Dual drug loaded superparamagnetic iron oxide nanoparticles for targeted cancer therapy

Abstract

The primary inadequacy of chemotherapeutic drugs is their relative non-specificity and potential side effects to the healthy tissues. To overcome this, drug loaded multifunctional magnetic nanoparticles are conceptualized. We report here an aqueous based formulation of glycerol monooleate coated magnetic nanoparticles (GMO-MNPs) devoid of any surfactant capable of carrying high payload hydrophobic anticancer drugs. The biocompatibility was confirmed by tumor necrosis factor α assay, confocal microscopy. High entrapment efficiency ∼95% and sustained release of encapsulated drugs for more than two weeks under in vitro conditions was achieved for different anticancer drugs (paclitaxel, rapamycin, alone or combination). Drug loaded GMO-MNPs did not affect the magnetization properties of the iron oxide core as confirmed by magnetization study. Additionally the MNPs were functionalized with carboxylic groups by coating with DMSA (Dimercaptosuccinic acid) for the supplementary conjugation of amines. For targeted therapy, HER2 antibody was conjugated to GMO-MNPs and showed enhanced uptake in human breast carcinoma cell line (MCF-7). The IC₅₀ doses revealed potential antiproliferative effect in MCF-7. Therefore, antibody conjugated GMO-MNPs could be used as potential drug carrier for the active therapeutic aspects in cancer therapy.

Introduction

Magnetic nanoparticles (MNPs) are emerging as promising candidates for applications in biomedical research encompassing of drug delivery [1], magnetic resonance imaging [2], cell mechanics [3], hyperthermia [4], tumor progression [5], in vivo tracking of stem cells, nucleic acid and cell separation [6], [7], due to their ultra fine sizes, biocompatibility and superparamagnetic behavior [1], [8]. For biomedical application the MNPs are perfect model for high level of accumulation in the target tissues or organ due to their host cell tropism, biophysical nature [9], and low toxicity [10]. The pharmaceutical drugs are loaded to the surface of the MNPs, which are released at the target site with external localized magnetic field gradient, thereby offering a possibility of nonspecific toxicities by administering lower but more accurately targeted dose [11].

The size and surface charge chemistry of MNPs are predominantly important for monitoring systemic tumor response to drug treatment [12]. The inherent aggregation behavior of MNPs occurring due to the high surface-to-volume ratio and attractive forces between the magnetites is a crucial limiting factor which reduces the intrinsic superparamagnetic properties [13], and triggers the opsonization process [9]. Therefore, to minimize the aggregation, it is essential to engineer the surface of the MNPs. Synthetic and natural polymers (dextran, polyethyleneglycol (PEG), and poly(vinylpyrrolidone) (PVP), streptavidin, poly-l-lysine (PLL), polyethylene imide (PEI) etc.) have been employed to modify the surface of the MNPs [13]. To facilitate the anchoring and attachment of the polymers to MNPs several monomeric species like bisphosphonates [14], dimercaptosuccinic acid [15], and aloxysilane [16], were examined, but these agents could not allocate colloidal stability at physiological pH [17]. Therefore, coating the MNPs with large molecules (polymers or surfactants) containing long-chain hydrocarbons, helps to prevent the aggregation of particles in biological solution for more effective stabilization. [18].

Various research groups mostly use long-chain polymer oleic acid (OA) and its salts [19], for the stabilization of iron oxide nanoparticles. Jain et al. have developed aqueous dispersible oleic acid-pluronic (F-127) stabilized iron oxide nanoparticle, encapsulating hydrophobic drug [1]. However, there is potential concern about the toxic effects of pluronic (F-127), towards human erythrocytes [20], and elevation of cholesterol and triglycerides level in the blood plasma [21]. Consequently, with an aim of achieving colloidal stability of the magnetic nanoparticles without use of any surfactant, a synthetic lipid, glyceryl monooleate (GMO) approved by food and drug administration (FDA) is considered in our study. GMO is an unsaturated monoglyceride amphiphilic lipid [22], used as emulsifier, flavouring agent and excipient for antibiotics. It forms different types of lyotropic liquid crystals which is water and temperature dependant [23] furthermore, its bioadhesiveness, high viscosity property increases the contact time at the targeted tissues providing a platform for sustained release of drugs by slow drug diffusion in solubilized form [24] thus, enhancing the therapeutic efficacy [25] for drug delivery [26]. The heterogeneous structure of GMO in water permits incorporation of hydrophilic and hydrophobic drugs unaffecting the phase structure [27]. GMO has a similar long-chain polymer structure as that of oleic acid (OA), mainly used in the formulation of MNPs. Keeping in view these properties of GMO, we have coated the MNPs with GMO replacing OA.

We hypothesize that, drug loaded GMO-MNPs will be an ideal drug delivery system for the treatment of cancer, where the hydrophobic drug would partition into the GMO coating, providing aqueous dispersibility with no loss of magnetization. Hence, we developed an aqueous based ultrafine stable GMO-MNP formulation for drug delivery without the use of any surfactant. The formulated GMO-MNPs were well characterized by different techniques, such as, transmission electron microscopy (TEM), dynamic laser light scattering (DLS), fourier transmission infrared spectroscopy (FT-IR), superconducting quantum interference device (SQUID), reverse-phase high pressure liquid chromatography (RP-HPLC) for their particle size, zeta potential, magnetization and entrapment efficiency respectively. The cytotoxicity and inflammatory response of the GMO-MNPs was determined through confocal microscopy, tumor necrosis factor (TNF-α) assay by enzyme-linked immunosorbent assay (ELISA) method using human breast carcinoma cell lines (MCF-7). Further, for the conjugation of any peptide or protein, the GMO-MNPs were functionalized with DMSA (2, 3 meso mercapto succinic acid). The targeted therapeutic purpose of the GMO-MNPs was achieved through the conjugation of HER2 antibody.