**1. Introduction**

Microbubbles are small gas bubbles (diameter 1–10 µm) that are clinically used as ultrasound contrast agents for non-invasive diagnostic imaging of blood perfusion [1]. Targeted microbubbles are employed for molecular imaging of inflammation, tumors, and cardiovascular disease [2]. Other types of microbubbles are being developed specifically for drug delivery [3]. All of these applications make use of the compression and expansion of the microbubble gas core upon ultrasound insonification. These microbubble vibrations produce a nonlinear response, including super- and subharmonic oscillations, which can be differentiated from the surrounding tissue to form a contrast-enhanced image [1]. Additionally, this acoustic response can induce bioeffects on nearby cells–resulting in enhanced uptake or extravasation of drug molecules [4]. Successful translation to the clinical use of microbubbles for molecular imaging and enhanced drug delivery is currently challenged, however, by the microbubbles' unpredictable acoustic behavior.

To stabilize the gas core, microbubbles are usually coated with a phospholipid monolayer, proteins, or polymers. For a schematic representation, the reader is referred to recent reviews on microbubbles [5,6]. The coating reduces surface tension and gas diffusion [7].

**Citation:** Langeveld, S.A.G.; Beekers, I.; Collado-Lara, G.; van der Steen, A.F.W.; de Jong, N.; Kooiman, K. The Impact of Lipid Handling and Phase Distribution on the Acoustic Behavior of Microbubbles. *Pharmaceutics* **2021**, *13* , 119 . https://doi.org/10.3390/ pharmaceutics13010119

Received: 18 December 2020 Accepted: 14 January 2021 Published: 19 January 2021

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If phospholipids or polymers are used as microbubble coating, a ligand can be attached for molecular imaging [8], and they can be loaded with a drug for localized delivery [3]. The physicochemical properties of the microbubble coating, such as the shell elasticity and viscosity, are related to the acoustical properties, such as the resonance frequency and the damping coefficient [9,10]. Therefore, the composition of the microbubble coating can affect the acoustical properties. For instance, the use of a phospholipid molecule with a longer acyl chain length, 1,2-distearoyl-*sn*-glycero-3-phosphocholine (DSPC; C18), resulted in a higher shell elasticity and more acoustic stability than the use of a shorter acyl chain length phospholipid, 1,2-dipalmitoyl-*sn*-glycero-3-phosphocholine (DPPC; C16) [11]. Besides the shell elasticity, acyl chain length has also been shown to affect the half-life of microbubbles, with longer acyl chain length resulting in more stable size distribution and ultrasound signal over time [12].

Since microbubbles are generally coated with a mixture of phospholipids and a PE-Gylated emulsifier, the physicochemical properties are determined by the miscibility and lipid phase behavior. Molecules in the microbubble coating can be in the liquid expanded (LE) or liquid condensed (LC) phase, resulting in distinctive microstructures. These microstructures can be altered by using different types of phospholipids [13], changing the ratio between phospholipid and emulsifier, or heating and cooling of the microbubble coating [14]. Microstructures formed by lipid phase separation have been shown to affect the subharmonic response to ultrasound [15]. The effect of lipid phase separation on the subharmonic response to ultrasound has been characterized previously in three types of microbubbles with different levels of lipid phase separation: 20%, 50% or 80% of the microbubbles had LC phase domains. Each microbubble type had a peak subharmonic response at a different microbubble size, suggesting that microstructures in the coating affect the acoustical properties of a microbubble [15]. The microbubble coating can also be altered by the distribution of the phospholipid and PEGylated-emulsifier molecules over the microbubble coating, depending on the lipid handling prior to microbubble production by probe sonication. The use of organic solvent resulted in a more homogeneous ligand distribution than the use of aqueous solutions only [16]. The effect of lipid handling on the acoustic response of microbubbles, however, has not been investigated.

For both ultrasound molecular imaging and drug delivery, it is important that all microbubbles respond uniformly and predictably to ultrasound. Currently available microbubbles respond to ultrasound in a heterogeneous way [11,17], even when they are the same size [18]. While it is thought this variability in response could be due to the microstructures in the microbubble coating, this is challenging to confirm because it can only be investigated by looking at single microbubbles. Different approaches have been used to record a single microbubble's response to ultrasound, including an ultra-highspeed camera to image the microbubble during insonification [19], recording the acoustic response [15] or optical scattering [20], and photo-acoustic techniques [21]. Until recently, however, no techniques were available to image both the lipid phase distribution in 3D and the acoustic response of the same microbubble. In this regard, the challenge lies in the time scale (µs) and optical resolution (µm) needed to record the lipid phase distribution and response to ultrasound of a single microbubble.

The purpose of this study was to relate the effects of lipid handling and phase distribution before microbubble production to the acoustic behavior of phospholipid-coated microbubbles. Cholesterol can modify the lateral molecular packing of phospholipids in a monolayer, resulting in a single liquid phase [22–24]. While microbubbles with cholesterol in their coating have been produced before [25,26], the effect of cholesterol on the lipid phase separation in microbubbles has not been studied. To determine this effect in the microbubble coating, we made microbubbles by probe sonication with DSPC as the main lipid and varying concentrations of cholesterol. The lipid phase distribution and ligand distribution in the microbubble coating were imaged using high-axial-resolution 4Pi confocal microscopy. To assess the acoustic response and variability in the acoustic behavior, we used a unique system combining a confocal microscope with the Brandaris

128 ultra-high-speed camera. With this system, the lipid phase separation (in nanometer resolution) and acoustic response to ultrasound (in nanosecond resolution) were captured at a single microbubble level.
