Free gas micro-/nano-bubble water: a novel dispersion system to prepare ultrasound imaging vehicles

Materials

ePC, PC-98T, C_PC>98 %, injection grade), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), were obtained from Shanghai Advanced Vehicle Technology Pharmaceutical L.T.D. Co. (AVT, Shanghai, China); PEG_4k was provided from Guangdong Guanghua Sci-Tech Co., Ltd. (Shantou, China); SF₆ with the purity of 99.99 % was purchased from Anhui Qiangyuan Gas Co., Ltd. (Wuhu, China); Bazedoxifene acetate was purchased from Acesys Pharmatech (Fairfield, NJ, USA). All other solvents and reagents were analytical purity and used without any purification.

Generation of SF₆ MNBs water

The schematic diagram of experiment setup for generation of SF₆ MNBs water was shown in Figure 1a referred to our previous work [21]. According to Figure 1, the distilled water (A) was pumped while SF₆ gas (B) was adopt through a gas flow regulator (M) into a gas-liquid mixed rotary pump (C) (15WSC04, Daile, Shanghai, China) under a pressure of 0.3–0.4 MPa to make more dissolved gas in water, and after water was released under atmospheric pressure, free gas bubbles (F) will be generated. Large bubbles could be cleared through the buoyancy of stagnant flow in a gas-liquid separator, and the released nanobubbles were transported into a reservoir whose outflow was the standby product of MNBs water. The whole procedure was operated under isothermal conditions at 25 °C. The ultimate production of MNBs water can be collected after the system stably circulated for 5 min.

Meanwhile, the degassed water prepared by boiling for 30 min and cooling to room temperature was used as the control.

Characterization of SF₆ MNBs water

Prepare shelled bubbles using SF₆ MNBs water

Characterization of shelled bubbles

Ultrasound imaging evaluation in vitro

Ultrasound imaging capacity of the obtained bubbles were studied in vitro. Samples were added into wells of a custom-made 3 % (w/v) agarose mold. The imaging performance were evaluated on VisualSonics micro-imaging Vevo^® 2,100 systems (Fuji Film Visual Sonics Inc., USA) and a transducer of MS250 was employed. Frequency was set at 21 MHz and the acquisition contrast gain was 30 dB. All parameters were not changed throughout all imaging acquisition. Before sampling, degassed water was scanned prior to injection to confirm a clear background signal. All samples were bolus injected and be started to image immediately. The mean power data in B-mode were analyzed according to the cross section of whole well, which was set as the Region of Interest (ROI).

Statistical analysis

The Student’s t-test was used for processing statistical analysis and p value was determined via Prism 7.0 (GraphPad Software, Inc.). If p<0.05, difference was considered to be statistically significant.

SF₆ MNBs water exists tyndall phenomenon

We employed the Tyndall phenomenon to test whether there have particles in the system over a long period of time (∼48 h), the degassed water and the obtained SF₆ MNBs water were placed in vials, respectively. Results are shown in (Figure S1, Supplementary Materials), which shows the Tyndall phenomenon observed in SF₆ MNBs water groups at different times. Each time point has two photos which represent the directions of laser from right to left and from left to the right, respectively. We can see no matter which direction of the laser, SF₆ MNBs waters groups (right vial in each photo in Figure S1) in every time point display the phenomenon of Tyndall, but not the groups of degassed water (left vial in each photo in Figure S1), which suggests the micro-/nano-paritcles exist in SF₆ MNBs waters over 48 h. Since the experiment was carried out in sunlight, the intensity of sunlight affected the observation of Tyndall phenomenon by naked eyes. Therefore, the results only qualitatively indicate the presence of colloidal particles within two days.

SF₆ MNBs water can separate out nanobubbles

When the silicon wafer is immersed into SF₆ MNBs water, the bubbles will be adsorbed onto the substrate [22]. We use AFM to observe whether there are separated bubbles from water. Results of SF₆ MNBs water group are shown in Figure 2. From the obtained two-dimensional and three-dimensional images in Figure 2, it can be seen that there are height changes in the range of 2×2 μm on silicon substrate, but in degassed water group it is not observed any height change, no difference from the substrate image (data not shown). Thus, the existence of gas bubbles in SF₆ MNBs water was indirectly validated.

Figure 2:

2D and 3D AFM images of SF₆ MNBs water scanning range is 2×2 μm on the wafer. Degassed water cannot be detected.

The addition of NaCl or bazedoxifene will increase the size of particles in SF₆ MNBs water

In this experiment, we firstly determined the particle size distribution of SF₆ MNBs water and degassed water. Furtherly, if bubbles presents, the introduction of molecules such as inorganic ionic compound NaCl or organic molecular compound bazedoxifene acetate should vary the size distribution. Figure 3 shows the results particle size distribution map of SF₆ MNBs water and addition of NaCl (Figure 3a) or bazedoxifene (Figure 3b). From Figure 3a, we can see most of the particle size distributed at 141.77 nm, accounting for 94.688 % of total scattering intensity. After adding sodium chloride, the particle size is distributed at 122.42–396.06 nm, indicating the particle size shifts to the right and becomes wider after NaCl addition. The introduction of the NaCl electrolyte has an effect on the bubbles distribution which results the particle size becomes larger and the distribution becomes wider. Figure 3b displays the particle size distribution of the same concentration of bazedoxifene dissolved in MNBs water and degassed water, MNBs water is still placed in Figure 3b as a refer. After the bazedoxifene added, the particle size is distributed at 164.18–255.00 nm, accounting for 66.640 % of total scattering intensity, also with size getting larger. Besides, the drug has a particle size peak at 58–68 nm when dissolved in degassed water, which may due to impurities or insoluble components in drug powder.

Figure 3:

Size distribution map and correlation curves (a) is size distribution of pure SF6 MNBs water and SF6 MNBs water with NaCl added. (b) Is size distribution of bazedoxifene dissolved in SF6 MNBs water and degassed water, respectively. (c) Is the correlation data of SF6 MNBs water. (d) Is the correlation data of SF6 MNBs water with NaCl added. (e) Is the correlation data of SF6 MNBs water with bazedoxifene dissolved into.

Since the polydispersity index (PDI) of the MNBs water is measured to be 1, the particle size cannot be expressed by Z-Average, and can only be observed by plotting the particle size distribution curve with Intensity as the vertical axis, with further verification by Raw Correlation Data. Figure 3c–e displays the correlation coefficient curves of MNBs water (c) and its sodium chloride solution (d) and its bazedoxifene solution (e). It can be observed that the decay curves of the three are not smooth, indicating that particles larger than 1,000 nm may exist in the system, which could be micrometer-sized bubbles within the system or larger clusters of nanobubbles. The absolute values of the slopes for the three are (c)>(e), (c)>(d), showing DPI of MNBs water increases after the addition of sodium chloride and bazedoxifene, especially the group of addition of sodium chloride (Figure 3d), where the decay angle increases and the PDI increases, from which, the clustering and merging of nanobubbles can be inferred.

The addition of NaCl or bazedoxifene can actually cause bubbles aggregation or form bubble clusters [11]. Uchida et al. reported the effect of NaCl on the particle size and stability of nanobubbles in water. It was found that low concentration of NaCl can stabilize bubbles due to shielding effect, while high concentration causes bubbles to disappear [23], which was speculated the high concentrations of NaCl can thin double layers of the bubbles [24]. Although NaCl would decrease the negative zeta potential, the hydration effects caused by NaCl, may disrupt the hydrogen bonding network between MNBs water molecules [25]. In a word, the results in this section indirectly confirmed the presence of nanoscale free bubbles in the MNBs water.

Surface tension is decreased in SF₆ MNBs water

In order to examine the adsorptions of lipids onto the bubble surface, surface pressure of SF₆ MNBs water and upon addition of DPPC with increase of time were measured. The distilled water was comparatively operated. The results are shown in Figure 4a, it can be seen that the surface pressure at equilibrium of SF₆ MNBs water is lower than that of distilled water, which is exactly consistent with the previous reports [26]. It is confirmed that surface tension of the nanobubbles dispersion was decreased [27]. The breakdown of hydrogen bonds between water molecules and ionization in the water are the main factors causing these changes [26]. Therefore, the addition of same concentration of DPPC in the bubbles dispersion would decrease the surface tension, whilst change of surface pressure could be contrarily increased in the distilled water. Immediate reduction of the surface pressure of group of DPPC added SF₆ MNBs water represents excess molecules at air-water surface were less than that in group DPPC added degassed water, like the surface is stretched, which indicates there might be an overall transport of monomer from the air-water interface to the numerously existed microscopic interface in bulk [28]. Therefore, microscopic gas/water interface in bulk liquid may lower their surface tension more efficiently and the low molecular weight surfactants including the phospholipids are prone to be absorbed on the interface resulting in stabilize the bubble [29].

Figure 4:

Surface pressure curves of different dispersions (a) is surface pressure vs. time change. The degassed water, SF6 MNBs water, and DPPC added into each were determined. (b) Represents the surface pressure vs. surface area vary. Including degassed water, SF6 MNBs water, and with DSPC added into were determined in sequence. Test temperature is 25 °C.

Figure 4b represents the results of the Π-A isotherm curve of DSPC added degassed water and SF₆ MNBs water. It has been reported the surface pressure of DSPC at air-water macro interface decreases with area at 20 °C, and the phospholipid film appears in different states [30], that is, the phospholipid membrane is in a gaseous phase (gas phase, G) at 0–1.0 mN/m, is liquid expanded phase (LE) about 1.0–4.0 mN/m, and becomes liquid condensed phase (LC) at 4.0–60 mN/m, then gets into solid state (Solid phase, S) when 60–75 mN/m, and finally collapse at 75 mN/m. It can be seen from Figure 4b that as the area decreases, the monolayer phospholipid molecules in both systems undergo G, LE, LC to S, and in degassed water system it has collapse at 70 mN/m. Moreover, in the states of LC and S, the surface pressure of MNBs water system is much lower than that of degassed water at the same area, which furtherly indicates that phospholipid molecules number at macroscopic gas-liquid interface is reduced or the area occupied by a single molecule becomes larger. It may due to the phospholipid molecules transferred into the bubble interface in bulk phase.

Therefore, the presence of nanobubbles in bulk phase can effectively reduce the surface pressure. And some low-molecular-weight surfactants, including phospholipids, are adsorbed to the gas-liquid interface then stabilizing the bubbles [29]. Although phospholipid molecules are water insoluble, the presence of nanobubbles in water provides a nano gas-liquid interface that facilitates their self-assembly at interface.

SF₆ MNBs water has the potent capacity to prepare nano-sized shelled bubbles

The mean particle diameter and the PDI were determined by photon correlation spectroscopy. We examined whether shaking times alter the size or the PDI of bubbles and results are presented in Figure 5. It can be seen from Figure 5a that size differences are significant between the different formulations. Compared with the ratio of 1:10 for m (ePC): m (PEG_4k), the sample of group of m (ePC): m (PEG_4k)=1:1, has lower size and PDI values. Meanwhile, the shaking time also influences the size distribution. In formulation of m (ePC): m (PEG_4k)=1:10, MA method results in the largest size and highest PDI value when shaking for 60 s. The reason may be that increase of shaking time would enhance the possibility of bubbles collision leading to large bubbles yielded. MA method can lead to a heterogeneous size distribution of the microbubbles, which was influenced by the formulation composition, shaking strength and time [31]. Group of SF₆ MNBs water has no significant differences with the group of shaking 20 s in both size and size distribution, but has lower values than those of shaking 60 s. Considering MNBs water preparation method only requires static incubation, which eliminates the size distribution differences caused by shaking, results here suggest incubation of SF₆ MNBs water and lyophilized lipids is potential to obtain nano-sized and even shorter distributed shelled bubbles.

Figure 5:

Size (a) and size distribution (b) of different formulations and shaking time 1:1 and 1:10 in each horizontal axis represented the ratios of ePC and PEG_4k by weight. Shaking different times after reconstruction were investigated.

Incubation of SF₆ MNBs water and lyophilized lipids results with less foams

Figure 6 shows the appearance and TEM results of selected formulation of m (ePC): m (PEG_4k)=1:1 and meanwhile shaking time for 20 s. It can be seen there are large foams floating in the upper vials, which may be caused by low concentration and low repulsion of PEG_4k [32]. But it appears more foams in MA group, which may relate with the shaking strength that mechanical energy generated by the shaking will provide numbers of interfaces [33] to form bubbles in the system. It thus can be inferred that shaking is easy to generate large bubbles or even foam, which will affect the common use of the product.

Figure 6:

Appearance and TEM results of lipid bubbles obtained by SF₆ MNBs water and MA method. Formulation of m (ePC): m (PEG_4k)=1:1 was selected to investigated. (a) Is the appearance of lipid bubbles prepared by SF₆ MNBs water and (b) and (c) are the TEM. (d) Is the appearance of lipid bubbles obtained by shaking method (20 s) and (e) and (f) are its TEM.

From the TEM results in Figure 6b we can see bubbles prepared by SF₆ MNBs water are round shape, and there are mainly two types of bubbles, the large ones are about 200 nm, while the small micelles are about 50 nm. Figure 6c has been supplemented with TEM results of bubbles obtained using the same method but from a different field of view. As seen in Figure 6e, particles obtained by the shaking method can be seen conspicuous large particles with different sizes which internally distributed with voids that may be caused by gas. In addition, the large particles manifest with a circular or elliptical shapes. Bubble collision caused by the shaking may be one reason of ellipse. The MA method also yielded some nanobubbles with sizes around 200 nm, although no distinct gas core was observed (as shown in Figure 6f). However, as seen in Figure 6d, the MA method tends to cause collisions between bubbles, leading to fusion and the potential formation of excessive foams.

The PDI obtained by DLS method (Figure 6b) represents the behavior of the particle population, such as group of 1:1 MA method with shaking 20 s, which have a low PDI, while TEM only indicate differences in structure. It is true that the particle size distribution appears to have a certain width (Figure 6b), but multiple TEM images may be needed for statistical analysis to indicate the PDI. Secondly, TEM measurements are conducted under dry and vacuum conditions, which may lead to slight alterations in particle size. Particularly when the gas content is relatively high, the escape of gas could result in morphological changes, such as an increase in size and a more rounded shape. Additionally, for the micro-/nano-bubble system, the rising of microbubbles may also cause the possibility of deviation in the results during sampling and DLS measurement. These possibilities may contribute to discrepancies between the size distributions obtained from TEM (Figure 6) and those from DLS (Figure 5).

Lipid bubbles obtained by SF₆ MNBs water has no significant difference in ultrasound imaging level with those obtained by MA method in the initial 5 min in vitro

Ultrasound imaging was evaluated by the custom-made 3 % (w/v) agarose mold. The schematic setup of ultrasound transducer was shown in Figure 7a. Formulation of m (ePC): m (PEG_4k)=1:10 was selected, 1 mL of the lipid bubbles obtained by the two preparation methods (shaking time was 60 s), were added into phantom, respectively. Ultrasound imaging data were collected at 1, 2, 5 min after the samples injection. The representative B-mode and contrast images was displayed in Fig. S2 (Supplementary Material) and the B-mode mean power at each point was analyzed in Figure 7b, which quantitatively demonstrates the ultrasound imaging ability of each sample during the first 5 min. It can be seen that the gray scale of the bubbles obtained by the two methods decreases with time, and there were no significant differences between the SF₆ MNBs water method and the MA method (p>0.05) in the initial 5 min in vitro, which indicates bubbles prepared by SF₆ MNBs water has a promising application in enhanced ultrasound imaging. Although it has been reported that ultrasound imaging ability of the nano-sized lipid bubbles obtained by MA method is weak [34], the initial infusion of nanobubbles may contact each other closely in agarose mold, which creates a larger acoustically reflective interface.

Figure 7:

The ultrasound contrast enhancement ability of lipid bubbles (a) schematic setup of the ultrasound imaging in vitro. (b) Gray-scale in B mode changes with time of lipid bubbles obtained by SF6 MNBs water and MA method. The formulation was m (ePC): m (PEG4k)=1:10, shaking time is 60 s, #p>0.05 (n=3, Mean ± SD).