Acoustic Droplet Vaporization of Perfluorohexane Emulsions Induced by Heterogeneous Nucleation at an Ultrasonic Frequency of 1.1 MHz.

Droplets made of liquid perfluorocarbon undergo a phase transition and transform into microbubbles when triggered by ultrasound of intensity beyond a critical threshold; this mechanism is called acoustic droplet vaporization (ADV). It has been shown that if the intensity of the signal coming from high ultrasonic harmonics are sufficiently high, superharmonic focusing is the mechanism leading to ADV for large droplets (>3 µm) and high frequencies (>1.5 MHz). In such a scenario, ADV is initiated due to a nucleus occurring at a specific location inside the droplet volume. But the question on what induces ADV in the case of nanometer-sized droplets and/or at low ultrasonic frequencies (<1.5 MHz) still remains. We investigated ADV of perfluorohexane (PFH) nano- and microdroplets at a frequency of 1.1 MHz and at conditions where there is no superharmonic focusing. Three types of droplets produced by microfluidics were studied: plain PFH droplets, PFH droplets containing many nanometer-sized water droplets, and droplets made of a PFH corona encapsulating a single micron-sized water droplet. The probability to observe a vaporization event was measured as a function of acoustic pressure. As our experiments were performed on droplet suspensions containing a population of monodisperse droplets, we developed a statistical model to extrapolate, from our experimental curves, the ADV pressure thresholds in the case where only one droplet would be insonified. We observed that the value of ADV pressure threshold decreases as the radius of a plain PFH droplet increases. This value was further reduced when a PFH droplet encapsulates a micron-sized water droplet, while the encapsulation of many nanometer-sized water droplets did not modify the threshold. These results cannot be explained by a model of homogeneous nucleation. However, we developed a heterogeneous nucleation model, where the nucleus appears at the surface in contact with PFH, that successfully predicts our experimental ADV results.

Ultrasound-triggered delivery of paclitaxel encapsulated in an emulsion at low acoustic pressures.

We investigated the in vitro ultrasound-triggered delivery of paclitaxel, a well known anti-cancerous drug, encapsulated in an emulsion and in the presence of CT26 tumor cells. The emulsion was made of nanodroplets, whose volume comprised 95% perfluoro-octyl bromide and 5% tributyl O-acetylcitrate, in which paclitaxel was solubilized. These nanodroplets, prepared using a high-pressure microfluidizer, were stabilized by a tailor-made and recently patented biocompatible fluorinated surfactant. The delivery investigations were performed at 37 °C using a high intensity focused ultrasound transducer at a frequency of 1.1 MHz. The ultrasonic pulse was made of 275 sinusoidal periods and the pulse repetition frequency was 200 Hz with a duty cycle of 5%. The measured viabilities of CT26 cells showed that paclitaxel delivery was achievable for peak-to-peak pressures of 0.4 and 3.5 MPa, without having to vaporize the perfluorocarbon part of the droplet or to induce inertial cavitation.

Perfluorocarbon nanodroplets stabilized by fluorinated surfactants: characterization and potentiality as theranostic agents.

We aim to produce emulsions that can act as contrast agents and drug carriers for cancer imaging and therapy. To increase tumor detection and decrease drug side effects, it is desirable to take advantage of the enhanced permeability and retention effect that allows nanoparticles to accumulate in tumor tissues. To do so, the emulsion droplets need to be small enough and stable over time in addition to enhancing image contrast and carrying a drug payload. In the present study, we have investigated the properties and potentiality as theranostic agents of perfluorocarbon emulsions stabilized by a biocompatible fluorinated surfactant called FTAC. To obtain better control of our system, the synthesis of those surfactants was studied and their physico-chemical properties were explored in different configurations such as micelles, in the perfluorocarbon droplet shell and at water/air and water/perfluorocarbon interfaces. The originality of this work lies in the determination of numerous characteristics of emulsions and fluorinated surfactants including surface tension, interfacial tension, critical micelle concentration, adiabatic compressibility, density, size distribution (aging studies), and ultrasonic echogenicity. These characterization studies were conducted using different types of FTAC and several perfluorocarbons (perfluoropentane, perfluorohexane, and perfluorooctyl bromide). We have also shown that a hydrophobic drug could be encapsulated in the FTAC-stabilized perfluorocarbon droplets thanks to triacetin addition. Finally, the perfluorocarbon emulsions were detectable in vitro by a clinical 3 T MRI scanner, equipped with a double frequency 19F/1H transmit-receive coil.

Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p<0.001, n=3×4), with evidence of toxicity only at MRs 50-100. Nebulization of SC12CDClickpropylamine nanocomplexes using the Aeroneb(®) Pro resulted in VMDs of ∼4 µm and FPFs of ∼57% at all MRs. SC12CDClickpropylamine-siRNA-mediated luciferase knockdown was found to be 39.8±3.6% at MR=20 before and 35.6±4.55% after nebulization, comparable to results observed using unnebulized commercial transfection reagent, RNAiFect. CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics.