In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets.

OBJECTIVE: Phase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets (NDs) using different analytical procedures. METHODS: Perfluorobutane NDs were prepared by condensation of precursor fluorescently labeled lipid-shelled microbubbles (MBs) and were characterized in terms of size distribution, gas core content and in vitro stability in blood, as well as for their acoustic vaporization behavior using a custom-made setup. In particular, the in vivo behavior of the NDs was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the in vivo persistence of NDs: (i) ultrasound contrast imaging of vaporized NDs, (ii) gas chromatography-mass spectrometry to determine the perfluorobutane core content and (iii) fluorescence intensity measurement in the collected blood samples. RESULTS: The Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentrations from MB and ND suspensions were obtained, indicating an exceptionally low rate of MB breakage and spontaneous nanodroplet vaporization. As expected, these nanoscale droplets have longer circulation times compared with clinically approved MBs, and only slight variations in half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal areas of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. CONCLUSION: These results further contribute to our understanding of both the in vitro and in vivo behavior of sono-responsive nanodroplets, which is key to enabling efficient clinical translation.

Preparation of perfluorocarbon emulsions by premix membrane emulsification for Acoustic Droplet Vaporization (ADV) in biomedical applications.

Perfluorocarbon (PFC) droplets are used in acoustic droplet vaporization (ADV), a phenomenon where droplets vaporize into gas microbubbles under exposure to ultrasound. The size and the size distribution of a phase change contrast agent is an important factor in determining the ADV threshold and the biodistribution. Thus, high throughout manufacturing of uniform-sized droplets, required to maintain spatial control of the vaporization process, remains challenging. This work describes a parametric evaluation of a novel process using premix membrane emulsification (PME) to produce homogeneous PFC emulsions at high rate with moderate pressure using Shirasu Porous Glass (SPG) membranes. In this study, we investigated the effect of several process parameters on the resulting pressure and droplet size: membrane pore size, flow rate, and dispersed phase type. The functionality of the manufactured emulsions for ADV was also demonstrated. Vaporization of the PFC emulsions was obtained using an imaging ultrasound transducer at 7.813 MHz, and the ADV thresholds were determined. Here, the pressure threshold for ADV was determined to be 1.49 MPa for uniform-sized perfluorohexane (PFHex) droplets with a mean size of 1.51 µm and a sharp distribution (CV and span respectively of 20% and 0.6). Thus, a uniform-sized droplet showed a more homogeneous vaporization with a uniform response in the focal region of the transducer. Indeed, polydispersed droplets had a more diffuse response outside the focal region due to the presence of large droplets that vaporize at lower energies. The ADV threshold of uniform-sized PFC droplets was found to decrease with the droplet diameter and the bulk fluid temperature, and to increase with the boiling temperature of PFC and the presence of an oil layer surrounding the PFC core.

Microfluidic preparation of various perfluorocarbon nanodroplets: Characterization and determination of acoustic droplet vaporization (ADV) threshold.

Over the last two decades, liquid perfluorocarbon nanodroplets (PFC-NDs), also known as Phase Change Contrast Agents (PCCAs), that are capable of vaporizing into gaseous echogenic microbubbles via an external stimulus, have gained much attention for diagnostic and therapeutic applications. In the present work, a microfluidic platform is evaluated for the preparation of various size-controlled nanodroplets. Here, two major lines of investigations were carried out. The first was to define the microfluidic device settings for the preparation of nanodroplets depending on the nature of the encapsulating shell such as lipids, fluorinated surfactants and PLGA biopolymers as well as the liquid perfluorocarbon core (perfluoropentane, perfluorohexane). Specifically, the effect of the microfluidic system parameters, such as total flow rate and flow rate ratio on PFC-NDs attributes including size and uniformity was assessed. Secondly, a custom-made set-up, based on echogenicity signals from produced bubbles, was designed and successfully applied to determine the Acoustic Droplet Vaporization (ADV) threshold of PFC-NDs. Finally, the influence of various formulation parameters on the vaporization outcome was investigated depending on the PFC type and the encapsulating shell composition (soft versus hard shells). This study indicates the usefulness of this novel formulation platform enabling the rapid design and optimization of narrowly dispersed nanodroplets at a reliable yield and ultimately accelerate nanomedicines development.