ppm (H, Fig. 2b). Therefore, DSPEG is 16.40 for PEG-PMMI-CholC6 (Table 1). PEG chain attachment was further validated by differential scanning calorimetry (DSC) analysis. As shown in Fig. 3, PEG-PMMI-CholC6 presented a sharp melting temperature (T m) at 58 °C when it was heated from 25 °C to 300 °C. This is much lower than the melting temperature of PMMI-CholC6 (140 °C), which can be attributed to the decreased hydrogen bonding in the PMMI-CholC 6 main chain due to the attachment of PEG and thus the decreased crystallinity of the obtained polymer (Table 1). Table 1 also presents the inherent viscosities (η inh) of the polymers, which were determined using an Ubbelohde viscometer at 35 °C in DMSO. As shown in Table 1, the viscosity of the PEGmodified polymer is clearly higher than that of its parent PMMI-CholC6 because of its increased molecular weight. The weight- and number-average molecular weights of the synthesized polymers were determined by gel permeation chromatography (GPC) (Fig. 4), and the results are summarized in Table 1. pH-dependent phase transition PMMI is a weak polyelectrolyte (pK a ≈3.85) with good solubility in water. The PMMI-CholC6 polymer, which is partially substituted with lipophilic cholesteryl moieties (DSchol =4.85), showed pH-dependent solubility in water [18]. The PMMI-CholC6 was easily dissolved in aqueous solution at pH>4 because the hydrophilicity of the polymer is relatively high due to the deprotonation of carboxylic acid

groups. However, it precipitated from aqueous solution at pH