Ultrasound-assisted synthesis of pH-responsive nanovector based on PEG/chitosan coated magnetite nanoparticles for 5-FU delivery

Highlights

• Ultrasound-assisted synthesis of pH-responsive nanovector through W/O/W multiple emulsion.

• Small and uniformly sized Fe₃O₄ nanoparticles were synthesized by co-precipitation method.

• Double-coated SPIONs were prepared from chitosan and different types of PEGs.

• The effect of PEG structure on the physical properties of nanoparticles was investigated.

• 5-FU release profile represents faster release of drug at lower pH.

Abstract

pH-responsive magnetic carriers at the nanoscale are one of the most important agents for the targeted treatment of cancer. In this study, Fe₃O₄ nanoparticles were prepared by co-precipitation method and functionalized with three types of PEG using ultrasound waves. PEGlated particles were modified with chitosan shell through ultrasound-assisted double emulsion method. The prepared material which was used as a pH responsive carrier for pinpointed 5-FU delivery. The chemico-physical properties of prepared nanoparticles have been investigated. Results demonstrated that pure Fe₃O₄ had a mean diameter of 20 nm with the regular spherical shape which was increased after modification step depending on the type of PEG. 5-FU loading properties and releasing behaviors studies in different pHs which showed that 5-FU can be efficiently loaded in the Fe₃O₄@Cs-PEG. Also, in the case of release, the amount of 5-FU released at pH = 5.8 is noticeably higher compared to the released amount at pH = 7.4 in all three samples at any distinct time. For instance at pH = 7.4, 27% of the 5-FU was released from the Fe₃O₄@Cs-PEG2 during 48 h; as the pH decreases to 5.8, the cumulative amount of 5-FU released enhanced to 52%. The in vitro MTT assay results demonstrated that the cell viability decreases in all synthesized nanoparticles as the pH medium of MCF-7 culture became to 5.8. For example, cell viability of Fe₃O₄@Cs-sPEG decreased from 44 ± 2% to 36 ± 1.9% at a concentration of 5 (μg/ml) as the pH varied from 7.4 to 5.8.

Introduction

Magnetite nanoparticles are one of the most functional vectors in therapeutic and diagnostic approaches. They play an important and undeniable role in drug delivery systems because of great magnetization values and small size [1]. Compared to body cells, the nanoparticles have a much smaller size and can be entered into the cells, which by intracellular or extracellular interaction with genes, enzymes and receptors may lead to cell death [2], [3]. Since the series of intracellular events that leads to the formation of cancer cells occur at the nanoscale, nanotechnology can be used to diagnose and treat them. Functional ability and capability to respond to the magnetic field make magnetic nanoparticles to be introduced as a useful carrier for the targeted diagnosis and treatment of cancer. Recently, great attention has been concentrated on Fe₃O₄ nanoparticles due to their great biocompatibility, drug targeting, and imaging [4], [5]. However, these nanoparticles tend to accumulate a lot because of powerful magnetic dipole-dipole interactions. Therefore, in order to enhance the stability, they were usually modified by oxides, metallic nanoparticles, organosilanes, surfactants and polymers [6]. Surface-modification not only improves the stability of magnetic nanoparticles but also provides the conditions for grafting special targeting agents and stimuli-responsive polymer on the surface of the carrier for drug delivery systems. Magnetic nanovectors are formed from a magnetic core to ensure an appropriate response to the magnetic field and organic shell to provide the desired sensitive part towards environmental conditions. Moreover, the multi-valent system can form from grafting different polymers which may lead to significantly improved the efficiency of drug loading [7]. However, the permeability of the drug through the cell membrane in the drug loaded nanoparticles compared to the free drugs is lower.

Chitosan is one of the most biocompatible and biodegradable polymers with positively surface charge containing reactive groups (hydroxyl and amine) [8]. Because of the positively charged of chitosan, cellular uptake and cell adhesion of chitosan to negatively charged cell membranes are very high which is very favorable for the treatment of solid tumor [9]. Therefore, the carrier made of chitosan has been extensively used in pharmaceutical and biological applications. However, due to great mucoadhesive properties, the magnetic nanoparticles modified with chitosan in the process of blood circulation absorbed into normal cells [10]. Clearly, this process is absolutely inappropriate for a targeted drug delivery therapy. According to studies published so far, few studies have addressed this point to modify magnetic nanoparticles with chitosan.

Poly(ethylene glycol) (PEG) is a hydrophilic polymer which is widely used in in vitro and in vivo studies [11], [12]. By modifying the surface of magnetic nanoparticles with PEG, reticuloendothelial system (RES), enzymatic degradation, and toxicity of particles decreases and water solubility and stability of nanoparticles significantly improves, which leads to an increase in the circulation half-life in the body. In addition to the above-mentioned points, many scientists have focused on new approaches to modify iron nanoparticles via chitosan [13].

In this study, chitosan was used as pH-responsive section and PEG was utilized to improve RES and circulation half-life of the functionalized Fe₃O₄ nanoparticles. Many methods have been proposed to modify Fe₃O₄ nanoparticles with chitosan including co-precipitation, polymer microgel template, and blending procedures. Unfortunately, these methods result in reaching particle size to the micrometer scale and losing their application in intracellular drug delivery [14]. To overcome this problem, a new approach was adopted so that as-synthesized Fe₃O₄ nanoparticles were first modified with different kinds of linear and star-shaped PEG via Michael addition.

Ultrasound, as a novel technology, can lead to agitate nanoparticles in a solution media and reduce the size of particles which provide greater controllability on the morphology of the nanoparticles, especially on the magnetic specimens such as Fe₃O₄ [15], [16], [17]. Aggregated magnetic nanoparticles can be broken apart and well dispersed in the media.

In order to physically coat pH-responsive chitosan and loading 5-FU to synthesized Fe₃O₄@PEG, W/O/W multiple emulsion was applied which ultrasound irradiation plays an important role in this process. The double emulsions are prepared by a two-step emulsification process using two surfactants: a hydrophobic one (Span) designed to stabilize the interface of w/o internal emulsion and a hydrophilic one (Tween) for the external interface of the oil globules for w/o/w emulsions. Synthesized Fe₃O₄@PEG in W/O state was dispersed ultrasonically and added to the aqueous solution contained chitosan, thus, long-term stable chitosan coated Fe₃O₄@PEG (Fe₃O₄@Cs-PEG) nanoparticles are prepared through ultrasound treatment.

The chemico-physical properties of Fe₃O₄@Cs-PEG were characterized by Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), scanning electron microscope (SEM), and dynamic laser light scattering (DLS). The ability of Fe₃O₄@PEG/Cs as carrier to load and release of 5-Fu was investigated by in vitro measuring.