Journal of Research Analytica ISSN 2473-2230

Research Article

CE-UV Analysis of Biochemically Active Compounds loading onto Chitosancoated Magnetic Nanoparticles Meissam Noroozifar1, Zafar Iqbal2 and Edward P.C. Lai2* University of Sistan and Baluchestan, Zahedan, Sistan & Baluchestan, Iran Department of Chemistry, Carleton University, Ottawa, Ontario, Canada

1 2

Corresponding author: E-mail: [email protected] (E.P.C.L)

*

Abstract Chitosan and polydopamine were successfully coated on Fe3O4 magnetic nanoparticles. These chitosan- and polydopaminecoated magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, vibrating sample magnetometry, and scanning electron microscopy. They were evaluated as new carriers for their potential capacity to load various biochemically active compounds. Their % loadings were determined by capillary electrophoresis with UV detection. The results showed that chitosan has a high loading capacity for aromatic compounds, which is a promising approach towards drug delivery applications.

Keywords: Nanoparticles; Chemotherapy; CE-UV analysis; Chitosan Introduction Prostate cancer is the most common type of cancer worldwide among men. In the United States it is responsible for more male deaths than other cancers except lung cancer [1]. Numerous phase I/II studies have been conducted to evaluate the safety and efficacy of neoadjuvant chemotherapy before prostatectomy [2]. Mitoxantrone (MTX) is a chemotherapy drug that is classified as an antitumor antibiotic used to treat advanced prostate cancer by interfering with the growth and reproduction of cancer cells [3]. A multi-agent regimen of MTX was reported to have antineoplastic activity as evidenced by reductions in prostate-specific antigen [4]. MTX is also a therapeutic compound approved by health authorities for the treatment of secondary progressive multiple sclerosis [5]. A novel capillary electrophoresis (CE) with chemiluminescence detection method was developed for the determination of MTX in commercial drug, human urine and plasma. It was based on sensitization of the reaction between potassium ferricyanide and luminol in sodium hydroxide medium, affording a detection limit of 1.0 × 10-8 M [6]. Received : May 05, 2016; Accepted: May 20, 2016; Published: May 23, 2016 Citation: Noroozifar M, Iqbal Z, Lai EPC. CE-UV Analysis of Biochemically Active Compounds loading onto Chitosan-coated Magnetic Nanoparticles. J Res Anal. 2016; 2(3):66-72. Copyright: ©2016 OLOGY Group.

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Prednisolone is a corticosteroid widely used as secondary hormonal treatment for castration-resistant prostate cancer [7]. It has many uses as treatment for a variety of cancers, such as leukemia, lymphoma, and multiple myeloma [8]. In chemotherapy, prednisolone stops the growth of cancer cells either by killing the cells or by stopping them from dividing [9]. It also produces its anti-cancer effects through inhibiting the inflammatory processes that may be caused by cancer [10]. Prednisolone can be combined with docetaxel for the treatment of hormone-refractory metastatic prostate cancer [11]. A modified low-dose regimen of prednisolone and mitoxantrone was conducted in palliative patients with androgen-independent prostate cancer to manage the toxicity [12]. Prednisolone was orally administered 5-50 mg daily and mitoxantrone was intravenously infused using 7-12 mg/m2 in 100 ml of 0.9% NaCl over 10 minutes [13,14]. Recently nontoxic Fe3O4 magnetic nanoparticles (MNPs) have attracted a wide range of applications in medicine, especially for enhancing the delivery of anticancer drugs with limited adverse effects [15] and for advancing the concept of nanomedical theranostics in conjunction with MRI imaging [16]. Coating or functionalization of MNP surfaces can be beneficial for drug delivery, targeted therapy, magnetic resonance imaging, transfection, and cell/protein/DNA separation [17]. MNPs were previously coated with different materials (such as amorphous silica, octadecylsilane and natural/synthetic polymers) to remove pollutants in water by different researchers [18-21]. Chitosan (CH) is a biodegradable and aqueous compatible polymer that has attracted great attention in the pharmaceutical development of nanoparticles for encapsulation of drugs and biological substances. A considerable amount of work has been published on chitosan and its potential use in drug delivery systems due to its properties for safe use as a pharmaceutical excipient [22]. CH-coated MNPs were prepared in one step by reverse microemulsion precipitation [23]. A CH coating demonstrated strong affinity for binding certain biochemically active pharmaceuticals [24]. In the present work the chemical functionality of chitosan-coated magnetic nanoparticles is investigated to examine its capacity to carry therapeutic drugs. An external magnetic field can potentially be applied to guide the loaded particles where magnetically mediated drug delivery is applicable [25-27]. After collection of these particles in a targeted tumor area, biochemical methods can be used to sustain the drug release over a prolonged period of time through degradation and corrosion of chitosan [28]. Differences in pH between healthy and tumor microenvironments provide pH responsive release of drugs from the particles at tumor site [29]. Chitosan can also be modified by alumino-silicate to achieve

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the sustained release [30]. Polydopamine (PDA) is intrinsically nontoxic and biocompatible. Coating with PDA is emerging as a useful surface functionalization due to the ability of PDA to form a nanometer-scale organic thin film on virtually any material surface to which proteins, peptides, oligonucleotides, metal ions or synthetic polymers are able to be attached [31]. PDA coating can reduce the in vivo toxicity of biomaterials (such as poly-L-lactic acid surfaces and quantum dots) that contact tissue or blood. Dopamine polymerization has recently been demonstrated to be a simple and versatile surface modification method, applicable to a variety of nanoparticle drug carriers irrespective of their chemical reactivity and the types of ligands [32]. Unidirectional uptake and release of charged molecules through PDA microcapsules was achieved by controlling pH value [33].

Experimental Materials Bisphenol A (BPA), chitosan (CH) with high molecular weight, chitosan with low molecular weight, disodium hydrogen phosphate (Na2HPO4), dopamine (DA), iron(II) chloride tetrahydrate (FeCl2.4H2O), iron(III) chloride hexahydrate (FeCl3.6H2O), mesityl oxide (MO), metformin hydrochloride (MF.HCl), mitoxantrone (MTX), naphthalene acetic acid (NAA), phenformin (PF), prednisolone (PRED), pyrrole (Py), sodium dodecyl sulfate (SDS) and triclosan (TC) were all obtained from Sigma-Aldrich (Oakville, ON, Canada). HPLC-grade methanol (MeOH) and ethanol (EtOH) were purchased from Caledon (Georgetown, ON, Canada). Coating magnetic nanoparticles with polydopamine and chitosan MNPs (Fe3O4) were prepared using a method previously described by Wang et al. [34] through co-precipitation of Fe2+ and Fe3+ ions in solution by excess NaOH under sonication. MNPs were coated with polydopamine (PDA) using a procedure reported by Wen et al. [35] with sonication. Coating of MNPs or MNPs@PDA with chitosan (high and low molecular weights) was carried out as follows: 30 mL of Tris buffer (10 mM, pH=9.2) including 50 mM SDS was sonicated in a 200 mL beaker for 10 min. Then, either 2 ml of CH(HM) 1% m/m in 1% acetic acid (AA) or 20 ml CH(LM) 0.1% m/m in AA was added and the solution was sonicated for 10 min. Then, the solution was added to 1.0 g of MNPs in Tris buffer and the suspension was sonicated for 20 min. Finally the black coated MNPs were separated by a magnet, washed with DDW and EtOH (5 times each), and dried at room temperature for 24 hours. CE-UV analysis A modular system was built in our laboratory for all capillary electrophoresis (CE) separations with ultraviolet (UV) detection at 200 nm. The pH 8.5 background electrolyte (BGE) inside the capillary was 20 mM Na2HPO4 in deionized distilled water (DDW), running under an applied voltage of 17–20 kV at ambient temperature. A Lambda 1010 UV detector (Bischoff, Leonberg, Germany) was set up at a wavelength of 200 nm to detect the migration of all particles and compounds. The

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PeakSimple software (SRI Instruments, Torrance, California, USA) was used to acquire the detector output signal.

Results and Discussion FTIR spectroscopy Different types of PDA- and CH-coated MNPs were first characterized by FTIR. As shown in Figure 1, the characteristic peak of MNPs at 592 cm-1 appears weakened in all these spectra due to suppression by the coatings. CH-coated MNPs are distinguishable from MNPs by characteristic peaks ranging from 900 to 1500 cm-1. Also, the appearance of two peaks around 2900 cm-1 indicates a thick CH coating on the MNPs. In comparison, PDA-coated MNPs were characterized by two peaks between 800 and 900 cm-1. Magnetic property The polydopamine-coated magnetic nanoparticles (MNPs@ PDA) and chitosan-coated magnetic nanoparticles (MNPs@ CH) were tested for their magnetic properties in air and aqueous suspension. Their strong attraction to the external magnet is illustrated in Figure 2. The nanoparticles were also characterized by vibrating sample magnetometer [36]. As shown in Figure 3, the relative magnetization of MNPs was only slightly decreased after coating with chitosan to form MNPs@CH. Capillary electrophoresis analysis CE-UV analysis of organic compounds (200 µg.mL-1) was performed before and after in-vitro binding with MNPs@CH (10 mg.mL-1) in 20 mM Na2HPO4 (pH 8.5 ± 0.2). A mixture including bisphenol A (BPA), metformin (MF), naphthalene acetic acid (NAA), phenformin (PF) and quinine sulfate (QS) was electrokinetically injected for 3 s at 17 kV and analyzed under 20 kV. Figure 4 shows that quinine sulfate and phenformin can be loaded, due to binding, more efficiently than bisphenol A and metformin. Surprisingly, naphthalene acetic acid cannot be loaded at all. It is important to note that metformin is significantly less loaded than phenformin. Determination of % loading Loading of biochemically active compounds (including bisphenol A, metformin, naphthalene acetic acid, phenformin and quinine sulfate) from a standard solution was investigated with three different kinds of MNPs (10 mg.mL-1) in 20 mM Na2HPO4 (pH 8.5 ± 0.2). This mixture of model compounds was studied for the reason of characterizing the different hydrophobic and ionic properties of MNPs after coating by chitosan and polydopamine. All % binding results are presented in Table 1 as the mean (± standard deviation) of triplicate measurements (n=3). Phenformin bound up to 95% with MNPs@CH by hydrophobic interaction whereas metformin bound only up to 21%. The NAA results were not surprising at all because it had previously been reported that the ionic interaction between drugs (insulin, diclofenac sodium, and salicylic acid) and chitosan was low

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Transmittance (%)

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30 20 10 0 400

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Figure 1: FTIR spectra of magnetic nanoparticles (MNPs), polydopamine-coated magnetic nanoparticles (MNPs@PDA), low-molecular-weight (LM) chitosancoated magnetic nanoparticles (MNPs@CH), and chitosan-polydopamine-coated magnetic nanoparticles (MNPs@PDA).

Figure 2: Magnetic properties of MNPs@PDA and MNPs@CH in air and aqueous suspension.

Relative magnetizaton (esu/g)

80 60 40 20 7000

2000

0 -20

-3000

-8000

-40 -60 -80

Applied field (Oe) Figure 3: Vibrating sample magnetometry: (a) MNPs, and (b) MNPs@CH.

[37]. A plausible explanation of the low ionic interaction seems to be a maximum of twelve water molecules that are tightly coordinated to the chitosan repeating unit [38]. From the chemical point of view, chitosan represents a weak base with pKa 6.5 [39]. The antimicrobial efficiency of high MW chitosan had also been explained by the binding of the cationic chitosan to the anionic molecules (teichoic acids) at the outer surface of the bacterial membrane [40-42].

aggregates had previously been attributed to hydrophobic domains inherent to chitosan itself [43]. This knowledge could be useful for the interpretation of high loading results for aromatic drugs (phenformin and quinine sulfate) onto the hydrophilic chitosan, especially the MNPs@CH prepared using 5 ml of low MW CH (1% in AA).

On the other hand, the appearance of intermolecular hydrophobic

MNPs, MNP@CH and MNPs@PDA were characterized by CE-

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Characterization of particles

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+17 kV injection voltage +20kV running voltage

1 mAU detector signal

Before

BPA 3.093 PE 2.303 ME 2.156

NAA5.140

OS 2.593

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Figure 3: Vibrating sample magnetometry: (a) MNPs, and (b) MNPs-CH.

NAA 5.070 MF 2.076 BPA 2.990 PF 2.

0

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2.603 4

6

8

Migration time (min) Figure 4: CE-UV analysis of organic compounds (200 µg.mL-1) before and after in-vitro binding with MNPs@CH (10 mg.mL-1) in 20 mM Na2HPO4 (pH 8.5±0.2): bisphenol A (BPA), metformin (MF), naphthalene acetic acid (NAA), phenformin (PF) and quinine sulfate (QS).

Organic compounds (200 ppm) Quinine sulfate (QS) Phenformin (PF) Bisphenol A(BPA) Metformin (MF) Naphthalene acetic acid (NAA)

% Binding with MNPs@CH (2 ml CH low MW 1% in AA ) 93(±1)% 95(±1)% 61(±1)% 21(±1)% 0(±1)%

% Binding with MNPs@CH (5 ml CH low MW 1% in AA) 100(±1)% 92(±1)% 46(±1)% 15(±1)% 0(±1)%

% Binding with MNPs@CH % Binding with MNPs@ (5 ml CH high MW PDA@CH (5 ml CH high 1% in AA) MW 1% in AA) 100(±1)% 100(±1)% 75(±1)% 89(±1)% 46(±1)% 60(±1)% 16(±1)% 22(±1)% 3(±1)% 0(±1)%

% Binding with MNPs@PDA 93(±1)% 60(±1)% 32(±1)% 6(±1)% 0(±1)%

Table 1: Loading of biochemically active compounds due to binding with different MNPs.

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UV (at 200 nm) as shown in Figure 5. Cationic chitosan-coated MNPs migrated faster than negatively-charged MNPs@PDA. Peaks appeared between 2.8 to 3.9 min (MNPs@CH) are due to the polydispersed particles. Fragmentation of long-chain high MW chitosan during coating would be a plausible explanation of this particle size distribution. Whereas a single and fairly sharp peak at 4.29 min represents the monodisperse nature of MNPs@PDA. Scanning electron microscopy (SEM) of the MNPs and MNPs@CH, as presented in Figure 6, supports the monodispersity of these nanoparticles. Mitoxantrone and prednisolone binding with particles

+17 kV injection voltage +20kV running voltage

4mAU detector signal

Mitoxantrone in CH3CN/phosphate buffer (pH=8.5) and prednisolone in ethanol/phosphate buffer (pH=8.5) were next subjected to binding tests based on CE-UV analysis (Figure 7). Both MNPs@PDA@CH (high MW) and MNPs@PDA were able to bind mitoxantrone very well (70%-75%) compared to prednisolone (45%-50%). One plausible explanation of the lower % binding for prednisolone (pKa=13.9) is its decrease in aromaticity relative to mitoxantrone (pKa=8.2 and 11.4) which has two hydrophobic phenyl groups. Chromatographic hydrophobicity measurements can be used in the summing of hydrophobicity values plus aromatic ring count [log DpH7.4

(or log P)+#Ar] to forecast property-based loading of the cancer drugs onto chitosan particles studied in this work [44]. Nanoparticles suspended in phosphate buffered saline (PBS) are compatible with intravenous infusion, as required by the treatment procedure [45]. Ultimately, understanding how nanoparticles are perceived from a biological perspective is crucial to their proper design with different immunogenic and immunosuppressive properties [46].

Conclusion The results reported in this study verify the feasibility of loading mitoxantrone and prednisolone onto chitosan-coated magnetic nanoparticles. Aromaticity of a drug compound proves to be a crucial factor in predicting the loading efficiency. The effect of different coverages on mitoxantrone and prednisolone loading will have to be considered systematically in our future work, as well as the role of polydopamine in modifying the properties of final nanoparticles. Nanoparticles in PBS suspension are generally compatible with intravenous infusion. The strong magnetic property of MNPs@CH and MNPs@CH@PDA suggests that these particles can potentially be applied for delivering bound cancer drugs to the prostate gland cells inside the patient under the guidance of an external magnetic field.

MNPs4.14 MNPs@CH 2.89 MNPs@PDA 4.29

0

4

8

Migration time (min)

Figure 5: Characterization of MNPs, MNP@CH and MNPs@PDA by CE-UV (at 200 nm).

a

b

c

d

Figure 6: Scanning electron microscopy images of: (a, b) MNPs with different magnifications, and (c, d) MNPs-CH with different magnifications.

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Running voltage: 20 kV Injection voltage: 17kV Injection current: 28 µA Running current flow: 34 µA Mitoxantrone: 1st injection for 3 sec Y-scale: 10 mV

Mitoxantrone

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HO

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Figure 7: CE-UV analysis of mitoxantrone (in CH3CN/phosphate buffer, pH 8.5) and prednisolone (in ethanol/phosphate buffer, pH 8.5) before binding tests with MNPs@PDA@CH and MNPs@PDA.

Acknowledgment Financial support of the Natural Sciences and Engineering Research Council (NSERC) Canada is gratefully acknowledged.

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