Superparamagnetic iron oxide – Loaded poly (lactic acid)-d-α-tocopherol polyethylene glycol 1000 succinate copolymer nanoparticles as MRI contrast agent

Abstract

We developed a strategy to formulate supraparamagnetic iron oxides (SPIOs) in nanoparticles (NPs) of biodegradable copolymer made up of poly(lactic acid) (PLA) and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) for medical imaging by magnetic resonance imaging (MRI) of high contrast and low side effects. The IOs-loaded PLA–TPGS NPs (IOs-PNPs) were prepared by the single emulsion method and the nanoprecipitation method. Effects of the process parameters such as the emulsifier concentration, IOs loading in the nanoparticles, and the solvent to non-solvent ratio on the IOs distribution within the polymeric matrix were investigated and the formulation was then optimized. The transmission electron microscopy (TEM) showed direct visual evidence for the well dispersed distribution of the IOs within the NPs. We further investigated the biocompatibility and cellular uptake of the IOs-PNPs in vitro with MCF-7 breast cancer cells and NIH-3T3 mouse fibroblast in close comparison with the commercial IOs imaging agent Resovist^®. MRI imaging was further carried out to investigate the biodistribution of the IOs formulated in the IOs-PNPs, especially in the liver to understand the liver clearance process, which was also made in close comparison with Resovist^®. We found that the PLA–TPGS NPs formulation at the clinically approved dose of 0.8 mg Fe/kg could be cleared within 24 h in comparison with several weeks for Resovist^®. Xenograft tumor model MRI confirmed the advantages of the IOs-PNPs formulation versus Resovist^® through the enhanced permeation and retention (EPR) effect of the tumor vasculature.

Introduction

Versatility in the use of supraparamagnetic iron oxide nanocrystals (SPIOs or IOs) has gained tremendous importance in the field of biomedical application. In cancer research, for example, IOs have been used for molecular imaging, tumor imaging, cancer hyperthermia therapy, and other techniques [1], [2], [3], [4]. IOs have been extensively investigated as a contrast agent for MRI, which are easily assimilated by the human body [5], [6], [7]. IOs are primarily used in MRI as liver signal erasers since they create a reduction in signal intensity in the reticuloendothelial tissues, in which they are greatly concentrated and thus result in greater T2 shortening, to produce more conspicuous area of liver not containing the reticuloendothelial tissues. However, long term retention of IOs in liver is a concern for the side effects of IOs imaging agent [8]. Several other side effects are also associated with administering of the IOs, which may create clinical complications [9].

Such problems could be addressed by two strategies. One is by coating of the IOs core and another is by formulating of the IOs in nanoparticles (NPs) of biodegradable polymers. Various coating techniques have been suggested to provide IOs with functionality of desired surface properties. For instance, thermally sensitive coatings provide the IOs with dual functionality for treatment of cancer by hyperthermia and through a release of the entrapped anti-cancer agent [10]. In another study, IOs with their surface coated with PEG and further linked to biotin or neutravidin along with a peptide enabled detection of specific cancer type that produces a proteolytic enzyme which enabled the peptide blocked biotin and neutravidin to self assemble and thus influences the T2 relaxivity in MRI. Such a coating strategy thus enables detection of the tumor as well as specification of the tumor type [11]. Moreover, coating IOs by hydrophilic or amphiphilic biodegradable polymer provides them with other functionality such as biocompatibility and long circulation in plasma.

Formulation of the IOs in nanoparticles (NPs) can be realized by using various FDA-approved biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) and various novel biodegradable copolymers such as poly(lactic acid)-poly(ethylene glycol) (PLEA) copolymer and poly(lactic acid)-d-α-tocopherol polyethylene glycol 1000 succinate (PLA–TPGS) copolymer [12], [13], [28]. Encapsulation by using preformed polymer can be done by several methods such as the micro/nanoemulsion method [14], [15], the nanoprecipitation method [16], [17] and the micelle formation [18], [19], [20]. Emulsion polymerization makes use of monomers that polymerize over the encapsulant, which is stabilized by using a surfactant [21], [22], [23], which is of great concern due to the presence of certain chemical precursors that were not removed and could have some cytotoxic effect [24]. The solvent displacement technique was used in the preparation of nanocapsules [24]. The nanocapsules were formed when the non-solvent replaces the solvent in the presence of a surfactant followed by deposition of the preformed polymer at the interface. The encapsulant was remained in an oily phase within the polymer layer. This process has been extended to the formation of nanospheres without the surfactant [16] and it has been gaining increasing importance over time for preparing nanoparticles. Various parameter optimization of this method has been done. For instance, it was suggested that the diffusion of the solvent into the non-solvent results in the phase transformation of the polymer and thus in the formation of the nanoparticles [25]. It was also shown how the nanoparticles formation could be affected by changing the concentration of the polymer in the organic phase or the concentration of the surfactant in the aqueous phase, which may influence the size of the formed nanoparticles [26]. Other research in the nanoprecipitation method included the choice of the solvent [27]. It was suggested that the solubility parameter of the solvent and the non-solvent system strongly influence the particle formation, in which the particle size was usually used as the primary criteria for optimization in the literature.

In the present research, we developed a strategy to formulate IOs in the NPs of PLA–TPGS copolymer, for enhanced MRI of higher image contrast and less system side effects. The amphiphilic nature and enhanced biocompatibility of this copolymer make it promising for nanoparticle delivery of diagnostic and therapeutic agents [28], [29]. We have exploited the different treatment between drug and the IOs in the nanoparticle encapsulation process. A crystalline drug after encapsulation could become in an amorphous state, resulting in approved solubility [30], [31], [32], whereas the IOs do not undergo a phase transition after encapsulation process. Rather these nanoparticles get entrapped within the polymeric matrix and retain their crystalline state. Thus diffusion of the solvent into the polymeric matrix plays a key role during the preparation of the nanoparticle encapsulation.

Two commonly used methods for polymeric nanoparticle preparation, namely the single emulsion method and the nanoprecipitation method, are employed in this research for the nanoparticle preparation in close comparison with optimization of the various synthesis parameters such as the emulsifier concentration in the aqueous phase, solvent/non-solvent ratio, loading of IOs/drug and more. The effect of synthesis parameters on the size and size distribution of the polymeric nanoparticles and the distribution of IOs within the polymeric matrix were studied by laser light scattering and transmission electron microscopy (TEM). The optimally synthesized nanoparticles were further investigated for in vitro cytotoxicity and cellular uptake of the IOs-loaded nanoparticles in close comparison with the commercial Resovist^®. Due to their desired particles size and their long half-life in the plasma, the IOs-loaded PLA–TPGS nanoparticles should have the property to accumulate in regions of leaky and high permeable vasculature and lymphatic vessels such as those in tumor, which is also referred to as the enhanced permeability and retention (EPR). The nanoparticle formulation of IOs can thus be exploited for passively targeted delivery of the imaging agent for clinical MRI with better contrast and less side effects [33].