Microbubbles decorated with dendronized magnetic nanoparticles for biomedical imaging: effective stabilization via fluorous interactions

Abstract

Dendrons fitted with three oligo(ethylene glycol) (OEG) chains, one of which contains a fluorinated or hydrogenated end group and bears a bisphosphonate polar head (CnX2n+1OEG8Den, X = F or H; n = 2 or 4), were synthesized and grafted on the surface of iron oxide nanoparticles (IONPs) for microbubble-mediated imaging and therapeutic purposes. The size and stability of the dendronized IONPs (IONP@CnX2n+1OEG8Den) in aqueous dispersions were monitored by dynamic light scattering. The investigation of the spontaneous adsorption of IONP@CnX2n+1OEG8Den at the interface between air or air saturated with perfluorohexane and an aqueous phase establishes that exposure to the fluorocarbon gas markedly increases the rate of adsorption of the dendronized IONPs to the gas/water interface and decreases the equilibrium interfacial tension. This suggests that fluorous interactions are at play between the supernatant fluorocarbon gas and the fluorinated end groups of the dendrons. Furthermore, small perfluorohexanestabilized microbubbles (MBs) with a dipalmitoylphosphatidylcholine (DPPC) shell that incorporates IONP@CnX2n+1OEG8Den (DPPC/Fe molar ratio 28:1) were prepared and subsequently characterized using both optical microscopy and an acoustical method of size determination. The dendrons fitted with fluorinated end groups lead to smaller and more stable MBs than those fitted with hydrogenated groups. The most effective result is already obtained with C2F5, for which MBs of ≈1.0 μm in radius reach a half-life of ≈6.0 h. An atomic force microscopy investigation of spin-coated mixed films of DPPC/IONP@C2X5OEG8Den combinations (molar ratio 28:1) shows that the IONPs grafted with the fluorinated dendrons are located within the phospholipid film, while those grafted with the hydrocarbon dendrons are located at the surface of the phospholipid film. Beilstein J. Nanotechnol. 2019, 10, 2103–2115. 2104 Introduction Microbubbles (MBs), that is, micrometer-sized gas particles dispersed in an aqueous medium, are clinically used as contrast agents for ultrasound imaging, including molecular imaging, and actively investigated for surgical ablation, targeted drug and gene delivery [1-10]. They are also being examined for use, in conjunction with focused ultrasound, and under magnetic resonance imaging guidance, for achieving blood/brain and blood/ tumor barrier crossing of drugs [11,12]. Medical MBs have a shell consisting of surfactants, phospholipids, or polymers and are usually stabilized by a fluorocarbon gas [13] that acts as an osmotic agent [14,15] and as a co-surfactant to phospholipids [16] and block co-polymers [17]. Nanoparticles can be attached to the bubble shells to extend their diagnostic and therapeutic potential by combining multimodal imaging, drug or gene delivery, and/or enhancement and control of the acoustic signal for energy deposition, as is required for sonothrombolysis or ablation surgery. MBs incorporating iron oxide nanoparticles (IONPs) are sought after as dual contrast agents for ultrasound and magnetic resonance imaging [18-20] and drug delivery [21,22]. The shells of the presently available MBs that incorporate IONPs are often made of polymers. For example, ultrasmall superparamagnetic iron oxide nanoparticles were embedded in the wall of poly(butyl cyanoacrylate)-based MBs, allowing the blood‒brain barrier penetration to be monitored [23]. Soft-shell colloids called lipospheres have also been reported for enhanced gene and drug delivery [24]. These lipospheres consist of gas-filled spheres coated by a film of soybean oil that encases the cargo of nanoparticles and is itself contained within a film of phospholipids [24]. Both polymer-shelled MBs and lipospheres have some advantages and some limitations [25]. In both cases, the shells can be custom-made to enhance stability, circulation duration, drugloading capacity and release rate, targeting the fusion with cell membranes [24]. Both types of constructs are generally more stable, but less echogenic than “true” gas microbubbles, due to the dampening effect of the polymer shell or oil contained in the phospholipid coating [24,25]. One important difficulty encountered in the preparation of such magnetic MBs is that IONPs rapidly aggregate in aqueous media [25]. Commercially available 50 nm magnetic IONPs coated with phospholipids allowed for the preparation of MBs that enabled transfection of neuroblastoma cells with a generic, fluorescent, small, interfering RNA under magnetic and ultrasound fields [26]. In the present work, we incorporated IONPs coated by dendritic phosphonates bearing oligo(ethylene glycol) (OEG) chains into the phospholipid shell of the MBs. OEG chains were selected in order to improve the dispersibility of the IONPs in water [27,28]. Dendritic phosphonates are effective anchoring agents Figure 1: a) Molecular structure of the dendrons investigated (CnX2n+1OEG8Den, X = F or H; n = 2 and 4); b) Schematic representation of a dendronized IONP showing the anchoring of the bisphosphonate function on the iron oxide. due to the covalent PO–metal bonds that stabilize aqueous dispersions of IONPs [27,29]. Such dendronized IONPs have been investigated for hyperthermia and magnetic resonance imaging owing to their increased stability in aqueous media and biocompatibility [27,28]. An even stronger anchoring agent consisting of a dendron structure bearing a bisphosphonate polar head provided increased colloidal stability in physiological media [30]. To the best of our knowledge, the implementation of dendronized IONPs in phospholipid-shelled MBs has not yet been reported. This approach is expected to combine some advantages over existing methods, including the ability to graft isolated IONPs instead of clusters at the MB surface, and allowing the microbubbles to go undetected, thus potentially minimizing the recourse to pegylated lipids. We report here the preparation of perfluorohexane (F-hexane)stabilized MBs with a shell of dipalmitoylphosphatidylcholine (DPPC) that incorporates IONPs grafted with OEG bisphosphonate-headed dendrons. Four dendrons were synthesized and investigated that feature two phosphonic acids and three OEG chains, including a longer one in the para position. The latter was fitted with a fluorinated (C2F5 or C4F9) or a hydrogenated (C2H5 or C4H9) end group (CnX2n+1OEG8Den, X = F and H; n = 2 and 4, Figure 1). First, we present the synthesis and the characterization of the IONPs grafted with the selected dendrons (IONP@CnX2n+1OEG8Den). Second, we report the adsorption kinetics of IONP@CnX2n+1OEG8Den at the interface between air or F-hexane-saturated air and water. Third, we discuss the size and stability characteristics of Beilstein J. Nanotechnol. 2019, 10, 2103–2115. 2105 Figure 2: Hydrodynamic diameter distributions of IONPs grafted with dendrons: C2H5OEG8Den (38 ± 1 nm, black), C2F5OEG8Den (37 ± 1 nm, red), C4H9OEG8Den (95 ± 12 nm, orange), C4F9OEG8Den (197 ± 15 nm, blue) in aqueous dispersions (Fe conc. 0.05 mg mL−1). F-hexane-stabilized DPPC-shelled MBs incorporating IONP@CnX2n+1OEG8Den. Fourth, we report an atomic force microscopy (AFM) study that reveals that the location of the dendronized nanoparticles in the phospholipid film strongly depends on the nature of the terminal group. Results and Discussion Synthesis and grafting of dendrons on iron oxide nanoparticles The OEG dendrons were synthesized as described in the Experimental section. Briefly, the piperazine scaffold was selected as an appropriate template to introduce the perfluoroalkylated or alkylated chain on a generation 1 bisphosphonic dendron bearing three OEG chains [31]. In order to facilitate the insertion and increase the visibility of the perfluoroalkylated (or alkylated) end group in the microbubble wall, the central OEG chain carrying the piperazine moiety was lengthened (Figure 1a). A multistep chemical sequence allowed for the production of bisphosphonate dendrons at a reasonable yield (55–80%). IONPs (mean diameter of 9.0 ± 0.9 nm) were synthesized by thermal decomposition of iron (II) stearate in the presence of oleic acid in dioctyl ether, which enables better control of the size, morphology and composition of the IONPs [32]. The four dendrons were grafted on the magnetic IONPs by direct exchange of the ligand (oleic acid) according to [33]. The excess dendron material was removed by ultrafiltration. The grafting of the dendrons on the IONPs was assessed by infrared spectroscopy (IR), which showed a significant reduction of the oleic acid alkyl bands (2926‒2850 cm−1) and the appearance of the OEG signal (1096 cm−1) (Supporting Information File 1, Figure S1). The grafting of the dendrons on the IONPs was also confirmed by dynamic light scattering (DLS, Figure 2). The hydrodynamic mean diameter of IONP@C2X5OEG8Den (X = F or H) was ≈37 nm, which is significantly larger than the mean diameter of the oleic-acid-covered IONPs (≈10 nm, Supporting Information File 1, Figure S2). This can be ascribed to the fact that a corona of OEG chains is now present around the nanoparticle and captures molecules of water, which contributes to a further increase of the hydrodynamic radius. The mean diameter of the IONP@C4X9OEG8Den materials was larger, namely ≈95 nm and ≈200 nm for X = H and F, respectively, revealing that aggregation occurs in aqueous media due to the hydrophobicity of the end group. Fortunately, this did not preclude performing the adsorption kinetics studies. Altogether, owing to their dendritic structure, the OEG chains were found to confer excellent dispersibility and stability to the IONPs [33]. Adsorption kinetics of dendronized nanoparticles at the gas/liquid interface The adsorption of the dendronized IONPs at the air/water and F-hexane-saturated air/water interface was first investigated using bubble profile analysis tensiometry. As described in our earlier reports [34,35], we first confirmed that F-hexane taken alone, when introduced into the