Development of an inhalable contrast agent targeting the respiratory tract mucus layer for pulmonary ultrasonic imaging

Abstract

Impaired mucociliary transport is a distinguishing sign of cystic fibrosis, but current methods of evaluation are invasive or expose young patients to ionizing radiation. Contrast-enhanced ultrasound imaging may provide a feasible alternative. We formulated a cationic microbubble ultrasound contrast agent, to optimize adhesion to the respiratory mucus layer when inhaled. Potential toxicity was evaluated in human bronchial epithelial cell (hBEC) cultures following a 24-hour exposure, compared to positive and negative control conditions. In vivo tolerability and pulmonary image enhancement feasibility were evaluated in mice, comparing oropharyngeal administration of contrast agent to saline control. When induced to flow across mucus plated on microscope slides, cationic microbubbles demonstrated greater affinity for target samples than standard microbubbles. Cationic microbubbles elicited no proinflammatory or cytotoxic response in hBECs, nor were any cross-links to the cilia observed. Unlike standard microbubbles, cationic microbubbles mixed into the mucus layer, without epithelial absorption, and were observed to move with the mucus layer by the action of mucociliary transport. When administered to mice, cationic microbubbles enhanced sonographic visualization of the trachea, and were well-tolerated with no adverse effects. This developmental work supports the safety and feasibility of a mucus-targeting contrast agent that may be useful for pulmonary ultrasound applications.

Keywords: Contrast agent; Cystic fibrosis; Microbubble; Mucociliary transport; Pulmonary; Ultrasound.

Fig. 1

Cationic TAP microbubbles (MB) remained bound to human mucus after washing with PBS (a). Standard, in-house MB contrast agent did not remain bound to human mucus after washing with PBS (b). Depicted in the lower right of each micrograph is a 5-micron scale bar (red). Note that both contrast agents were enriched for larger diameter MB before the binding experiment, as described in the Methods.

Fig. 2

Time-lapse movement of fluorescently labeled TAP microbubbles on a human bronchial epithelial cell culture. Panel (a): transmitted light video, panel (b): epifluorescence video. Panels show the same field of view, with fluorescently labeled particles similar in size and motion to those seen in the transmitted light images. Scale bar: 20 μm.

Fig. 3

Mucociliary transport rate measured across 8 fields of view by tracking the motion of TAP microbubbles (fluorescence speed) versus the motion of visible debris (transmitted light speed) on human mucociliary transport cultures.

Fig. 4

Cytotoxic (lactate dehydrogenase release) and proinflammatory response (interleukin-8 secretion) induced by exposing human bronchial epithelial cell cultures to TAP microbubble contrast agent, TAP microbubble vehicle, and saline. Abbreviations: LDH = lactate dehydrogenase, IL-8 = interleukin-8, TAP = 1,2-distearoyl-3-trimethylammonimumpropane, Il-1B = interleukin-1 beta, TNFa = tumor necrosis factor alpha. Error bars = standard deviations.

Fig. 5

Comparison of in vivo tracheal ultrasound following oropharyngeal administration of saline versus TAP microbubble. Panels: Anatomical slice of mouse trachea (a). Ultrasound b-mode (b, d) and contrast mode (c, e) of a mouse trachea post-saline (b, c) and post inhalation of TAP microbubbles (d, e). Images acquired using a FUJIFILM VisualSonics Vevo F2. Anatomic slice of mouse trachea (panel a) was acquired by computed tomography and is courtesy of IMAIOS “Micheau A, Hoa D, e-Anatomy, www.imaios.com, DOI: 10.37019/e-anatomy”.