Preparation of multi-functionalized Fe3O4/Au nanoparticles for medical purposes

https://doi.org/10.1016/j.colsurfb.2015.02.002Get rights and content

Highlights

  • The preparation of magnetite/gold nanoparticles is described.

  • A polyelectrolyte multilayer is adsorbed on the magnetic core prior to gold coating.

  • The polyelectrolyte multilayer formation improves the stability of the coating.

  • The magnetite/gold nanoparticles are able to load DOX and/or BEV.

  • The DOX and BEV can be simultaneously released from the particles.

Abstract

In this work, we investigate a route towards the synthesis of multi-functionalized nanoparticles for medical purposes. The aim is to produce magnetite/gold (Fe3O4/Au) nanoparticles combining several complementary properties, specifically, being able to carry simultaneously an antitumor drug and a selected antibody chosen so as to improve specificity of the drug vehicle. The procedure included, firstly, the preparation of Fe3O4 cores coated with Au nanoparticles: this was achieved by using initially the layer-by-layer technique in order to coat the magnetite particles with a three polyelectrolyte (cationic-anionic-cationic) layer. With this, the particles became a good substrate for the growth of the gold layer in a well-defined core–shell structure. The resulting nanoparticles benefit from the magnetic properties of the magnetite and the robust chemistry and the biostability of gold surfaces. Subsequently, the Fe3O4/Au nanoparticles were functionalized with a humanized monoclonal antibody, bevacizumab, and a chemotherapy drug, doxorubicin. Taken together, bevacizumab enhances the therapeutic effect of chemotherapy agents on some kinds of tumors. In this work we first discuss the morphology of the particles and the electrical characteristics of their surface in the successive synthesis stages. Special attention is paid to the chemical stability of the final coating, and the physical stability of the suspensions of the nanoparticles in aqueous solutions and phosphate buffer. We describe how optical absorbance and electrokinetic data provide a follow up of the progress of the nanostructure formation. Additionally, the same techniques are employed to demonstrate that the composite nanoparticles are capable of loading/releasing doxorubicin and/or bevacizumab.

Introduction

One of the main objectives in anticancer drug development is the design of delivery systems that can contribute to transport and release the therapeutic agents in a targeted and selective fashion to their site of action, thus decreasing adverse effects and enhancing efficacy and specificity. Nanoparticles (NPs) have been demonstrated to be a main component of recent strategies aimed at delivering conventional drugs, recombinant proteins, vaccines and, more recently, nucleotides. NPs modify the kinetics, body distribution and release of an associated drug, and can perform this function either passively (the drug is released by diffusion or matrix dissolution in an otherwise uncontrolled way) or actively (specific interactions are established between the NP functional groups and the target cell, which trigger their payload release only in specified conditions [1]). In the first case, NPs-based systems work by exploiting the special characteristics of tumor growth, specifically, the so-called EPR (enhanced permeability and retention) effect [2], [3], [4] as a passive form of targeting. As a result of the imperfect (leaky) vasculature and limited lymphatic drainage, EPR favors that nanoparticles smaller than 100 nm can enter the interstitia and be captured by the tumor cells [3], [5], [6]. In active targeting, specific ligands attached to the surface of the drug vehicles allow their binding to receptors over-expressed by tumor cells and not by normal cells [6].

The possibilities of driving the nanoparticles to the site of action and keeping them there during the drug release are enhanced if an external field can be used with that purpose. As an example, a growing interest exists in iron oxide nanoparticles as core of nanostructures which can be responsive to external magnetic fields. Additional advantages of those superparamagnetic particles in biomedical and clinical applications come from their enormous potential in such techniques as magnetic resonance imaging (MRI) contrast agents [7], magnetic hyperthermia treatment [8], [9] or tissue repair [10]. In spite of such applications, improvements are still needed concerning their biocompatibility, mechanisms of attachment to cells and biomolecules, colloidal and physical stability, or water dispersibility [11]. These modifications are essential for efficient delivery to cells.

A possible modification consists of producing a gold coating prior to loading the resulting composite nanoparticles with its drug payload [12], [13]. Justification for this is related to the fact that gold particles have low toxicity, high surface area, tunable stability, and significant chemical reactivity. Their presence on the magnetic cores may improve their capabilities for adsorbing, transporting and releasing the active chemicals [14], [15]. The resulting magnetic nanostructures would have the additional advantage of particular optical properties, specifically, increased absorbance in the visible spectrum, thus opening the possibility of using them in photothermal therapy.

Doxorubicin (DOX) will be used as drug payload. This is one of the most potent and widely used anticancer drugs, and it works by inhibiting the synthesis of nucleic acids within cancer cells [16]. However, it has a number of well known undesirable side effects such as cardiotoxicity and myelosuppression, leading to a very narrow therapeutic index. For this reason, a number of investigations have focused on ways to deliver DOX to cancer tissues in a controlled way, so as to reduce such side effects. For instance, DOX was conjugated to poly(lactic-co-glycolic) acid (PLGA) nanoparticles and tested in vivo by Yoo et al. [16]. Comparison with daily DOX injections was carried out by Brannon-Peppas and Blanchette [17], who showed a good suppression of tumor growth, comparable to but not better than standard treatment with injected DOX. In vitro tests have been described by Rudzka et al. [18] and Gómez-Sotomayor et al. [19] using magnetic nanostructures based on superparamagnetic maghemite or iron/magnetite nanoparticles.

Bevacizumab (Avastin®, BEV hereafter), a humanized recombinant monoclonal antibody, is the first anti-angiogenic protein approved by the FDA (USA) as an anti-(human vascular endothelial growth factor) (VEGF) agent. This is a blood circulating protein responsible for blood vessels growth, and up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. BEV has shown antitumor activity in a number of tumor types, especially when combined with standard chemotherapy treatments [20], [21]. Studies of the combination of bevacizumab to anthracycline-based cancer therapy like DOX have found promising results although, in some cases, the combination of DOX and BEV can result toxic [21], [22]. Wang et al. [23] studied the efficiency of a combination therapy of BEV and DOX on T-leukemia/lymphoma, and found that if both compounds are administered simultaneously the therapeutic effect of the drug in the treatment of this malignancy was greatly improved. A similar synergistic effect between DOX and BEV was described by Kim et al. [24] in the treatment of sarcoma patients. Likewise, improvements in the response of breast cancer to the combination of BEV and DOX compared to DOX alone were found by Lindholm et al. [25], whereas Kristian et al. [26] demonstrated that when DOX was given to breast cancer patients 24 h after BEV the drug efficacy was reduced in comparison to concomitant treatment.

In this work we describe the preparation and functionalization of superparamagnetic magnetite nanoparticles, coated with a gold shell, as delivery systems for DOX and BEV. Magnetite nanoparticles were coated with three polyelectrolyte (cationic-anionic-cationic) layers using the layer-by-layer technique [27], [28]. Then a gold layer was added using the same technique. We discuss the morphology of the particles and the electrical characteristics of their surface in the successive synthesis stages. Special attention is paid to the chemical stability of the final coating, and the physical stability of the suspensions of the nanoparticles in aqueous solutions. Subsequently, we study the possible application of the Fe3O4/Au nanoparticles as antitumor drug vehicles. The Fe3O4/Au nanoparticles were functionalized with Bevacizumab and Doxorubicin, and the nanoparticles containing DOX, BEV and DOX + BEV were characterized in terms of zeta potential. Finally, the release profiles of the two compounds, either jointly of separately, were examined.

Section snippets

Materials

Iron (II) sulfate heptahydrate (FeSO4·7H2O) was purchased from Fluka. Potassium nitrate (KNO3), sodium hydroxide (NaOH), polyethyleneimine (PEI, Mw  2000 g/mol), poly(styrenesulfonate) (PSS, Mw  2 × 106 g/mol), chloroauric acid (HAuCl4), sodium citrate tribasic dihydrate, sodium borohydride (NaBH4), doxorubicin hydrochloride (C27H29NO11·HCl), sodium chloride (NaCl), and hydrochloric acid (HCl) were from Sigma–Aldrich. Bevacizumab (Avastin® 25 mg/ml) was from Genentech/Roche (Hoffmann-la Roche, Basel,

Stability of the polymer layer

As described, in order to improve the gold particle attachment to magnetite cores, these were coated with several polyelectrolyte layers using the layer by layer technique [27], [28]. The stability of such layers with time was monitored by means of electrophoretic mobility, ue (Fig. 1). MNPs (both uncoated and coated with the different layers) were redispersed in 1 mM NaCl solutions at different pH values and then, the electrophoretic mobility of the samples was measured at different times Fig. 1

Conclusions

Multi-functionalized magnetite/gold nanoparticles were synthesized and investigated as mono/multi-drug delivery systems for doxorubicin and bevacizumab. The first step in the preparation of Fe3O4/Au nanoparticles was achieved using the layer-by-layer technique in order to coat the magnetite particles with a three polyelectrolyte (cationic-anionic-cationic) layer. The polyelectrolyte multilayer formation improves the stability of the coating as compared to a single layer of polyelectrolyte. With

Acknowledgements

Financial support from Junta de Andalucía (PE2012-FQM0694), and Ministerio de Economía y Competitividad (FIS2013-47666/C3-1-R), Spain, is gratefully acknowledged.

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