Molecular imprinted magnetic nanoparticles for controlled delivery of mitomycin C

Abstract

Controlled drug delivery system is a technique which has considerable recent potential in the fields of pharmacy and medicine. Mitomycin C is commonly used drug in the treatment of superficial bladder and breast cancers. In the present study, mitomycin C-imprinted magnetic poly(hydroxyethyl methacrylate)-based nanoparticles (MIMNs) were prepared using surfactant free emulsion polymerization for controlled delivery of mitomycin C. The MIMNs were characterized by fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscopy, electron spin resonance, and elemental analysis. The average particle diameter of MIMNs was about 200 nm.

Keywords::

• mitomycin
• C delivery
• molecular imprinting
• nanoparticles
• PHEMA

Introduction

Molecular imprinting is a powerful technique for constructing receptor with tailor-made binding sites, that is, molecularly imprinted polymers (MIPs) (Wizeman and Kofinas 2001). Molecular imprinting technique involves forming a prepolymerization complex between the template molecule and functional monomer. These are the polymers with specific chemical structures designed to interact with the template either by covalent chemistry (self-assembly) or noncovalent chemistry (Mosbach and Romström 1996, Sellergren 1997, Whitcombe et al. 1995, Kirsch et al. 2000). Once the prepolymerization complex is formed, the polymerization reaction occurs in the presence of a crosslinking monomer and an appropriate solvent, which control the overall polymer morphology and macroporous structure. After the removal of template molecule, the product is a heteropolymer matrix with specific recognition elements for the template (Bryne et al. 2002).

Recently, the potential of MIPs for new applications in the pharmaceutical field as carriers for drugs, amino acids, peptides, hormones, and proteins has gained increasing attention (Hiratani et al. 2005, Calarco et al. 2010, Arias 2008, Xiang et al. 2010). Molecular imprinting technique can provide polymeric materials with the ability to recognize specific bioactive molecules such as drugs and with a binding/delivery bahavior that can be made sensitive to the surrounding medium (Pişkin et al. 1985). Mitomycin C is a bifunctional alkylating agent which is a potent anticancer drug used in the treatment of superficial bladder cancer and breast cancers (Kiremitçi et al. 1986, Denizli et al. 1988a, 1988b). Because of its enhanced activity in hypoxic environment (Yıldırmaz et al. 2003a), mitomycin C has great potential for regional treatment of solid tumors since a significant percentage of viable cancer cells within a solid tumor can be hypoxic (Yıldırmaz et al. 2003b). However, use of mitomycin C is associated with a number of acute and chronic toxicities such as irreversible myelo-suppression and hemolytic-uremic syndrome, which limit its clinical applications. Therefore, efforts have been made to lessen the toxic effects of mitomycin C and improve its utility using various controlled delivery methods (Rauth et al. 1983).

Polymer-based carriers continue to attract considerable interest for drug targeting and delivery in the treatment of cancers and other pathological conditions (Rochwell and Hughes 1994, Cheung et al. 2005, Fournier et al. 2004). Efficient drug delivery systems should provide a desired delivery rate of therapeutic dose at the most appropriate place in the body, in order to prolong the duration of pharmacological action and reduce the side effects, minimize the dosing frequency, and enhance patient complience. The efficient delivery of therapeutic levels of drug to a target site while limiting nonspecific, systemic toxicity also requires optimization of the drug delivery materials, and the treatment protocol. There is a progressive increase in the number of articles and patents devoted to the application of MIPs in the construction of new drug delivery systems (Yerriswamy et al. 2010).

This study aims at forming mitomycin C-imprinted poly(hydroxyethyl methacrylate/N-methacryloyl-L-histidine methyl ester) (MIMNs) magnetic nanoparticles which deliver mitomycin C in a controlled way. For this purpose, MIMNs were prepared by means of surfactant free emulsion polymerization. The MIMNs were characterized thouroughly and delivery studies were conducted in vitro. In these studies, the effects of mitomycin C loading ratio, pH, and temperature on the mitomycin C delivery rate were determined in buffer medium.

Experimental

Materials

Mitomycin C was obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan). MAH was obtained from NanoReg (Ankara, Turkey) and used as received. Potassium persulfate (KPS) and magnetite nanopowder (Fe₃O₄, average size: 20–50 nm) were purchased from Sigma (St Louis, USA). Hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) were obtained from Fluka A.G. (Buchs, Switzerland), distilled under reduced pressure in the presence of hydroquinone inhibitor, and stored at 4°C until use. All other chemicals were of reagent grade and were purchased from Merck AG (Darmstadt, Germany). All water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure LP^® reverse osmosis unit with a high-flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure^® organic/colloid removal and ion exchange packed-bed system. Buffer and sample solutions were prefiltered through a 0.2 μm membrane (Sartorius, Göttingen, Germany). All glassware were extensively washed with dilute nitric acid before use.

Synthesis of mitomycin C-imprinted magnetic nanoparticles

The surfactant free emulsion polymerization was carried out as reported elsewhere with minor modifications (Öztürk et al. 2007, Türkmen et al. 2008, Karakoç et al. 2009). In the first step, MAH-mitomycin C complex was prepared. In order to prepare this complex, mitomycin C was dissolved in distilled water and MAH was added into this solution. The homogeneous mixture was shaken at 100 rpm for 2 h at room temperature. In the second step, the MIMNs were prepared by surfactant free emulsion polymerization. Briefly, the stabilizer, poly(vinyl alcohol) (PVAL, 0.5 g), was dissolved in 50 ml deionized water for the preparation of the continuous phase, followed by the addition of HEMA (0.6 mL), EGDMA (0.3 mL), MAH-mitomycin C complex (300 mg), and Fe₃O₄ nanopowder (100 mg). The reaction mixture was sonicated in an ultrasonic bath for 30 min. The initiator, KPS (0.5 mg/mL) was added to the monomer phase, and nitrogen gas was blown through the medium for about 1–2 min to remove dissolved oxygen. Polymerization was carried out in a shaking bath at 70°C, under nitrogen atmosphere for 24 h. After the polymerization, the MIMNs were washed with methanol and water several times to remove the unreacted monomers and other ingredients. For this purpose, the MIMNs were precipitated and collected with the help of a centrifuge (Zentrifugen, Universal 32 R, Germany) at the rate of 25,000 g for 1 h and resuspended in methanol and water several times. After that, the MIMNs were further washed with deionized water and were collected using a magnet.

Characterization studies

The average size and size distribution of MIMNs was determined by Zeta Sizer (Malvern Instruments, Model 3000 HSA, England).

Swelling ratio of the MIMNs was determined in distilled water. Dry nanoparticles were carefully weighed before being placed in 10 mL vials containing distilled water. The vials were put into an isothermal water bath (25°C) for 2 h. The nanoparticles were removed from the water periodically every 15 min, blotted using filter paper, and weighed. The weight ratio of dry and wet samples was calculated by using Eq. (1).

where W_o and W_s are weights of nanoparticles before and after uptake of water, respectively.

The nanoparticles were imaged in dry state by Scanning Electron Microscope (SEM, Phillips XL-30S FEG, the Netherlands). For this purpose, the samples were initially dried in air at 25°C for 7 days before being analyzed. A fragment of the dried nanoparticles was mounted on a SEM sample mount and was sputter coated with gold for 2 min. The sample was then examined in a SEM.

The size of the MIMNs was also analyzed by atomic force microscopy (AFM) (Digital Instruments, MMafm-2/1700 EXL). Scanning was performed at a scan rate of 1.001 Hz and scan size of 5000 μm. The tip loading force was minimized to avoid structural changes of the sample.

FTIR spectra of the nanoparticles were obtained using a FTIR spectrophotometer (Varian FTS 7000, USA). The dry nanoparticles (about 0.1 g) were mixed with KBr (0.1 g, IR Grade, Merck, Germany), and pressed into a tablet form and the spectrum was recorded.

Delivery studies

In vitro mitomycin C delivery studies were carried out in a continuous delivery system which is described in the United States Pharmacopeia XXII for achieving perfect sink conditions. The continuous delivery system consisted of a pipe with 20 cm length, 1 cm diameter, and total volume of 25 mL. The temperature-control jacket and the upper connector were all made from polyethylene. The delivery cell temperature was controlled by circulating water through the jacket. The MIMNs were placed in the delivery cell. Delivery medium consisted of different buffer systems in the range of 4.0–7.4. Mitomycin C concentration was measured at 364 nm using a double beam UV/Vis spectrophotometer (Shimadzu, Model 1601, Tokyo, Japan).

Results and discussion

The uses of MIPs in different drug delivery systems have been reported in the literature (Hiratani and Lorenzo 2002, Suedee et al. 2000, Norell et al. 1998, Allender et al. 2000, Suedee et al. 2002, Zhang et al. 2006, Alvarez-Lorenzo and Concheiro 2006). Hiratani et al have investigated the influence of the template-functional monomer proportion on the achievement of molecularly imprinted hydrogels with nanocavities with a high affinity for the sustained drug delivery (Hiratani et al. 2005). Puoci et al. have prepared MIPS via a novel precipitation polymerization for the controlled delivery of sulfosalazine (Puoci et al. 2004). Liu et al. designed new delivery system mitomycin C-polyethylene glycol-controlled release film in preventing the epidural scar adhesions after laminectomy in the rat model (Liu et al. 2010). Puoci et al. prepared irregular and nonswellable microparticles of 5-fluorouracil imprinted polymers by employing bulk polymerization as devices for controlled drug delivery (Puoci et al. 2007).

Characterization of nanoparticles

Surfactant free emulsion polymerization produced MIMNs with an average size of 200 nm in diameter with a polydispersity index of 0.196. The average particle size was an average of minimum 30 measurements, and the size distribution was recorded automatically by the software of the repeated measurements. It is apparent that the MIMNs are perfectly spherical with a relatively smooth surface and uniform as shown by the SEM image (Figure 1). The specific surface area was calculated as 970 m²/g. The small polydispersity index suggests that the nucleation step is fast compared with nanoparticles growth, and also the absence of a secondary nucleation step. In addition, the total monomer conversion was determined as 96.1% (w/w). The MIMNs were highly dispersive in water by ultrasonication due to hydroxyl groups on the surface of nanoparticles. The dispersion state of the nanoparticles was confirmed visually by the observed white color of the suspension. The aqueous dispersion of nanoparticles were stable for several weeks. The size of the MIMNs was also given by AFM in Figure 2. AFM image confirms that the nanoparticle size is about 200 nm. Some properties of the MIMNs are summarized in Table I.

Figure 1. Scanning electron microscopy image of MIMNs.

Figure 2. AFM image of MIMNs.

Table I. Some properties of the MIMNs.

Particle diameter	200 nm
Polydispersity index	0.196
g Factor	2.48
Zeta potential	− 8.85 mV
Specific surface area	970 m^2/g
MAH loading	24 μmol/g

The chemical structures of mitomycin C, MAH, and MIMNs were demonstrated in Figure 3. MAH is nontoxic, hydrophilic, and biocompatible material. MAH was selected as a preorganization complex and/or comonomer because it is: (i) commercially available at low cost, (ii) nontoxic, and (iii) it contains imidazole ring for mitomycin C complexation. The FTIR spectrum of MIMNs has the characteristic stretching vibration bands of hydrogen-bonded alcohol, O–H, around 3586 cm^{− 1}, carbonyl at 1645 cm^{− 1}, and amide II absorption bands at 1516 cm^{− 1}.

Figure 3. Chemical structures of mitomycin C and MIMNs.

In vitro mitomycin C delivery studies

The most important parameters that affect delivery rate from polymeric structures are the type of delivery mechanism and amount of substance loaded to polymeric structure (Denizli et al. 1988b). When gradually decreasing delivery rates are observed, delivery rate decreases in relation to square root of time. When delivery rates are independent of time, constant and linear delivery rates are observed (Yıldırmaz et al. 2003b). In controlled drug delivery systems, the mechanism that controls the delivery of the active agent and the application areas of the controlled delivery are taken into consideration. When the dynamic swelling graphs and the delivery rates of the MIMNs used in the study are examined, it is observed that at the beginning (in the first hour) the mitomycin C delivery was a swelling-controlled diffusion mechanism. In the mitomycin C delivery experiments the MIMNs swelled in the first hour by absorbing water. Later on, the polymeric chains in the structure gained activity because of swelling and the pore size changed. As a result mitomycin C started to deliver. In other words, while the solvent diffused into the structure, mitomycin C diffused out of the swollen structure.

The effect of mitomycin C loading rates on delivery

The maximum amount of drug that can be loaded in the polymeric structure is very important. If the maximum loading amount is exceded, the drug prevents the polymer chains from growing and thus interface with the polymer structure formation. In this study, mitomycin C loading ranged between 2 and 8 mg per gram of monomer mixture. The effect of mitomycin C loading amount on the mitomycin C delivery from the MIMNs is shown in Figure 4. The curves in Figure 4 show that increasing in the mitomycin C loading amount in the MIMNs accelerated the MIMNs delivery. This can be explained in the following way: by increasing the loading amount, the concentration gradient triggers and the diffusion process also increases. Another fact to be considered is that a considerable amount of the mitomycin C was leached from the structure during the 30-h delivery process. The delivery ratio of a MIMNs loaded with 8 mg/g mitomycin C was 92.5%. The mitomycin C delivery ratios for the MIMNs were reduced loaded with less amount of mitomycin C, that is, 78% for 4 mg/g and 54% for 2 mg/g loading amounts.

Figure 4. The effect of mitomycin C loading amount on mitomycin C delivery in MIMNs; Delivery medium: phosphate buffer, pH 6.0; T: 25°C.

Previously, a number of materials have been used as drug delivery carriers. To further assess the performance of MIMNs, the release parameters of the reported polymers are compared with other carriers. A similar observation was made by Yerriswamy et al. in case of the drug release studies on 5-fluorouracil drug through poly(vinyl caprolactam-co-vinyl acetate) microspheres crosslinked with N’, N’ methylene bisacrylamide prepared by free-radical emulsion polymerization (Yerriswamy et al. 2010). In vitro delivery studies indicated the delivery of 5-fluorouracil up to 10 h. Duan et al. prepared chitosan-based polymeric prodrug of mitomycin C for controlled release and they observed an obvious burst delivery within the initial 8 h, wherein 65% release of mitomycin C was observed (Duan et al. 2011). The burst release of mitomycin C might be due to free drug of weakly combined drug. Sing and Chauhan have synthesized HEMA- and acrylic acid-based 5-fluorouracil imprinted hydrogels and they observed that the recognition affinity of MIPS increased when the hydrogels were synthesized in a high template concentration (Singh and Chaukan 2008). They observed that the equilibrium release time of 5-fluorouracil from the poly(HEMA-AAc) hydrogels was 5 h. They also reported the 50% of total drug was released in the case of the MIPs. Blanco et al. encapsulated 5-fluorouracil in microspheres of poly(D,L-lactide) and poly(lactide-glycolide) using spray-drying technique (Blanco et al. 2005). The total release of 5-fluorouracil from poly(lactide-glycolide)-based microspheres took place at 28 h and from poly(D,L-lactide)-based microspheres it was at 129 h. Blanco et al. prepared poly(N-isopropylacrylamide) nanohydrogels by inverse microemulsion for the controlled release of anti-tumour drugs including 5-fluorouracil, methoxtrexate, and mitomycin C (Blanco et al. 2008). The average nanoparticulate diameter was 170 nm. Total release took place at 120 h for mitomycin C. Suisha et al. have formed thermoreversible gels by xyloglucan polysaccharide derived from tamarind seed as sustained release vehicle for the intraperotoneal administration of mitomycin C (Suisha et al. 1998). They showed that the in vitro release of mitomycin C from the enzyme-degraded xyloglucan gels is diffusion controlled over a time interval of 5 h. Hu et al. synthesized poly(trimethylene carbonate-dimethyl trimethylene carbonate) (Hu et al. 2011). These anticancer magnetic polycarbonate microspheres showed strong magnetic responsiveness and high mitomycin C-loading capacity. A steady release rate from the microspheres was sustained over the 7 days of measurement. The mitomycin C cumulative percentage release from these microspheres after 7 days reached 78%. Cao et al. synthesized intracellular-targeted conjugates of xyloglucan and mitomycin C with degradable peptide spacer and galactosamine (Cao et al. 2011). The total release was approximately 57% at 8 h and then the release of mitomycin C slowed down. Mitomycin C has been also used to modulate tissue response to surgical trauma in glaucoma surgery. Zimmermann et al. examined the pharmacokinetics behavior of mitomycin C-loaded collagen implant in vitro. In the delivery experiments, the mean total dose delivered by collagen implants was in the range of 0.049–2.29 μg (Zimmermann et al. 1994). Kurita et al. reported a method preparing finely powdered green tea and release profiles (Kurita et al. 2004). Fifty percent release time of mitomycin C was 18 min, and 96% mitomycin C was released after 60 min. Cirillo et al. have prepared molecularly imprinted nanoparticles as devices for the controlled delivery of 5-fluorouracil in biological fluids (Cirillo et al. 2009). They reported that the drug is completely released within 5 h. They also noted that monodispersed spherical particles allow to obtain isotropic release bahavior, also a better control of 5-fluorouracil release profile in comparison with carriers with irregular size and shape.

The effect of pH on mitomycin C delivery

Another parameter that affects the mitomycin C delivery rate from controlled delivery systems is pH. The microenviroment conditions are as significant as the structural characteristics of the polymer (particle size, pore size, pore distribution, etc). In this study, pH in the delivery medium was varied between 4.5 and 7.4. Figure 5 shows the effect of the pH of the delivery medium on mitomycin C delivery. For the MIMNs loaded with 8 mg/g mitomycin C when the pH was 4.5, 6.0, and 7.4, the delivery ratios were 62%, 78%, and 92%, respectively. Thus at higher pH (7.4), the initial delivery rate is faster than lower pHs (4.5 and 6.0). N-methacryloyl-L-histidine methyl ester (MAH) is an ionizable basic comonomer in the polymeric structure, so its delivery behavior greatly depends on the pH due to the ionization–deionization of the imidazole ring in the histidine. At lower pH values, the N-methacryloyl-L-histidine methyl ester comonomer is ionized and the attractive forces between the charged imidazole groups and mitomycin C is dominant. Therefore this leads to low delivery. Similar observation was reported by Johnson et al. in case of PHEMA-poly(L-histidine) hybrid materials for intracellular anticancer drug delivery (Johnson et al. 2012).

Figure 5. The effect of pH of the delivery medium on mitomycin C delivery in the MIMNs; Mitomycin C loading ratio; 8 mg/g; Temperature: 25°C.

The effect of temperature on mitomycin C delivery

The effects of temperature on mitomycin C delivery from the MIMNs at 25°C and 37°C were investigated in the study and the results were given in Figure 6. It is quite obvious that with an increase in the temperature the delivery amount also increases. The cumulative delivery amounts observed were 78% at 25°C and 93% at 37°C. One explanation for this behavior is that the chains in the polymeric structure gain more activity at the higher temperature and the imprinted nanocavities in the nanoparticle structure become larger, resulting in an increase in the mitomycin C delivery rate. Similar observation was reported by Li et al. in case of poly(NIPAM-GMA) microgels for drug delivery (Li et al. 2013). The anticancer drug 5-fluorouracil release from the poly(NIPAM-GMA) microgels is studied at 20°C and 37°C in PBS. Comparing the release profiles at different temperatures, the release amounts are 10% less at 20°C than at 37°C at all the measurement time intervals.

Figure 6. The effect of temperature on mitomycin C delivery rate from the MIMNs; Delivery medium: phosphate buffer, pH 6.0; Mitomycin C loading: 8 mg/g.

Conclusion

In the last years, MIPs were used in drug delivery system. In particular, magnetic nanoparticle drug carriers continue to attract considerable interest for drug delivery systems (Xiang et al. 2010, Hu et al. 2011, Poma et al. 2010). The technique to produce MIPs, using the noncovalent approach, involves arranging functional monomers around the templating ligands (Bereli et al. 2008, Bereli et al. 2011, Mosbach 1994, Ekberg and Mosbach 1989, Zhang et al. 2011). The target substance should form a prepolymerization complex with the monomer by noncovalent interactions. MAH was selected as the functional monomer for complexing with mitomycin C. The ability to correlate high binding affinity and specificity depends on the relative amount of cross-interactions including hydrogen bonding, hydrophobic interactions, π-π orbital interactions, ionic interactions, and van der Waals forces. The three-dimensional structure of the imprinted nanocavities in the MIMNs should also be stable enough to maintain the drug conformation, and it should be also flexible enough to facilitate the release of the template in the imprinted cavity (Singh and Chaukan 2008). MIMNs were prepared by surfactant free emulsion polymerization, and were successfully used as drug-carrier for the controlled release of mitomycin C. A major side effect of mitomycin C is its severe toxicity toward bone marrow and gastrointestinal system (Soyez et al. 1997). Drug delivery systems would reduce these side effects.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.