Optoacoustic/Fluorescent/Acoustic Imaging Probe Based on Air-Filled Bubbles Functionalized with Gold Nanorods and Fluorescein Isothiocyanate.

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.

Introduction

Imaging is one of the fastest-growing research areas today with many clinical and preclinical applications. Numerous imaging modalities are known, such as magnetic resonance imaging (MRI), positron emission tomography, single-photon emission computed tomography, fluorescence tomography (FT), optical coherence tomography, ultrasound (US), and optoacoustic or photoacoustic (OA) visualization.[1] During the recent years, there has been an active transition from anatomical imaging to molecular imaging, which allows a more detailed analysis of the problem, choosing optimal therapy methods, noninvasive monitoring of <span class="Disease">tumor response, progression, and transformation after therapy or relapse. It should be noted that parameters such as tissue penetration depth and spatial resolution (down to microns) are considered to be the most important. The use of contrast media can significantly improve the signal received during the imaging procedure.

US imaging has been a part of clinical practice for over 30 years.[2] It has significant advantages over MRI and CT, such as no radiation, no restrictions on use, simplicity and low cost, real-time image acquisition. Targeted US contrast agents are used to perform molecular imaging.[3] First introduced in 1968 for echocardiography, US contrast agents generated considerable research interest, leading to the development of several commercially available agents such as Albunex (Molecular Biosystems Inc., San Diego, CA), SonoVue (Bracco Suisse SA, Geneva, Switzerland), and Definity (Lantheus Medical Imaging, North Billerica, MA), which mainly consisted of microbubble suspensions.[4−9] In 2016, a contrast agent received the US Food and Drug Administration approval for non-cardiac contrast imaging.[10] The superior acoustic properties of gaseous core have made microbubbles the most popular core–shell structure for contrast enhancement in US imaging.

The average bubble size varies from 1 to 10 μm, making them a real blood pool agent.[11−14] Contrast agents based on bubbles with submicron and nanosize have recently been described and shown to have similar stability and acoustic properties.[15−17] Compared to other particles, bubbles have a special property of “exploding”/rupturing when exposed to ultrasonic radiation, causing the destruction of bubbles and the change in permeability of cell membranes. Thus, microbubbles are ideal objects for molecular imaging, and they can also be used as agents for theranostics because bubbles can be used as drug carriers for targeted delivery by US-induced cavitation and materials for gene delivery.[18−21]

The bubble shell can be made of different molecules, including <span class="Chemical">lipids, proteins, and <span class="Chemical">polymers.[22,23] The choice of compounds that the microbubble shell consists of can determine the surface properties of obtained microbubbles: if the molecules in a shell are packed loosely or if the shell is not compact enough, the gas from the core can diffuse through the shell. Even a combination of surfactants can be used as a base for bubble formation. Still, only a few surfactant combinations in certain molar ratios have been shown to be successful.[24] Thus, <span class="Chemical">sorbitan esters (<span class="Chemical">Span) and their ethoxylates (<span class="Chemical">polyethoxylated sorbitan esters, <span class="Chemical">Tween) are well-known nonionic surfactants with many applications, as emulsifiers, solubilizers, wetting agents, dispersants in pharmaceutics, and the food industry.[25−27] Recently, several potential applications of this surfactant pair were taken into consideration. For example, <span class="Chemical">Span 80–<span class="Chemical">Tween 80-based fluid-filled organogels were used as a matrix for drug delivery: <span class="Chemical">ciprofloxacin, a fourth-generation <span class="Chemical">fluoroquinolone, was incorporated within the organogels, and the diffusion-controlled drug release was described.[28] Also, in vitro skin permeation of neutral and pH-sensitive vesicles made with the use of <span class="Chemical">Span 60 and <span class="Chemical">Tween 20 surfactants as well as the assessment of the drug entrapment of <span class="Chemical">ibuprofen within the vesicles were described.[29] The use of <span class="Chemical">Span 60 only as a microsphere matrix was recently performed with <span class="Chemical">ibuprofen–<span class="Chemical">Span 60 microspheres preparation.[30] Also, it was known that the presence of <span class="Chemical">Tween 80 in the formulation would provide contrast enhancement for imaging and drug uptake enhancement.[31] Thus, the combination of <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 as a surfactant complex should be considered as the promising and biocompatible basis for contrast agent preparation in echo enhancement.

The first contrast agent based on surfactants, ST68, was described in 1993.[24] The authors used <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 surfactants to prepare the shell for air bubbles according to a sonication method/protocol.[24,32] Bubbles were later produced with their core filled with <span class="Chemical">sulfur hexafluoride (SF6) and <span class="Chemical">decafluorobutane (<span class="Chemical">C4F10) to improve the stability of the contrast medium. Also, submicron-sized ST68 substances were obtained, and surfactants with different <span class="Chemical">hydrocarbon lengths (<span class="Chemical">Span 40 and <span class="Chemical">Tween 40, <span class="Chemical">Span 60, and <span class="Chemical">polyoxyethylene 40 stearate, <span class="Chemical">Span 60 and vitamin E) [α-tocopherylpoly (ethylene glycol) succinate] were tested.[16,32−35] Still, additional detailed research on analysis and selection of synthesis conditions for obtaining optimal microbubble samples is needed to fully cover the optimal path of probe preparation. Thus, physicochemical investigations on the surface properties of described surfactants can be implemented: an increase in stability, storage, and circulation time can be achieved with the surface property control and modification of bubbles. Only a few examples of bubble modifications based on surfactants are known in the literature: obtaining layers on already formed bubbles by layer-by-layer deposition, a widely used technique for microcapsule composition and functionalization, introducing nanoparticles and quantum dots into the bubble structure after formation.[36−38] Shchukin et al. described the self-assembly of gas microbubbles coated with <span class="Chemical">Span 60 and <span class="Chemical">Tween 80, with a stepwise layer-by-layer deposition of oppositely charged polyelectrolytes.[36] The modification of microbubbles with CdTe quantum dots was obtained by Ke et al.: a possible combination of ultrasonic and fluorescent (FL) modalities has been well described.[37] However, it should be noted that all of the above studies were not further systematized and structured. To the best of our knowledge, there have been no examples of successful combinations of ultrasonic, OA, and FL imaging with a contrast agent based on microbubbles coated with <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 surfactants. However, such contrast agents should be able to provide the possibility of implementing multimodal imaging for <span class="Species">patients.

Previously, microbubbles were mainly used to visualize blood flow, blood clots, <span class="Disease">tumors, inflammations, and so forth.[39−41] Still, microbubbles’ size limits the range of their application; they penetrate poorly into <span class="Disease">tumor tissues and pass poorly through microcapillaries. Thus, an important task is to obtain microbubbles with a diameter of up to 1–2 μm. It should be noted that tiny bubbles (less than 300 nm) can hardly be visualized with US. The production of such agents will be complicated because of their low stability and rapid dissolution.

Thus, this study aims to achieve functional microbubbles with optimal properties as a potential submicron-/micron-sized contrast agent with a shell consisting of the <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 surfactants. In this study, the analysis and selection of a method for obtaining bubbles and modifying their structure to create a promising multimodal contrast agent that combines ultrasonic, OA, and/or FL methods were carried out.

Results and Discussion

Nowadays, US imaging is in widespread clinical use, and the most sensitive structures in the US modality include the gas core because of its acoustic backscattering properties; thus, micro- and nanobubbles are well-known probes as US contrast agents.[22]

Obtaining microbubbles is an essential task from both the chemical, biological, and physical aspects of the issue. There are numerous methods for obtaining and analyzing the resulting microbubbles. The use of bubbles depends on their size, stability, and shell structure. Hence, microbubbles were chosen as a promising basis for further shell functionalization to obtain multimodal contrast agents. In the course of this work, optimal conditions were selected for obtaining stable micron-sized bubbles. For further practical use, the structures were fully characterized and analyzed.

In addition to obtaining microbubbles with optimal size and stability, this study’s focus was to introduce labels in the form of FL dyes and nanoparticles with the ability to act as contrast agents in different imaging modalities into the microbubble structure. This modification allows the bimodal use of bubble patterns. In this work, microbubbles were visualized by the following methods: FT, raster-scanning optoacoustic mesoscopy (RSOM), acoustic characterization with the use of US in linear B-scan mode, and a potential application has also been shown thanks to the demonstration of the ultrasonic examination of the obtained microbubbles.

Optimization of the Experiment: the Selection of the Ratio of Initial Reagents, Synthesis Conditions, and Modification of Microbubbles

Surfactants Span and Twin were chosen as the basis because previous studies have shown their properties to be one of the most optimal for microbubble preparation. Reagent ratios reported in previous publications were used as the starting point for our study. Ultrasonic treatment of surfactant solutions was chosen as the primary approach for producing bubbles. This method for obtaining microbubbles was chosen because of its simplicity for the further widespread use and the possibility to vary the volume of the obtained material of microbubbles. The technique makes it possible to carry out synthesis in situ and to use the obtained microbubbles immediately.

The existing methods of microbubble production were tested to obtain probes with the shell consisting of surfactants. The only known research on collapse pressure characterization of microbubbles with the <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 shell was described by Signhal et al. in 1993 and was based on Langmuir trough: the molar ratio 1.7:1 of <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 was chosen as optimal for microbubble production, and variation of collapse pressure and inflection area per molecule of <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 was well described to obtain a stable probe.[24] Another intriguing study was demonstrated in 2005 by Shchukin et al., where layer-by-layer assembly was used for the functionalization of microbubbles with polyelectrolyte layers having opposite charges; thus, zeta-potential measurements were provided.[36] The molar ratio of <span class="Chemical">Span 60/<span class="Chemical">Tween 80 used in the article was 1:1.87. Only partial knowledge is known about contrast agent preparation by using the mentioned surfactants and further investigations on the preparation condition selection and further characterization of the obtained microbubbles were needed. Thus, previously used molar ratios presented above were chosen for further research as existing examples of preparation and for comparison complemented with molar ratios of 1:6, 1:3, 1:1, 2:1, and 5:1 to describe the surfactant solution system precisely and to achieve optimal conditions for microbubble shell preparation further.

Interestingly, existing probe preparation methods were not optimal under our preparation conditions described in the Experimental Section: low yields of microbubbles were observed, size distributions of the obtained samples were polydisperse, and microbubbles had low stability. Thus, it was decided to modify the probe preparation method to increase stability and reduce the obtained bubbles’ size.

To the best of our knowledge, the investigation of dynamic surface tension dependence on the molar ratio for <span class="Chemical">Span 60/<span class="Chemical">Tween 80 aqueous solutions was not previously performed. However, previous studies have tested surface tension as a characterization method to predict bubbles’ stability with different shell composition.[42] Although the use of pendant drop and rising drop for surface tension measurements was shown not to be sufficient as a stand-alone method for predicting the stability of nanobubbles under US, an overall trend was still observed with 0.2 L10/lipid molar ratio resulting in the most stable nanobubbles, which also had one of the lowest surface tension values. Thus, we decided to find an optimal molar ratio of surfactants, which would allow obtaining one the lowest surface tension values at a chosen age of the pendant drop and confirm results by microscopic inspection, dynamic light scattering (DLS), and determination of the mean size at room temperature, 37 °C, and after storage at 4 °C.

A set of <span class="Chemical">Span 60/<span class="Chemical">Tween 80 M ratios was tested (1:6, 1:3, 1:1,87, 1:1, 1.7:1, 2:1, and 5:1), and dynamic surface tension measurements for <span class="Chemical">Span 60 solution and <span class="Chemical">Tween 80 solution were performed for comparison. All measurements were performed at room temperature. Surface tension values obtained for the <span class="Chemical">Tween 80 solution (60.9 mg/mL) and <span class="Chemical">Span 60 solution (20 mg/mL) at early surface age (<1 min) of the pendant drop agree with previous reports.[43−46] As one can see in Figure a, the lowest value of the last 2 min surface tension average (31.6 mN/m) corresponded to a molar ratio of 1:3, which was chosen as the optimal one for air-filled bubble preparation. This molar ratio differed from the values presented previously because of modifying the method for obtaining bubbles. It should be noted that 1:6 and 1:1.87 ratios used in previous studies have also shown low surface tension values, hence, were chosen in addition to 1.7:1 for evaluating microbubble lifetime.

Figure 1

(a) Surface tension measurements for Span 60/Tween 80 aqueous solution with different molar ratios. Green star corresponded to 1:3 Span 60/Tween 80 M ratio, gray circle corresponded to 1:6 M ratio, blue dot corresponded to 1:1.87 M ratio, and purple dot corresponded to 1.7:1 M ratio. (b) Schematic representation of sonication procedure used for microbubble production: aqueous solution of surfactants mixed together (Span 60–Tween 80 solution) was obtained and then sonicated. The tip of the sonicator was placed at the air-solution interface to produce samples-contained microbubbles. (c) Lifetime of microbubbles with the shell obtained from aqueous solutions of selected surfactant molar ratios during storage at 4 °C.

(a) Surface tension measurements for <span class="Chemical">Span 60/<span class="Chemical">Tween 80 aqueous solution with different molar ratios. Green star corresponded to 1:3 <span class="Chemical">Span 60/<span class="Chemical">Tween 80 M ratio, gray circle corresponded to 1:6 M ratio, blue dot corresponded to 1:1.87 M ratio, and purple dot corresponded to 1.7:1 M ratio. (b) Schematic representation of sonication procedure used for microbubble production: aqueous solution of surfactants mixed together (<span class="Chemical">Span 60–<span class="Chemical">Tween 80 solution) was obtained and then sonicated. The tip of the sonicator was placed at the air-solution interface to produce samples-contained microbubbles. (c) Lifetime of microbubbles with the shell obtained from aqueous solutions of selected surfactant molar ratios during storage at 4 °C.

Surface tension analysis is a necessary but not sufficient condition to obtain optimal microbubbles. Therefore, the second stage in selecting the ratio was the analysis of the resulting microbubbles, the assessment of size and stability. Microbubbles were obtained with a set of various <span class="Chemical">Span 60–<span class="Chemical">Tween 80 M ratios (1:6, 1:3, 1:1.87, 1.7:1) with the sonication method described below, and optical microscopy (OM) testing along with DLS measurements were carried out for quick assessment of probe’s mean size and stability during storage at 4 °C. Interestingly, empirical criteria for the stability of sample-contained bubbles was found: the most stable probes demonstrated approximately the same size and concentration of bubbles through the entire volume of the solution in the glass vial; also, the presence of thick uniform foam contributed to the sample stability, compared with samples where it was absent.

For each of the 4 M ratios selected for microbubble production, OM images were carried out for the samples collected from the top and the bottom of the solution in the glass vial; as one can see in Figure S1, microbubbles produced from the solution with a molar ratio of 1:3 demonstrated similarity in size dispersion and absence of large bubbles in both images, as opposed to samples with other <span class="Chemical">Span 60–<span class="Chemical">Tween 80 ratios. The presence of bubbles larger than 10 μm does not allow us to consider such samples for use as US contrast agents because of the possibility of <span class="Disease">thrombosis in the organism, thus, it can be considered as a significant criterion of probe choice. Also, wide size dispersion was mentioned for other samples during OM.

Furthermore, additional characterization for probes is obtained and presented in Table S1. As one can see, clear evidence of the possibility of air-filled microbubble production with the solutions of each selected molar ratio was shown. However, the question is the lifetime of probes, when most of the microbubbles are present in the sample. Figure c demonstrates that microbubbles produced from the solution of 1:3 <span class="Chemical">Span 60/<span class="Chemical">Tween 80 M ratio remained stable for 47 days without significant changes in size dispersion, while other samples produced under conditions described below remained stable for no longer than 12 days.

Thus, according to the described set of characteristics, the molar concentration of 1:3 for <span class="Chemical">Span 60/<span class="Chemical">Tween 80 aqueous solution was determined to be the optimal basis for bubble production and evaluation of the US contrast agent with the shell consisting of surfactants.

Based on the obtained optimal molar ratio, the sample production conditions were examined for the sonication method: the power amplitude of sonotrode, time of sonication procedure, and temperature were varied. A power of 100 W and sonication for 180 s were found to be the most suitable conditions for the stable preparation of probes. Thus, all samples were prepared by using a modified sonication method presented in Figure a. Also, the temperature of the solution was raised to 50 °C to additionally reduce the surface tension and lower the mean size of the obtained bubbles to 1–2 μm.

Figure 2

(a) Scheme of microbubble production by the sonication method: surfactants (Span 60 and Tween 80) were diluted with deionized (DI) water, additionally functionalized with gold nanorods (AuNRs) and fluorescein isothiocyanate (FITC) (with the use of Tween–FITC conjugate), then obtained solutions were sonicated to obtain microbubbles. Air core provides the possibility to be used as an US contrast agent, while AuNRs provide OA imaging modality, and Tween–FITC provides FL imaging modalities. (b) To prove the morphology of AuNRs, transmission electron microscopy (TEM) images of nanoparticles were carried out (scale bar of 20 nm).

(a) Scheme of microbubble production by the sonication method: surfactants (<span class="Chemical">Span 60 and <span class="Chemical">Tween 80) were diluted with deionized (DI) <span class="Chemical">water, additionally functionalized with gold nanorods (AuNRs) and <span class="Chemical">fluorescein isothiocyanate (FITC) (with the use of <span class="Chemical">Tween–FITC conjugate), then obtained solutions were sonicated to obtain microbubbles. Air core provides the possibility to be used as an US contrast agent, while AuNRs provide OA imaging modality, and <span class="Chemical">Tween–FITC provides FL imaging modalities. (b) To prove the morphology of AuNRs, transmission electron microscopy (TEM) images of nanoparticles were carried out (scale bar of 20 nm).

To obtain a multimodal imaging probe, further functionalization of air-filled bubbles with the <span class="Chemical">Span 60–<span class="Chemical">Tween 80 shells is needed. OA imaging modality was achieved by functionalizing probes with AuNRs. AuNRs were obtained by a method similar to the standard method of reduction with <span class="Chemical">borohydride in a <span class="Chemical">CTAB emulsion, with an increased concentration of gold in the resulting solution. Results of TEM provided in Figure b proved the shape of nanoparticles used for microbubble functionalization. For the implementation of FL imaging modality, FITC was chosen and covalently bound to <span class="Chemical">Tween 80 for the conjugation of <span class="Chemical">Tween 80 and FITC. To the best of our knowledge, such a strategy was not performed before for the conjugation of FITC with the surfactant, and such binding was confirmed by mass spectrometry measurements, as one can see in Figure .

Figure 3

Mass spectrometry measurements for (a) Tween 80 aqueous solution and (b) Tween–FITC conjugate aqueous solution were carried out to confirm the covalent binding Tween–FITC conjugate.

Mass spectrometry measurements for (a) <span class="Chemical">Tween 80 aqueous solution and (b) <span class="Chemical">Tween–FITC conjugate aqueous solution were carried out to confirm the covalent binding <span class="Chemical">Tween–FITC conjugate.

Thus, as a result of the synthesis, four primary samples were obtained: microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 shell (ST MBs), microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween–FITC shell (ST–FITC MBs), microbubbles coated with the surfactants pair shell and functionalized with AuNRs (ST–AuNRs MBs), and microbubbles produced with the addition of the <span class="Chemical">Tween–FITC conjugate as a surfactant and AuNR additives (ST–FITC–AuNRs MBs). The next stage of work consisted of characterization and analysis of the obtained microbubbles, and, as a consequence, performing the proof-of-concept of their use as multifunctional contrast agents.

Samples Characterization

The first step in characterization is the visual analysis by using an optical microscope. OM and fluorescence lifetime imaging microscopy (FLIM) observations of air-filled probes can be seen in Figure : in all samples, the bubbles’ size was less than 10 μm, which meets the criteria for the size dispersion of practically used US contrast agents.

Figure 4

OM and FLIM images of air-filled bubbles. (a) OM of microbubbles coated with the Span 60 and Tween 80 shell (ST MBs), (b,c) OM and FLIM of microbubbles coated with the Span 60 and Tween–FITC shell (ST–FITC MBs), (d) OM of microbubbles coated with the Span 60 and Tween 80 shell functionalized with AuNRs (ST–AuNRs MBs), and (e,f) OM and FLIM of microbubbles coated with the Span 60 and Tween–FITC shell functionalized with AuNRs (ST–FITC–AuNRs MBs).

OM and FLIM images of air-filled bubbles. (a) OM of microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 shell (ST MBs), (b,c) OM and FLIM of microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween–FITC shell (ST–FITC MBs), (d) OM of microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 shell functionalized with AuNRs (ST–AuNRs MBs), and (e,f) OM and FLIM of microbubbles coated with the <span class="Chemical">Span 60 and <span class="Chemical">Tween–FITC shell functionalized with AuNRs (ST–FITC–AuNRs MBs).

Still, during OM observations, several drawbacks of the used method’s setup were found: the resolution of the optical microscope used during characterization may overestimate the size values for the obtained agents; also, larger probes popped up and occurred to be brighter. Thus, additional data are needed for accurate size distribution characterization.

FLIM demonstrated the micron size range of ST–FITC MBs and ST–FITC–AuNR MBs—sizes of all observed probes were less than 2 μm in the micron size range (Figure ). Fluorescence lifetime distribution over individual particles occurred to be narrow. It centered at 1.68 ns for ST–FITC MBs and 1.57 for ST–FITC–AuNR MBs (Figure S2). No correlations be<span class="Chemical">tween the particle size and fluorescence lifetime were observed, suggesting the dye’s homogeneous distribution over the MBs (Figure S3).

Two of the most common approaches in the characterization of microbubble size dispersion are OM and light-scattering methods such as DLS or nanoparticle tracking analysis (NTA); thus, we decided to additionally characterize the obtained probes with the use of DLS and NTA measurements. It is well known that both optical methods are widely used to detect and characterize particles in the submicron and even micron range, such as microparticles, protein or latex beads, exosome nanobubbles, and core–shell nanoparticles used for microbubble stabilization.[15,47−51] The values of the mean size obtained with both methods are presented in Table and demonstrated that all described probes had the submicron size range according to these methods.

Table 1

Microbubble Characterization at the Room Temperature (25 °C): Surface Tension Measurements of Solutions Used for Microbubble Preparation, DLS, NTA, and Zeta-Potential Measurements for Microbubbles

	last 2 min surface tension average of solution used for sample preparation (mN m–1)	mean size obtained by DLS measurements (nm)	mean size obtained by NTA (nm)	concentration (particles mL–1)	zeta potential (mV)
ST MBs	31.6 ± 0.3	560 ± 110	780 ± 290	1.6 × 108	–25 ± 4
ST–FITC MBs	33.8 ± 0.3	410 ± 60	570 ± 90	2.2 × 108	–26 ± 5
ST–AuNRs MBs	33.9 ± 0.4	550 ± 100	550 ± 220	5.7 × 108	43 ± 12
ST–FITC–AuNRs MBs	35.5 ± 0.2	520 ± 100	530 ± 330	5.2 × 107	42 ± 16

The DLS and NTA measurements tended to decrease the mean sample size than would appear according to the CLSM and FLIM results. Thus, additional characterization and analysis are required to perform in conjunction with DLS and NTA measurements. In contrast, only DLS and/or NTA measurements may differ from actual results, shifting into the submicron range. Under reporting of results can be associated with various issues. The first possible reason is the discrepancy be<span class="Chemical">tween the object of research—a microbubble of air in a shell of surfactants—with the analysis methods because optimal light scattering materials for such methods are dense particles such as metallic or <span class="Chemical">polymer materials. Accordingly, the underestimation of the bubble diameter can be explained by multiple internal reflections of light inside the bubble’s gas core, which cannot be observed in solid samples of similar size ranges. The second reason that should be considered is the possibility of the local heating of the sample when measured by microscopy, while DLS and NTA measurements were made at the preset room temperature. Then, we assumed that the bubble diameter should increase with increasing temperature, which can play a significant role during in vivo experiments. To confirm the above hypothesis, measurements of DLS were carried out also at the body temperature (37 °C), and the obtained results can be seen in Table .

Table 2

Comparison of DLS Measurements for Bubbles Carried out at Room Temperature (25 °C) and the Body Temperature (37 °C)

	mean size obtained by DLS at 25 °C (nm)	mean size obtained by DLS at 37 °C (nm)
ST MBs	560 ± 110	1650 ± 200
ST–FITC MBs	410 ± 60	800 ± 110
ST–AuNRs MBs	550 ± 100	1010 ± 80
ST–FITC–AuNRs MBs	520 ± 100	1210 ± 90

Indeed, the increase in size affects the measured diameter. Thus, it is necessary to take into account all factors when analyzing the size of bubbles and also remember about the effect of temperature on the increase in size, as well as the possibility of bubble <span class="Disease">rupture during overheating. The most reliable estimates of the size range of the agents were obtained using confocal fluorescence imaging because the use of OM for characterization required comparable optical resolution to prevent oversizing of the observed bubbles.

However, because of the peculiarities of NTA, the estimate of the concentration of the object for bubbles is quite fair e because the calculation of the number of bubbles is based on the direct observation of bubbles and tracking their trajectories during measurement: for all samples, except for ST–FITC–AuNRs MBs, the concentration was about 108 particles mL–1, while the ST–FITC–AuNRs MB sample had a lower concentration value (Table ), this can be described as the effect of functionalization on the surface tension and thus on the sample properties. As can be seen from Table , functionalization slightly increased the values of the surface tension of the solutions used for the further preparation of microbubbles, and the introduction of functional additives can explain this behavior; hence, functionalization (and the associated increase in the surface tension of the solutions used for probe production) can affect other properties of the bubbles, such as average size and concentration.

The zeta potential of the ST MBs and ST–FITC MBs showed moderate stability, and the functionalization of probes with AuNRs revealed good stability of the ST–AuNR MBs and ST–FITC–AuNR MBs and changes in the bubble charge: from negative to positive, as can be seen in Table . It is also important to mention that zeta potential measurements for the obtained samples did not reveal any significant changes within the storage period of 47 days, and the obtained values remained the same within observational errors, as shown in Table S2.

For a further introduction into clinical practice, studies of microbubble stability are required: thus, OM, DLS observations, and zeta potential measurements were carried out to assess the presence of a significant number of microbubbles in the sample and changes in size dispersion during storage. Therefore, in a refrigerator, no changes in the properties of the samples were observed within 47 days.

Also, observations of OM and CLSM for samples dried at room temperature (25 °C) for 24 h on a glass plate showed that the bubbles retained their rounded shape and gas inside, and a significant number of bubbles in the samples survived under these conditions as can be seen in Figure S4. Such results can also indirectly indicate the stability of the samples obtained. A drop of each probe sample with bubbles inside formed a thin film on the glass plate’s surface without microbubble destruction, which was observed during the OM studies. Still, placing a slide on top of a slide with dried films caused the bubbles to be destroyed, as seen in CLSM images.

Thus, functionalized probes with a micron size dispersion of fewer than 2 μm and stability for 47 days during storage were obtained. The next step was the evaluation of the functionality of the obtained samples for use in various imaging modalities: FL, OA, and US.

OA, FL, and US Imaging Characterization: a Promising Way for Multimodality

Next, potential applications of obtained probes were tested for FL, OA, and acoustic/US imaging modalities.

The results of FT measurements can be seen in Figure : samples with microbubbles functionalized with FITC only (ST–FITC MBs) demonstrated high fluorescence intensity, which was higher compared with the fluorescence intensity for <span class="Chemical">Tween–FITC aqueous solution because of the scattering and multiple reflections of light, which is a characteristic of a gas bubble. Both for aqueous solution of FITC and AuNRs and microbubbles functionalized with such additives, fluorescence quenching was observed; fluorescence intensity did not depend on the concentration and was significantly lower compared with sample-contained FITC only, still, the presence of bubbles in the sample led to a certain increase in intensity. Therefore, FL imaging modality can be significantly achieved with the ST–FITC MB probe.

Figure 5

Fluorescence imaging of obtained probes: comparison of total radiant efficiency dependencies on the concentration of FITC used for microbubbles functionalized with Tween–FITC (ST–FITC MBs) and both Tween–FITC and AuNRs (ST–FITC–AuNRs) with corresponding solutions used for probe preparation. The inlet shows fluorescence imaging of a plate with solutions containing Tween–FITC conjugate, ST–FITC MBs, and ST–FITC–AuNR MBs in concentrations used in the plot.

Fluorescence imaging of obtained probes: comparison of total radiant efficiency dependencies on the concentration of FITC used for microbubbles functionalized with <span class="Chemical">Tween–FITC (ST–FITC MBs) and both <span class="Chemical">Tween–FITC and AuNRs (ST–FITC–AuNRs) with corresponding solutions used for probe preparation. The inlet shows fluorescence imaging of a plate with solutions containing <span class="Chemical">Tween–FITC conjugate, ST–FITC MBs, and ST–FITC–AuNR MBs in concentrations used in the plot.

All functionalized samples revealed tangible absorbance properties, as can be seen in Figure a, probes functionalized with AuNRs (ST–AuNRs MBs and ST–FITC–AuNRs MBs) significantly absorbed light at a wavelength of 532 nm, which correlates with the laser excitation line for application in OA imaging (additional comparison of extinction characteristics for probes functionalized with AuNRs and the aqueous solution of AuNRs and its correlation with the concentration of samples can be seen in Figure S5), and ST–FITC MBs demonstrated appropriate extinction at the wavelength corresponded to the fluorescence excitation maximum for FITC (490 nm), which was used in FL modality. The ST MB sample did not contain any functionalization; thus, it did not absorb noticeably at marked wavelengths.

Figure 6

(a) Extinction spectra of obtained bubbles. Orange dot and its vertical line correspond to the fluorescence excitation maximum for FITC (490 nm), green dot, and its vertical line correspond to the OA excitation (532 nm). (b–d) RSOM measurements of AuNRs only, ST–AuNR MBs, and ST–FITC–AuNR MB samples. Scale bar represented in mm both in Z and Y axes, respectively. (e–g) Schematic representation of the agarose phantoms used for each measurement of AuNRs only, ST–AuNR MB, and ST–FITC–AuNR MB samples.

(a) Extinction spectra of obtained bubbles. Orange dot and its vertical line correspond to the fluorescence excitation maximum for FITC (490 nm), green dot, and its vertical line correspond to the OA excitation (532 nm). (b–d) RSOM measurements of AuNRs only, ST–AuNR MBs, and ST–FITC–AuNR MB samples. Scale bar represented in mm both in Z and Y axes, respectively. (e–g) Schematic representation of the <span class="Chemical">agarose phantoms used for each measurement of AuNRs only, ST–AuNR MB, and ST–FITC–AuNR MB samples.

Because AuNRs are well-known probes used for OA imaging implementation, even enhanced plasmonic photothermal therapy by AuNR delivery using microbubbles can be achieved; such a functional additive was chosen for the OA modality implementation.[52−55] Thus, ST–AuNR MBs and ST–FITC–AuNRs were tested comparing the AuNR aqueous solution by RSOM measurements (Figure b).

For control aqueous solution containing AuNRs only, the response primarily in low frequencies (with a range of 11–33 MHz) was achieved in the entire sample volume in the phantom; such a result was in good correlation with the known literature data. The presence of microbubbles in ST–AuNR MB and ST–FITC–AuNR samples led to the occurrence of a high-frequency OA signal in the range of 33–99 MHz with a central frequency of 50 MHz and its localization in the upper part of samples because of bubbles floating. The signal amplification to the high-frequency region can be associated with the micron-size range of the obtained probes.

Thus, for the ST–AuNR MB sample, the response in low-frequency ranges corresponding to the free AuNRs distributed over the entire sample volume and high-frequency response in the upper part of the droplet introduced by the presence of microbubbles and for ST–FITC–AuNR MBs, only high-frequency response were obtained; the quenching of FITC fluorescence can explain the lack of response for AuNRs and FITC distributed over the entire sample volume at the OA excitation wavelength of 532 nm, where AuNRs has excellent extinction properties, while the FITC probe without AuNRs demonstrated comparable emission properties at the same wavelength. Thus, the ability of OA contrast enhancement with the use of AuNR-containing probes was proven because of the excellent acoustic properties provided by the gas core of the microbubbles; additionally, a frequency of 50 MHz was validated as the optimal for OA imaging modality and suggested for further characterization for US imaging. Also, it is known that a frequency of 50 MHz for US imaging can be used in dermatology for epidermis and dermis visualization.[56,57]

Acoustic characterization revealed the possibility of use of the obtained probes as US contrast agents: US enhancement of 48 dB was demonstrated. All samples revealed significant acoustic response to the US at a frequency of 50 MHz compared with DI <span class="Chemical">water only without any acoustic response obtained, as shown in Figure .

Figure 7

Acoustic response of probes: (a) schematic representation of the experiment of US imaging at a frequency of 50 MHz and US imaging of (b–e) ST MBs, ST–FITC MBs, ST–AuNR MBs, and ST–FITC–AuNR MB sample, respectively. Scale bar represented in mm both in Z and Y axes, respectively.

Thus, the response of probes functionalized with AuNRs or FITC demonstrated the possibility of use in OA or FL imaging, respectively. All microbubble samples demonstrated excellent acoustic response for US imaging, provided by the gas core of the bubble.

Summary and Conclusions

Thus, the ultrasonic method for the synthesis of microbubbles of air with a shell based on surfactants was optimized. The optimum molar ratio of <span class="Chemical">Span 60/<span class="Chemical">Tween 80 surfactants was found based on surface tension measurements to obtain stable air-filled bubbles in the micron size range. Further, the functionalization of a probe was made for the possibility of use in OA imaging (by implementing AuNRs in the bubbles shell) and in FL imaging, while the gaseous core of a probe provided acoustic properties for use in US imaging. For the first time, a covalent label—a dye—FITC was introduced into the structure of surfactants, which makes it possible to easily visualize the resulting microbubbles using confocal microscopy, which also made it possible to most accurately determine the size of the resulting microbubbles.

A detailed analysis of the optimal methods for characterizing air microbubbles based on surfactants was carried out. It has been shown that confocal and FLIM microscopy are the optimal methods for determining bubbles’ size. Light scattering data underestimate the samples’ actual size, while the optical microscope lacks resolution and overestimates the size.

Multimodal imaging probes can be obtained with the samples described above: the highest FL signal was demonstrated with the ST–FITC MB sample, while ST–AuNR MBs and ST–FITC–AuNR MBs demonstrated significant OA properties at the high-frequency region and its center frequency of 50 MHz. A remarkable acoustic signal for all samples was obtained at a frequency of 50 MHz, proving the possibility of use of the obtained probes as contrast agents for US imaging because of the air core properties. Also, storage stability for up to 47 days and the possibility of using it under physiological conditions demonstrate the possibility of further using the obtained samples in clinical practice.

Gas-filled surfactant-shelled microbubbles functionalized with FITC–AuNRs provided the possibility of use as a successful multimodal contrast agent for most clinical relevant methods, such as US, fluorescence, and/or OA imaging. The <span class="Chemical">Span 60–<span class="Chemical">Tween 80 combination of surfactants can be considered as a model system for testing of multimodal probes because of ease and low cost of production. Suggested types of contrast agents are relevant because of the rapidly growing numbers of preclinical and clinical devices combined with such imaging modalities.

Experimental Section

Materials

Sorbitan mono<span class="Chemical">stearate (<span class="Chemical">Span 60, MW 431), <span class="Chemical">polyoxyethylene sorbitan monooleate (<span class="Chemical">Tween 80, MW 1307) were purchased from Croda (Croda International Plc, UK) and used without further purification. <span class="Chemical">Chloroauric acid (<span class="Chemical">HAuCl4·3H2O), FITC isomer I (<span class="Chemical">C24H11NO5S), <span class="Chemical">hexadecyltrimethylammonium bromide (<span class="Chemical">CTAB) (<span class="Chemical">C19H42BrN), <span class="Chemical">sodium borohydride (<span class="Chemical">NaBH4) from <span class="Disease">Aldrich, silver nitrate (<span class="Chemical">AgNO3), <span class="Chemical">N,N-dimethylformamide (<span class="Chemical">DMF anhydrous 99,8%) <span class="Chemical">triethylamine (99%), and <span class="Chemical">agarose were purchased from Sigma-<span class="Disease">Aldrich. DI <span class="Chemical">water with specific resistivity, higher than 18.2 MΩ m from a Milli-Q Integral 3 <span class="Chemical">water purification system (Millipore, MA), was used to make all solutions.

AuNR Synthesis

The small Au nanorod samples were prepared by the seed-mediated growth method in aqueous solutions through changing the seed-to-Au(III) molar ratio in the growth solution. During the preparation, <span class="Chemical">cetyltrimethylammonium bromide (<span class="Chemical">CTAB) was employed as the stabilizing surfactant, which is similar in structure to the surfactants used for microbubble preparation, which allowed Au nanorods to be incorporated into the bubble structure without causing a significant change in properties. The seed solution was made by adding a freshly prepared, ice-cold <span class="Chemical">NaBH4 solution (0.6 mL, 0.01 mol) into a mixture solution composed of <span class="Chemical">HAuCl4 (0.25 mL, 0.01 mol) and <span class="Chemical">CTAB (9.75 mL, 0.1 mol) under vigorous stirring. The resultant solution was stirred for 2 min and then kept at room temperature for at least 2 h before use. The growth solution was made by the sequential addition of <span class="Chemical">HAuCl4 (0.5 mL, 0.1 mol), <span class="Chemical">AgNO3 (0.1 mL, 0.01 mol), and HCl (0.2 mL, 1.0 mol) into <span class="Chemical">CTAB (9 mL, 0.1 mol). A freshly prepared <span class="Chemical">ascorbic acid solution (0.8 mL, 0.1 mol) was then added under rapid stirring. Once, the resultant solution became colorless, the seed solution (2 mL) was then added into the growth solution under vigorous stirring. The resultant solution was kept under stirring for 2 min and then left undisturbed overnight.

Tween–FITC Preparation

The synthesis of the complex of FITC with <span class="Chemical">Tween 80 was carried out in a solution of <span class="Chemical">dimethylformamide (0.5 mL) in the presence of <span class="Chemical">triethylamine (0.01 mL), successively dissolving <span class="Chemical">Tween 80 (337 mg, 0.257 mmol) and excess FITC (20 mg, 0.051 mmol), left for 12 h at stirring, and washed.

Surface Tension Measurements

Custom software, implemented in Matlab (Mathworks, Natick, MA) and described in detail previously, was used to capture and process pendant drop images for surfactant characterization.[58,59] The actual update rate of the surface tension measurements, droplet volume, and surface area was 2 s, constrained by the time needed to perform the necessary calculations on a particular computer used by us (Dell Latitude 7280). At the preprocessing step, the pendant droplet boundary was determined by converting a grayscale image into a binary image by using the threshold value calculated by Otsu’s method.[60] For each row of pixels, a midpoint be<span class="Chemical">tween boundary points of the droplet was determined. The vertical centerline position that divides the droplet into two symmetrical halves was then obtained by averaging midpoint values obtained for each row of pixels. The surface tension of the drop was then found by solving the Young–Laplace equation at each time point by using the system identification theory that minimizes the difference be<span class="Chemical">tween the theoretically predicted and the imaged shape of the interface.[61] For each measurement, a pendant drop was formed, and surface tension was obtained in real time for 10 min at room temperature using described Matlab software. The measurement was repeated 3 times. The final result represents the average surface tension and standard deviation of the last 2 min of the 3 repeated measurements.

Mass Spectrometry Measurements

The analysis was performed using a rapifleX MALDI-TOF/TOF MS system (Bruker Daltonik GmbH, Germany) time-of-flight mass spectrometer with matrix laser desorption/ionization (MALDI-TOF/TOF). The operating mode was the following: reflector mode, positive ionization, analysis range m/z 400–3000, accelerating voltage 20 kV, SmartBeam III laser, laser frequency 10 kHz, and frequency 200 Hz. Before analysis, the instrument was calibrated using a mixture of peptides “Peptide Calibration Standard II” (Bruker Daltonik GmbH, Germany). The mixture included peptides with a mass range of 700–3200 Da. <span class="Chemical">2,5-Dihydroxybenzoic acid (Bruker Daltonik GmbH, Germany) with purity >99.0% was used as a matrix. A matrix solution with a concentration of 20 mg/mL was prepared in a mixture of 30% <span class="Chemical">acetonitrile/70% <span class="Chemical">water/0.1% <span class="Chemical">trifluoroacetic acid. Aqueous solutions of the samples were mixed with the matrix in a ratio of 1:1, and 1 μL of the mixture was applied to the plate.

Microbubble Preparation

Microbubbles were obtained by the modified sonication method.[32,33,36,62] Briefly, <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 were dissolved in DI <span class="Chemical">water in the desired molar ratio to obtain <span class="Chemical">Span–<span class="Chemical">Tween solution. Then, a portion of <span class="Chemical">Span–<span class="Chemical">Tween solution (2 mL) was used to prepare air-filled bubbles coated with the shell consisting of <span class="Chemical">Span 60 and <span class="Chemical">Tween 80 (ST MBs). To functionalize bubbles with AuNRs, AuNR solution (1 mL) was added to the <span class="Chemical">Span–<span class="Chemical">Tween solution (1 mL) before the bubble preparation (ST–AuNR MBs and ST–FITC–AuNR MBs). For samples labeled with FITC (ST–FITC MBs, ST–FITC–AuNRs MBs), the <span class="Chemical">Tween 80–FITC solution (0.5 mL) was added to the portion of the required solution (2 mL). All samples were stored in a glass vial and heated to a temperature of 50 °C to lower the solution’s surface tension. Each sample was sonicated for 180 s at the maximum power of 100 W using the Bandelin SONOPULS HD4100 sonicator with the TS103 sonotrode probe (Bandelin Electronic GmbH & Co KG, Germany). The tip of the sonotrode was placed at the interface be<span class="Chemical">tween the phases of liquid solution and air. After sonication, each sample was stored at 4 °C for 30 min for further stabilization.

Optical Microscopy

OM was carried out on Olympus CX33 (Olympus Corporation, Japan), CLSM was carried out on a Axio Observer. Z1/7 with Plan-Apochromat 40×/1.3 Oil DIC (UV) VIS-IR M27 objective (Carl Zeiss Microscopy GmbH, Germany). FLIM measurements and image processing were carried out on a MicroTime 200 STED microscope (PicoQuant GmBH, Germany), where a 402 nm laser was used as the excitation source and a 425 nm bandpass filter was applied. Measurements were made at a pulse rate of 80 MHz, a pulse duration of 40 ps, and optical power of 85 μW for microbubbles functionalized with AuNRs and FITC and optical power of 59 μW for microbubbles functionalized with FITC only. Fluorescence lifetime images were acquired in the time domain. The laser beam was focused on microbubbles with a 100 × 1.4 <span class="Chemical">NA oil immersion objective (UPlanSAPO, Olympus, Japan). According to a dwell time of 0.2 ms with a pixel size of 0.200 μm, the total image acquisition time was 40 s for an image size of 400 × 400 pixels—80 × 80 μm.

Transmission Electron Microscopy

Micrographs of the samples were obtained on a Titan Themis Z transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) with an operating accelerating voltage of 100 kV. Samples were prepared by applying 3–5 μL of the solution onto a formvar-coated <span class="Chemical">copper mesh with a diameter of 3.05 mm, which was then dried in air.

DLS Measurements

DLS and zeta-potential measurements were performed on the ZetaSizer Nano ZS analyzer (Malvern Panalytical, UK); all measurements were repeated three times. For zeta-potential measurements, all samples were diluted 20 times in DI <span class="Chemical">water and placed in U-cuvette; for DLS measurements, all samples were diluted 40 times in DI <span class="Chemical">water and placed in a plastic cuvette; and Zetasizer Software 8.00 was used for results processing. DLS measurements were carried out at 25, 37 °C, and zeta-potential measurements were carried out at 25 °C.

NTA Measurements

The NTA characterization was performed immediately after sample preparation. Prior to analysis, if necessary, to achieve a proper microbubble concentration, samples were diluted 1:10 in DI <span class="Chemical">water. The sample was then injected into the test cell and illuminated by a 45 mW blue laser (488 nm wavelength, NanoSight model NS-300, Malvern, Salisbury, UK). The scattered light was video recorded using the built-in high sensitivity sCMOS camera for 60 s at 25 frames per second with 0.1 ms shutter speed. The recording was repeated 5 times for each sample keeping the camera level at 1. Each video consisted of 1498 frames that captured at least 2000 valid particle tracks. Approximately 10–30 particles were observed in the field of view during video capture, which corresponds to a concentration of ∼2–9 × 108 particles per milliliter. The videos were analyzed by NTA software (version 3.2) to produce the size distribution of microbubbles, its mode, mean, and standard deviation. The following parameters were used during the analysis: the minimum track length and blur size were set to auto, and the detection threshold was set to 30. The cell’s temperature was measured automatically by the NTA instrument and stayed within 296.15–297.15 K during room temperature measurements or at 37 °C throughout the nanoparticle tracking by using the integrated temperature control unit. The viscosity of DI <span class="Chemical">water at this temperature is nearly constant and equal to ∼0.91 cP at room temperature and 0.7 cP at 37 °C.

Extinction Spectra Measurements

Extinction spectra were measured using a multifunctional microplate reader Tecan Infinite M Nano+ (Tecan Trading AG, Switzerland) at room temperature (25 °C), where samples were placed in a plastic 96-well plate. All samples were diluted in DI <span class="Chemical">water, with the following concentrations (0.01, 0.02, 0.1, 0.2, and 0.5, 1) compared with the original one.

FT Measurements

For FT measurements, each sample was diluted in DI <span class="Chemical">water and added in a 96 well plate with the following concentrations (0.01, 0.02, 0.1, 0.2, 0.5, and 1) compared with the original one. The plate with samples was then imaged by the IVIS CT Spectrum In Vivo system (Xenogen Corp., CA) at room temperature (25 °C). Sequence images were acquired with the excitation/emission pair for FITC (490/525 nm). Exposure time is auto, FOV = C. Photons were quantified with Living Image software (Xenogen Corp., CA).

RSOM Measurements

RSOM system Explorer P50 (iTheraMedical GmbH, Germany) was used to collect OA signals from microbubbles. The OA signals were collected by a custom-made spherically focused LiNbO3 detector (center frequency—50 MHz; bandwidth—11–99 MHz; focal diameter—3 mm; focal distance—3 mm). The samples were irradiated by a frequency-doubled flashlamp-pumped Nd:YAG laser (532 nm, pulse length—2.5 ns; 200 μJ pulse–1; repetition rate—1–2 kHz). Light from the laser is delivered through a glass fiber 2-arm bundle (spot size—3.5–5 mm). The scanning head is mounted to 2 motorized stages (field view up to 12 × 12 × 4 mm). The samples were tested in the <span class="Chemical">agarose phantom: for the preparation of a phantom, <span class="Chemical">agarose (100 mg) was diluted in DI <span class="Chemical">water (10 mL) at room temperature, then the solution was stirred intensively at a temperature of 373.15 K and then degassed to avoid the presence of small air bubbles in the solution. Briefly, for a phantom formation, a droplet of <span class="Chemical">agarose (43 μL) was placed on the bottom of the reservoir, and then after 20 s, a droplet of a sample (7 μL) was injected into the upper third of the formed <span class="Chemical">agarose droplet, forming a liquid reservoir of the sample inside the phantom. Additional storage at fridge conditions (4 °C) was applied for 15 min to solidify the phantom, and then, the phantom with the sample was covered with a layer of DI <span class="Chemical">water (1.5 cm) to carry out the measurements. The scan head was coupled to the sample by a <span class="Chemical">water-filled reservoir, and the samples were scanned over the field of view (8 × 8 mm) with the predefined depth (4 mm).

Acoustic Characterization

DUB SkinScanner equipment (taberna pro medicum GmbH, Germany) was used for acoustic characterization as high-frequency US (HIFU) imaging system, and a linear B-scan applicator with a frequency of 50 MHz was placed at the interface of the desired solution, avoiding the air bubble appearance be<span class="Chemical">tween the applicator and the sample. The custom-made plastic cuvette was made with the following parameters (30 × 35 × 5 mm) and two protrusions on the sides (5 × 35 × 2 mm) for the ease of the applicator placement sample–air interface boundary. The received signals from the applicator were processed using DUB SkinScanner software (taberna pro medicum GmbH, Germany). All measurements were carried out at body temperature (37 °C).