Elsevier

Acta Biomaterialia

Volume 66, 15 January 2018, Pages 200-212
Acta Biomaterialia

Full length article
A new formulation of poly(MAOTIB) nanoparticles as an efficient contrast agent for in vivo X-ray imaging

https://doi.org/10.1016/j.actbio.2017.11.011Get rights and content

Abstract

Polymeric nanoparticles (PNPs) are gaining increasing importance as nanocarriers or contrasting material for preclinical diagnosis by micro-CT scanner. Here, we investigated a straightforward approach to produce a biocompatible, radiopaque, and stable polymer-based nanoparticle contrast agent, which was evaluated on mice. To this end, we used a nanoprecipitation dropping technique to obtain PEGylated PNPs from a preformed iodinated homopolymer, poly(MAOTIB), synthesized by radical polymerization of 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) monomer (MAOTIB). The process developed allows an accurate control of the nanoparticle properties (mean size can range from 140 nm to 200 nm, tuned according to the formulation parameters) along with unprecedented important X-ray attenuation properties (concentration of iodine around 59 mg I/mL) compatible with a follow-up in vivo study. Routine characterizations such as FTIR, DSC, GPC, TGA, 1H and 13C NMR, and finally SEM were accomplished to obtain the main properties of the optimal contrast agent. Owing to excellent colloidal stability against physiological conditions evaluated in the presence of fetal bovine serum, the selected PNPs suspension was administered to mice. Monitoring and quantification by micro-CT showed that iodinated PNPs are endowed strong X-ray attenuation capacity toward blood pool and underwent a rapid and passive accumulation in the liver and spleen.

Statement of Significance

The design of X-ray contrast agents for preclinical imaging is still highly challenging. To date, the best contrast agents reported are based on iodinated lipids or inorganic materials such as gold. In literature, several attempts were undertaken to create polymer-based X-ray contrast agents, but their applicability in vivo was limited to their low contrasting properties. Polymer-based contrast agents present the advantages of an easy surface modification for future application in targeting. Herein, we develop a novel approach to design polymer-based nanoparticle X-ray contrast agent (polymerization of a highly iodine-loaded monomer (MAOTIB)), leading to an iodine concentration of 59 mg/mL. We showed their high efficiency in vivo in mice, in terms of providing a strong signal in blood and then accumulating in the liver and spleen.

Introduction

Over the past decades, many advances have been made in the early-stage pathology diagnostic field by non-invasive in vivo visualizations over a wide imaging scale, from cells to human whole-body. Currently, the medicine field possesses an arsenal of biomedical imaging tools that prevent patients from undergoing an unpleasant experience or surgery to obtain an accurate diagnosis. Regardless of the imaging modalities used [1], [2], [3], [4], such as X-ray computed tomography (X-ray CT), magnetic resonance imaging (MRI), positron emission tomography (PET) or single photon emission computed tomography (SPECT), in vivo imaging techniques aim to provide reconstructed 2D or 3D images where morphology, anatomy, and physiologic functions of a region of interest (ROI) can be explored and examined. Although all these imaging techniques contribute to reveal pathologic tissues among the healthy ones, each technique differ from the other with regard to their principle, resolution, sensitivity, depth of tissue penetration, quantification, etc., which make them quite complementary [1], [2], [3], [4].

Historically, X-ray imaging is the oldest imaging modality, which was considerably developed by Cormark and Hounsfield and has been applied to preclinical research and medicine [5], [6], [7], [8], [9], [10]. CT scanners appear to be a good compromise between low cost of operation, efficiency for achieving high resolution images, low energy consumption, and easy availability compared to other imagers. Contrast agent-free CT imaging is devoted to skeleton visualization, and with the help of a particulate contrast agent, the technique can be extended to soft tissue imaging (e.g. gastrointestinal tract, kidneys, liver, spleen), cardiorespiratory system, head, bones, and tumor tissues [1], [6], [8], [10], [11], [12]. In general, these tools remain complex and limited to preclinical imaging [13], [14].

It is well known that X-ray images of soft tissues suffer from a lack of contrast enhancement due to their similar X-ray absorption ability. To overcome this limitation, radiopaque contrast agents (X-ray CA) are adminstered. Current X-ray CAs are composed of low-molecular-weight water-soluble molecules. However, they are not compatible with X-ray imaging application in small laboratory animals due to fast excretion by renal clearance and the limitation of injecting high volume due to the small blood volume of animal. This implies the use of concentrated dose of X-ray CA to ensure contrast enhancement during image-guided procedure, which potentially causes nephrotoxicity and high viscosity and osmolality. Literature already showed the scope of CA alternatives for micro-CT, including nanoparticles (NPs) of inorganic elements [7] such as noble metal (gold, silver, platinum), heavy metal (thorium, bismuth, tantalum, etc.), or lanthanide (gadolinium, ytterbium, etc.) and soft NPs made of lipids [4] (nano-emulsion, micelle, liposome, emulsion) or polymeric materials (nanosphere, nanocapsule, dendrimer) in which active compounds can be encapsulated or embedded.

Soft NPs were actually found to be an appealing material for X-ray probes because: i) there is a wide range of biocompatible designs and chemical constituents safer than metallic NPs that are suitable for in vivo environment and ii) they provide a protective shield surrounding the contrasting core and thus prevent cargo from leaking and from fast clearance [4], [6], [7], [8], [9], [10], [11], [12], [15], [16], [17], [18], [19]. Typically, NPs-based X-ray CAs must fulfill additional requirements to constitute a real advanced type of X-ray CA [1], [4], [8], [16], [19], [20], [21]. They should have a core exhibiting a high attenuation ability compared to the dose injected by means of loading molecules with a high content of contrasting element. This will reduce osmolality and viscosity close to those of physiologic fluids, thus rendering the intravenous injection more tolerable [7]. They should have a specific biodistribution (which will avoid incompatibility with concentrated CA formulation) and good pharmacokinetics profile (longer retention time to enable imaging and possible excretion of CA without an adverse effect from CA or from its degradation products) [22], [23], [24], [25], [26]. Basically, in vivo NPs fate is dictated by physicochemical properties, mostly surface and size. These are considered as two key parameters because stealth surface functionality obtained by decoration with PEG, active targeting properties obtained by decoration with specific ligands, and a size smaller than 200 nm will avoid vessel embolism and would reduce the opsonization of NPs by escaping the immune system and preventing excretion by renal clearance. This size range would ensure stability of NPs in physiological media until they reach and accumulate in the targeted tissues [22], [23], [24], [25], [26], [27], [28], [29], [30], [31].

Among all the above mentioned nanoscale assemblies, polymeric nanoparticles (PNPs) are promising NPs-based CA because of the control over their architectures – including morphology, size, surface charge, functionalities and rigidity – and their compositions, imparting proper bioavailability, biostability and long persistence in systemic compartment and generating high-loaded iodine biomaterials [2], [32], [33]. There are several synthesis and formulation techniques that allow preparation of PNPs-based probes: these include heterogeneous polymerization process (in emulsion, suspension, or dispersion) to yield in situ PNPs and post-synthesis process (emulsion solvent -diffusion, -evaporation, salting out, solvent displacement, supercritical fluid) for bulk of preformed synthetic polymers and biopolymers [34], [35], [36], [37], [38]. Over the last couple of decades, researchers have focused their attention on the preparation of iodinated biodegradable [39], [40] and/or biocompatible [41], [42], [43], [44] PNPs and macromolecules as X-ray probes.

Unfortunately, there is still a need in determining how to obtain a balance between high iodine content on polymer structure and suitable design to aim at the preparation of an injectable and efficient X-ray CA. Radiopaque PNPs from the polymerization of iodinated derivatives of 2-hydroxyethylmethacrylate (HEMA) monomer, such as 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) monomer (MAOTIB) with an iodine content of 62 wt%, seem to be an outstanding alternative to achieve this goal. Most of the studies on this MAOTIB monomer relied on heterogeneous polymerization and bulk process, leading to micron-sized particles or copolymeric NPs with consequently low iodine loading on macromolecule backbone and slight in vivo contrast enhancement [45], [46], [47], [48], [49], [50]. Therefore, Poly(MAOTIB) appears to be a good candidate for the synthesis of X-ray CA, but is yet to be optimized and used for that purpose.

In the present paper, we investigated a straightforward approach to produce biocompatible, radiopaque, and controllable size distribution polymer-based nanospheres as X-ray micro-CT CA. In addition, in contrast with the research in the field of polymer-based X-ray CA, the current study show its applicability in vivo with high contrast measured in blood and soft tissues. To this end, we used an adapted nanoprecipitation dropping technique to obtain PEGylated PNPs from a preformed iodinated homopolymer, poly(MAOTIB), synthesized by radical polymerization of the MAOTIB monomer. As mentioned in the literature, since the development of nanoprecipitation or solvent displacement method by Fessi et al. [51], many authors have studied this technique because it is a low cost, simple, and reproducible method [35], [52], [53]. Furthermore, nanoprecipitation is reported to be very efficient technique to formulate monodisperse and nanoscale colloids by altering key parameters such as the polymer and surfactant weight ratio. In our case, it appears as the most suitable method to enable the balancing of size distribution and iodine content. In the present study, we adapted the dropping technique (Fig. 1) in which an organic solution of our polymer is poured dropwise in a non-solvent containing the surfactant. After diffusion of the solvents in each other, hydrophilic poly(MAOTIB) NPs were obtained because of a PEGylated stabilizer coating. PEGylated surfactant was used to control the size, stealth and stabilizing properties for in vivo media. The main novelty of the work comes from the innovative formulation and optimization of poly(MAOTIB); thus, it can be applied efficiently as a micro-CT CA, thereby allowing to conduct the in vivo study of its biodistribution and pharmacokinetics.

In a nutshell, the strength of such PNPs formulation lies not only on its X-ray attenuation properties but also on the control of the design of PNPs to impart suitable physicochemical features for in vivo use. Formulation parameters such as component amount, phase volume ratio, and stirring have been reported to severely affect the size distribution of PNPs. Consequently, in the present study, the role of polymer loading as the inner core of the nanoconstruct and the surfactant-to-polymer weight ratio were studied and elucidated to optimize the nanoprecipitation process and identify the best alternative between high iodine content within the injectable PNPs suspensions, size distribution and colloidal stability. Several poly(MAOTIB)-based PNPs were thus produced with iodine concentration from 15.5 to 62 mg I/mL and with the surfactant-to-polymer weight ratio varying from 30 to 80 wt%. Discrimination among all suspensions relied on iodine content needed for good X-ray attenuation ability and on size distribution measurement by DLS. An optimal suspension was subjected to further characterization such as SEM and in vitro study under physiological conditions to assess iodine content, cell interactions, and colloidal stability after exposure to biological media. These studies revealed that the formulation was composed of 163 nm spherical PNPs with 59 mg I/ml with an excellent performance in simulated in vivo media. In vivo assays on mice after intravenous administration indicated that the passive accumulation drove PNPs into hepatic and splenic compartments in which adequate opacification allowed clear delineation of both soft tissues. Complete micro-CT follow-up was carried out to evaluate pharmacokinetics profile of the selected suspension.

Section snippets

Materials

All chemicals were commercially available agents and were used as received. 2,3,5-Triiodobenzoic acid (TIB), 4-dimethylaminopyridine (DMAP), 2-hydroxylethyl methylmethacrylate (HEMA), benzoyl peroxide (Luperox® A75), triethylamine (NEt3), thionyl chloride, cyclohexane, dichloromethane (DCM), ethyl acetate, methanol, tetrahydrofuran (THF), and toluene were purchased from Sigma-Aldrich (France). Non-ionic surfactant (Kolliphor ELP® Castor oil PEG-35) was donated by Laserson (Etampes, France).

Results and discussion

After syntheses and purification, poly(MAOTIB) was formulated into PNPs through the nanoprecipitation process. As mentioned in Table 1, three quantities of polymer and six SPR were used to render three series of suspensions, namely PNPs-1, PNPs-2, and PNPs-3, with their own theoretical iodine content (see Section 2.2.3), and thus radio-opacity ability, from 15.5 to 31 and finally to 62 mg of Iodine/mL with specific size distributions depending on the amount of surfactant involved.

Before

Conclusion

This study presents the preparation of a biocompatible injectable CA for X-ray micro-CT imaging of soft tissues. A novel method based on nanoprecipitation was used to form hydrophilic and stealth nanoparticles from poly(MAOTIB) homopolymer produced by bulk radical polymerization of the MAOTIB monomer (62 wt% of the molecular weight is iodine). The effects of polymer and PEGylated surfactant weight ratio and the polymer content were elucidated to determine the best alternative between iodine

Acknowledgements

The authors acknowledge the ICS Characterization Platform for the use of ATG and DSC and thank Mrs. M. Legros and C. Saettel for the analyses. In vivo imaging of this study was performed on the imaging facilities of CERMEP-Imagerie du vivant, Bron, F-69677, France. The authors thank the technical staff of the platform.

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