European Journal of Pharmaceutics and Biopharmaceutics
Research paperPerfluorocarbon-loaded lipid nanocapsules as oxygen sensors for tumor tissue pO2 assessment
Graphical abstract
Influence of component weight proportion on the PFCE-loaded lipid nanoparticles properties and in vitro/in vivo sensitivity of the formulated sensor to pO2.
Introduction
Hypoxia is a characteristic feature of locally-advanced solid tumors resulting from an imbalance between oxygen supply and consumption [1]. Hypoxia has been shown to play a key role in tumor resistance to radiation therapy [2] and also in decreasing the efficacy of certain cytotoxic drugs [3]. Numerous studies have demonstrated that tumor hypoxia, in addition to diminishing therapeutic efficacy, plays a major role in malignant progression [4].
Methods to determine tumor oxygenation are therefore of crucial importance for the prediction of therapeutic outcomes. Many current methods, such as Eppendorf electrodes [5], near-infrared spectroscopy [6], electron paramagnetic resonance imaging [7], susceptibility magnetic resonance imaging based methods [8] or 19F magnetic resonance imaging oximetry [6], [9], have been used in preclinical studies but have either been highly invasive, non-quantitative, or have lacked spatial resolution. As far as clinically available devices are concerned, MR-based techniques may be of interest, whereby proton-based-imaging can be used to access blood oxygen change using the BOLD (Blood Oxygen Level–Dependent) sequence [8], currently widely used in clinics for functional magnetic resonance imaging (fMRI) studies. Nevertheless, this imaging technique is based on relaxivity changes that allow the depiction of oxygenation variations, but it does not enable the quantitative assessment of oxygen concentration [10]. However, recent studies have shown a high correlation between oxygen changes assessed using BOLD-imaging and oxygen levels measured using 19F-MRI of a fluorinated oxygen probe in a preclinical model [11].
Currently, the major limitation of 19F-oximetry is the lack of equipment of clinical MR to perform fluorine imaging. However, such a limitation can easily be removed since it only requires the acquisition of a specific 19F-tuned probe. Fluorine oximetry is based on the influence of oxygen tension on the relaxivity of certain fluorinated probes. In the early 1980s, Clark et al. [12] showed a linear relation between longitudinal relaxivity of fluorinated probes with oxygen tension; this opened the field for 19F-MR oximetry [13], [14], [15]. Numerous fluorine probes were tested as oxygen sensors, and numerous protocols have been proposed to optimize the injection of the fluorinated probes. Zhao et al. proposed a multiple deposit of the probe along three orthogonal directions in order to ensure that the interrogated regions are representative of the whole tumor [16]. However, as reported by these authors [11], the deposit induced signal voids on the anatomical proton images that can jeopardize the information that can be extracted from the anatomical data set.
To overcome this drawback, we propose the development of lipid nanocapsules to carry the fluorinated probe. Lipid nanocapsules are engineered using FDA-approved constituents (i.e., Solutol and Lipoid) that are prepared by a solvent-free and low-energy process [17], they are able to deliver drugs to a target [18] and, amazingly, they are characterized by an intrinsic effect on Multi-Drug-Resistance via the inhibition of P-gp [19]. According to the preparation process used, this nano-scaled platform can encapsulate hydrophilic [20] and lipophilic [21] compounds and therefore can encapsulate the lipophilic fluorinated O2 probe, the perfluoro-15-crown-5-ether (C10F20O5) in this case. This compound was chosen since all twenty fluorines are MR equivalent and therefore provide a single and intense resonance [22]. The second advantage of lipid nanocapsules is that they can be efficiently and homogeneously infused in tumors using the convection-enhanced delivery (CED) technique [23], a technique initially introduced in the early 1990s [24] to deliver macromolecules to the brain but which is nowadays used in clinics to improve tumor therapy [25].
Section snippets
Materials
Lipoïd® S75-3 (soybean lecithin – 69% phosphatidylcholine and 10% phosphatidylethanolamine) and Solutol® HS15 were supplied by Lipoïd GmbH (Ludwigshafen, Germany) and BASF (Ludwigshafen, Germany), respectively. Perfluoro-15-crown-5-ether (C10F20O5) was provided by Chemos GmbH (Regenstauf, Germany). NaCl was purchased from Prolabo (Fontenay-sous-bois, France). Deionized water was obtained from a Milli-Q plus® system (Millipore, Bilerica, USA). I2 and KI were supplied by Sigma Aldrich Chemie GmbH
PFCE-LNC formulation
The morphology of LNC results in an oily core surrounded by lecithins and a non-ionic surfactant [34]. The formulation protocol is based on a phase-inversion process due to the specific properties of the non-ionic surfactant, PEG-HS present in Solutol. Then, PEG-HS gives an oil-in-water emulsion (O/W) at room temperature and a water-in-oil emulsion (W/O) at high temperature [17].
This inversion process was tested to obtain PFCE nanocapsules (PFCE-LNC), using PFCE as the oil in the formulation.
Conclusions
Nanoscaled platform capable of measuring brain tumor pO2 using fluorine MRI was synthesized. Despite the high density and the high lipophilicity of the fluorinated pO2 sensor, that is, perfluoro-15-crown-5-ether, the low-energy, phase-inversion process used to produce lipid nanocapsules could be implemented. The resulting nanocapsules are composed of a perfluoro-15-crown-5-ether core surrounded by a chaotic arrangement of polyethylene glycol hydroxystearate that does not interfere with O2
Acknowledgements
Authors acknowledge financial support from “Comité Inter-Régional Grand Ouest de La Ligue Contre le Cancer” – CIRGO and “La Ligue nationale contre le cancer – Equipe Labellisée 2012”. Authors would like to thank Dr. David Rees for editing the manuscript.
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