Novel Ultrasonic Shearing for Monodisperse and Size-Controllable Nanobubble Manufacturing in Therapeutic Delivery Systems

Abstract Ultrasound responsiveness and unique physicochemical properties make nanobubbles (NBs) promising nanocarriers and imaging agents for disease treatment. However, limitations in synthesis precision and monodispersity currently hinder their clinical translation as drug delivery systems. This paper aims to study the effects of manufacturing parameters of the novel manufacturing method termed “ultrasonic shearing” (USS) on NB characteristics and to determine optimal parameters for production. USS is an alternative method to current NB technologies that improves productivity and precision while allowing production reproducibility. USS combines the shearing capabilities of a rotor–stator component and the acoustic wave propagation of the ultrasound (sonotrode) component. The complete assembly addresses NB production challenges of size control and polydispersity through uniform emulsification of reagent solution to reduce NB generation from larger, heterogeneous nanodroplets and the decrease in uneven acoustic energy distribution caused by acoustic attenuation, which would otherwise result in large, polydisperse NB populations. The manufacturing parameters that were investigated included rotational speeds of the shearing component, amplitude and pulse cycle intervals of the ultrasonic component, and total synthesis duration. Optimal settings were determined by evaluating the diameter, polydispersity index (PDI), and zeta potential, accomplished through dynamic light scattering (DLS) measurements. Favorable NB production was further validated by evaluating NB stability and ultrasound responsiveness to replicate the stimuli utilized for therapeutic NB activation. It was hypothesized that the use of USS for NB synthesis would produce NBs with predictable and scalable diameters, PDIs, and zeta potentials. Amplitude and synthesis duration had the most influence on NB characteristics, where reduced amplitudes contributed to decreased diameter and polydispersity but increased absolute zeta potential, and reduction of synthesis duration produced unstable NB populations. Amplitude strongly affects NB characteristics, as acoustic waves induce NB formation—high amplitudes form NBs and subsequently stimulate their growth, thereby contributing to larger diameter populations. Reducing synthesis duration does not allow sufficient time for the reagent solution to emulsify, resulting in a heterogeneous mixture that yields unstable and unpredictable NB populations. The optimal synthesis parameters for minimizing NB diameter and PDI were 2000 rpm, 25% amplitude, a 1 s ON/1 s OFF pulse cycle, and a 2.5 min synthesis duration. Stability displayed an NB lifecycle of 6 days after the first dose of low-intensity pulsed ultrasound (LIPUS) stimulation and 14 days after the second dose of LIPUS stimulation, indicating NBs can release therapeutics sequentially.