The performance of PEGylated nanocapsules of perfluorooctyl bromide as an ultrasound contrast agent

Abstract

The surface of polymeric nanocapsules used as ultrasound contrast agents (UCAs) was modified with PEGylated phospholipids in order to escape recognition and clearance by the mononuclear phagocyte system and achieve passive tumor targeting. Nanocapsules consisted of a shell of poly(lactide-co-glycolide) (PLGA) encapsulating a liquid core of perfluorooctyl bromide (PFOB). They were decorated with poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine (DSPE-PEG) incorporated in the organic phase before the solvent emulsification–evaporation process. The influence of DSPE-PEG concentration on nanocapsule size, surface charge, morphology, hydrophobicity and complement activation was evaluated. Zeta potential measurements, Hydrophobic interaction chromatography and complement activation provide evidence of DSPE-PEG presence at nanocapsule surface. Electronic microscopy reveals that the core/shell structure is preserved up to 2.64 mg of DSPE-PEG for 100 mg PLGA. In vivo ultrasound imaging was performed in mice bearing xenograft tumor with MIA PaCa-2 cells, either after an intra-tumoral or intravenous injection of nanocapsules. Tumor was observed only after the intra-tumoral injection. Despite the absence of echogenic signal in the tumor after intravenous injection of nanocapsules, histological analysis reveals their accumulation within the tumor tissue demonstrating that tissue distribution is not the unique property required for ultrasound contrast agents to be efficient.

Introduction

Ultrasonic imaging is a widely available, non-invasive and cost-effective diagnostic modality, but the weak difference of echogenicity between different tissues often hampers a clear diagnostic. In order to better visualize specific tissues, ultrasound contrast agents (UCA) are frequently used. The last generation of ultrasound contrast agents consists of gaseous perfluorocarbons (PFC) bubbles encapsulated within polymer shells [1], [2], [3], [4]. The combination of a fluorinated gas, displaying a low solubility in blood and a biodegradable and non toxic polymer, increases the plasmatic half-life of UCA (>5 min) [3], [4].

Recently, polymeric contrast agents have been developed [5], [6] substituting the gaseous PFC by a liquid one. These new UCAs consist of nanocapsules of poly(lactide-co-glycolide) (PLGA) encapsulating a liquid core of perfluorooctyl bromide (PFOB). They have shown promise for blood pool contrast [7]. Liquid PFOB yields capsules that are more resistant to pressure changes and mechanical stresses. In nanoemulsions, PFOB has been used as a blood pool UCA for the ventricular function and wall motion abnormalities [8], [9]. PLGA was chosen because it is approved by the FDA for use in sutures and drug delivery devices and several authors have reported it is biodegradable and has a good biocompatibility [10]. Additionally, the polymeric shell improved the stability of the capsules as compared to UCA stabilized by a monomolecular layer of surfactant [7].

However, because of their rather hydrophobic surface, these nanocapsules are quickly eliminated by the mononuclear phagocyte system and end up in the liver [11]. To avoid a rapid clearance from the systemic circulation, surface modification of these polymeric nanocapsules has been considered to render them stealth. The ultimate goal is to achieve passive tumor targeting taking advantage of the well-known enhanced permeation and retention effect (EPR) [12], [13]. It has been shown that covering particle's surface with poly(ethylene glycol) (PEG) is very efficient [14], [15]. By its hydrophilic nature PEG provides protection from blood proteins adsorption and uptake by the mononuclear phagocyte system is drastically reduced [16]. PEGylation can be achieved by different methods such as physical adsorption, covalent grafting or using PEG copolymers [17], [18], [19].

In a previous paper, an easy method based the work of Fahmy et al. [20] was developed to modify the surface chemistry of PLGA microcapsules encapsulating the PFOB using PEGylated phospholipids [21] Using the very same method, PFOB nanocapsules were decorated by PEGylated phospholipids in order to escape recognition and clearance by the mononuclear phagocyte system and achieve passive tumor targeting.