Recent efforts in the area of acoustic droplet vaporization with the objective of designing extravascular ultrasound contrast agents has led to the development of stabilized, lipid-encapsulated nanodroplets of the highly volatile compound decafluorobutane (DFB). We have developed two methods of generating DFB droplets, the first of which involves condensing DFB gas (boiling point of −1.1° to −2° C) followed by extrusion with a lipid formulation in HEPES buffer. Acoustic droplet vaporization of micron-sized lipid-coated droplets at diagnostic ultrasound frequencies and mechanical indices were confirmed optically. In our second formulation methodology, we demonstrate the formulation of sub-micron sized lipid-coated nanodroplets based upon condensation of pre-formed microbubbles containing DFB. The droplets are routinely in the 200 – 300 nm range and yield microbubbles on the order of 1 – 5 microns once vaporized, consistent with ideal gas law expansion predictions. The simple and effective nature of this methodology allows for the development of a variety of different formulations that can be used for imaging, drug and gene delivery, and therapy. This study is the first to our knowledge to demonstrate both a method of generating ADV agents by microbubble condensation and formulation of primarily sub-micron droplets of decafluorobutane that remain stable at physiological temperatures. Finally, activation of DFB nanodroplets is demonstrated using pressures within the FDA guidelines for diagnostic imaging, which may minimize the potential for bioeffects in humans. This methodology offers a new means of developing extravascular contrast agents for diagnostic and therapeutic applications.

Recently, an interest has developed in designing in biomaterials for medical ultrasonics that can provide the acoustic activity of microbubbles, but with improved stability in vivo and a smaller size distribution for extravascular interrogation. One proposed alternative is the phase-change contrast agent. Phase-change contrast agents (PCCAs) consist of perfluorocarbons (PFCs) that are initially in liquid form, but can then be vaporized with acoustic energy. Crucial parameters for PCCAs include their sensitivity to acoustic energy, their size distribution, and their stability, and this manuscript provides insight into the custom design of PCCAs for balancing these parameters. Specifically, the relationship between size, thermal stability and sensitivity to ultrasound as a function of PFC boiling point and ambient temperature is illustrated. Emulsion stability and sensitivity can be ‘tuned’ by mixing PFCs in the gaseous state prior to condensation. Novel observations illustrate that stable droplets can be generated from PFCs with extremely low boiling points, such as octafluoropropane (b.p. −36.7°C), which can be vaporized with acoustic parameters lower than previously observed. Results demonstrate the potential for low boiling point PFCs as a useful new class of compounds for activatable agents, which can be tailored to the desired application.

Currently available microbubbles used for ultrasound imaging and therapeutics are limited to intravascular space due to their size distribution in the micron range. Phase-change contrast agents (PCCAs) have been proposed as a means to overcome this limitation, since droplets formed in the hundred nanometer size range might be able to extravasate through leaky microvasculature, after which they could be activated to form larger highly echogenic microbubbles. Existing PCCAs in the sub-micron size range require substantial acoustic energy to be vaporized, increasing the likelihood of unwanted bioeffects. Thus, there exists a need for PCCAs with reduced acoustic activation energies for use in imaging studies. In this article, it is shown that decafluorobutane, which is normally a gas at room temperature, can be incorporated into metastable liquid sub-micron droplets with appropriate encapsulation methods. The resulting droplets are activatable with substantially less energy than other favored PCCA compounds. Decafluorobutane nanodroplets may present a new means to safely extend ultrasound imaging beyond the vascular space. (E-mail: padayton@bme.unc.edu)

Recent efforts using perfluorocarbon (PFC) nanoparticles in conjunction with acoustic droplet vaporization has introduced the possibility of expanding the diagnostic and therapeutic capability of ultrasound contrast agents to beyond the vascular space. Our laboratories have developed phase-change nanoparticles (PCNs) from the highly volatile PFCs decafluorobutane (DFB, bp =-2 °C) and octafluoropropane (OFP, bp =-37 °C ) for acoustic droplet vaporization. Studies with commonly used clinical ultrasound scanners have demonstrated the ability to vaporize PCN emulsions with frequencies and mechanical indices that may significantly decrease tissue bioeffects. In addition, these contrast agents can be formulated to be stable at physiological temperatures and the perfluorocarbons can be mixed to modulate the balance between sensitivity to ultrasound and general stability. We herein discuss our recent efforts to develop finely-tuned diagnostic/molecular imaging agents for tissue interrogation. We discuss studies currently under investigation as well as potential diagnostic and therapeutic paradigms that may emerge as a result of formulating PCNs with low boiling point PFCs.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. We have recently shown that FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, we examined the distribution of cell cycle markers in dFmr1 brains compared with wild-type throughout larval development. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 NB generate significantly more neurons than controls. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS.