Ultrasound is a widely-used imaging modality in clinics as a low-cost, non-invasive, non-radiative procedure allowing therapists faster decision-making. Microbubbles have been used as ultrasound contrast agents for decades, while recent attention has been attracted to consider them as stimuli-responsive drug delivery systems. Pioneering microbubbles were Albunex with a protein shell composed of human serum albumin, which entered clinical practice in 1993. However, current research expanded the set of proteins for a microbubble shell beyond albumin and applications of protein microbubbles beyond ultrasound imaging. Hence, this review summarizes all-known protein microbubbles over decades with a critical evaluation of formulations and applications to optimize the safety (low toxicity and high biocompatibility) as well as imaging efficiency. We provide a comprehensive overview of (1) proteins involved in microbubble formulation, (2) peculiarities of preparation of protein stabilized microbubbles with consideration of large-scale production, (3) key chemical factors of stabilization and functionalization of protein-shelled microbubbles, and (4) biomedical applications beyond ultrasound imaging (multimodal imaging, drug/gene delivery with attention to anticancer treatment, antibacterial activity, biosensing). Presented critical evaluation of the current state-of-the-art for protein microbubbles should focus the field on relevant strategies in microbubble formulation and application for short-term clinical translation. Thus, a protein bubble-based platform is very perspective for theranostic application in clinics.

Microbubbles have already reached clinical practice as ultrasound contrast agents for angiography. However, modification of the bubbles’ shell is needed to produce probes for ultrasound and multimodal (fluorescence/photoacoustic) imaging methods in combination with theranostics (diagnostics and therapeutics). In the present work, hybrid structures based on microbubbles with an air core and a shell composed of bovine serum albumin, albumin-coated gold nanoparticles, and clinically available photodynamic dyes (zinc phthalocyanine, indocyanine green) were shown to achieve multimodal imaging for potential applications in photodynamic therapy. Microbubbles with an average size of 1.5 ± 0.3 μm and concentration up to 1.2 × 10⁹ microbubbles/mL were obtained and characterized. The introduction of the dye into the system reduced the solution’s surface tension, leading to an increase in the concentration and stability of bubbles. The combination of gold nanoparticles and photodynamic dyes’ influence on the fluorescent signal and probes’ stability is described. The potential use of the obtained probes in biomedical applications was evaluated using fluorescence tomography, raster-scanning optoacoustic microscopy and ultrasound response measurements using a medical ultrasound device at the frequency of 33 MHz. The results demonstrate the impact of microbubbles’ stabilization using gold nanoparticle/photodynamic dye hybrid structures to achieve probe applications in theranostics.

Microbubbles are intravascular contrast agents clinically used in diagnostic sonography, echocardiography, and radiology imaging applications. However, up to date, the idea of creating microbubbles with multiple functionalities (e.g., multimodal imaging, photodynamic therapy) remained a challenge. One possible solution is the modification of bubble shells by introducing specific compounds responsible for such functions. In the present work, air-core microbubbles with the shell consisting of bovine serum albumin, albumin-coated gold nanocages, and zinc phthalocyanine were prepared using the sonication method. Various physicochemical parameters such as stability over time, size, and concentration were investigated to prove the potential use of these microbubbles as contrast agents. This work shows that hybrid microbubbles have all the necessary properties for multimodal imaging (ultrasound, raster-scanning microscopy, and fluorescence tomography), which demonstrate superior characteristics for potential theranostic and related biomedical applications.

Liquid/surfactant/gas interfaces are promising objects for nanoengineered multimodal contrasts, which can be used for biomedical imaging in preclinical and clinical applications. Microbubbles with the gaseous core and shell made of lipids/proteins have already acted as ultrasound (US) contrast agents for angiography. In the present work, microbubbles with a shell composed of Span 60 and Tween 80 surfactants functionalized with fluorescein isothiocyanate and gold nanorods to achieve a multimodal combination of US, fluorescence, and optoacoustic imaging are described. Optimal conditions for microbubble generation by studying the surface tension of the initial solutions and analyzing the size, stability, and charge of the resulting bubbles were found. By controlling and modifying bubbles’ surface properties, an increase in stability and storage time can be achieved. The functionalization of bubbles with gold nanoparticles and a dye by using an optimally selected sonication protocol was performed. The biomedical application’s potential in imaging modalities of functionalized microbubbles using a medical US device with a frequency of 50 MHz, fluorescence tomography, and raster-scanning optoacoustic mesoscopy measurements was evaluated. The obtained results are important for optimum stabilization and functionalization of gas/liquid interfaces and the following applications in the multimodal biomedical imaging.