ISSN 1998-0124 CN 11-5974/O4

2019, 12(1): 000–000 https://doi.org/10.1007/s12274-020-2732-x

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Temperature-regulated self-assembly of lipids at free bubblesinterface: A green and simple method to prepare micro/nanobubbles Juan Jin1, Fang Yang1 (), Bin Li1, Dong Liu2,3, Lihong Wu4, Yan Li1, and Ning Gu1 ()

1 State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Sciences and Medical

Engineering, Southeast University, Nanjing 210096, China 2 West Anhui University, Lu’an 237012, China 3 Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resources, Lu’an 237012,

China 4 CSPC Zhongqi Pharmaceutical Technology (SJZ) Co., Ltd., Shijiazhuang 050035, China © Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 Received: 10 January 2020 / Revised: 24 February 2020 / Accepted: 25 February 2020

ABSTRACT Micro/nanobubbles play an essential role in ultrasound-based biomedical applications. Here, a green and simple method to fabricate micro/nanobubbles was developed by the temperature-regulated self-assembly of lipids in the presence of free bubbles. The self-assembly mechanism of lipids interacting with gas-water interfaces was investigated, and the ultrasound imaging of the obtained lipid-encapsulated bubbles (LBs) was further confirmed. Above the phase transition temperature (Tm), fluid lipids transform from vesicles to micelles, and further assemble to the free bubbles interface to be a compressed monolayer, resulting in lipid shelled microbubbles. Cooling below Tm induces the lipid shell to glassy state and stables the LBs. Moreover, increasing the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2K) content in lipids formulation can further manipulate the shell curvature and reduce the LBs size into nanobubbles. LBs with diameters of 1.68 ± 0.11 μm, 704 ± 7 nm and 208 ± 6 nm were successfully prepared. The in vitro and in vivo ultrasound imaging results showed that all of the LBs had excellent echogenicity. The nanosized LBs revealed elongated imaging duration time and greater microvascular details for the liver tissue. Avoiding the organic solvent and complicated multiple preparation process, this method has great potential in construction of various multifunctional micro/nanobubbles with size control for theranostic applications.

KEYWORDS lipid, self-assembly, gas–liquid interface, microbubbles, nanobubbles, ultrasound imaging

1 Introduction Because of their acoustic response, micro/nanobubbles have been widely employed in biomedical fields such as ultrasound imaging, drug and gene delivery, and disease therapy. A bubble consists of a gaseous core and stabilizing shell, usually made of one or more surfactants (including lipids, proteins, and polymers) [1]. Among the variety of different shells, lipid shells are very flexible and provide an excellent response to ultrasound radiation [2]. Furthermore, lipids are the most versatile shell materials due to their good biocompatibility and easy modifi-cation. Currently, a higher number of lipid-shell microbubbles, such as Definity, Imagent and SonoVue, are approved for clinical ultrasound imaging [3].

In recent years, different methods have been developed to prepare lipid-encapsulated bubbles (LBs), including sonication [4], thin-film hydration [5], and microfluidics [6], among others. The use of organic solvent is often required to disperse lipids in the solution, which has raised environmental and toxicity concerns during preparation, and safety concerns of the trace organic residues for biomedical applications. Besides, complicated

processes are usually needed to obtain LBs, including lipid dispersion, the subsequent organic solvent removal, mix with gas and posttreatments. Microfluidics techniques can generate microbubbles with uniform size but suffer from low production rates [6]. Improving the degree of bubble characteristics and production rate inevitably increases requirements for equipment and preparation processes. Moreover, the micrometer-sized LBs are often fabricated to be mainly applied for ultrasound contrast imaging of large blood vessels [7]; the imaging of microvascular details is limited due to their large size. Therefore, it would be highly desirable to design a green and efficient route to prepare LBs with size control from micrometer to nano-meter levels for biomedical applications.

Phospholipid molecules, natural amphiphilic surfactants, can self-assemble into organized structures such as micelles, vesicles and lamellar structures in solution. The stability of these assemblies can be modulated by the phase behavior of lipids, which in turn can be adjusted by temperature [8]. Temperature often plays an important role in the self-assembly of lipids into different structures [9, 10]. In the presence of gas– water interface, molecules would interact with the interface

Address correspondence to Ning Gu, guning@seu.edu.cn; Fang Yang, yangfang2080@seu.edu.cn

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and assemble into ordered state, which could be designed to produce micro/nano structures [11–15]. Free bubbles in bulk water provide excellent gas–liquid interfaces for the assembly of lipids. In recent years, the scale-up generation of free bubbles and their application in water treatment [16, 17], aquaculture [18], and seed germination [16] at industrial levels has been reported. Free bubbles can be generated through a variety of methods, such as swirl flow, ejector, cavitation, pore, and sonication [19]. We previously developed a pressure-driven method to prepare free bubbles by repeatedly compressing gas into water [20]. Given the easy generation of free bubbles, we hypothesize that micro/nanobubbles can be prepared simply and effectively through the self-assembly of lipids at free bubbles interface.

In this work, the self-assembly of lipids in solution was regulated by temperature in the presence of free bubbles, and LBs were successfully prepared. The assembly mechanism was investigated in details. Nanobubbles were further prepared by regulation of lipid formulation. The potential of different sized LBs as ultrasound contrast agents was examined and compared in vitro and in vivo. The described approach provides a simple and green route with industrial preparation prospects to fabricate microbubbles and nanobubbles, which will broaden the application of LBs-mediated ultrasound-assisted diagnosis and treatment in the future.

2 Experimental

2.1 Materials

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmit- oyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-gly- cero-3-phosphocholine (DMPC) and egg phosphatidylcholine (ePC; PC-98T) were purchased from Shanghai Advanced Vehicle Technology Pharmaceutical Ltd., Co. (AVT, Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2K, purity > 99%) was obtained from Southeast Pharmaceuticals Co., Ltd. (Suzhou, China). Sulfur hexafluoride (SF6) with purity of 99.99% were purchased from Anhui Qiangyuan Gas Co., Ltd. (Wuhu, China). 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Beyotime Biotechnology (Haimen, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-2000)]-fluorescein isothiocyanate (DSPE- PEG2000-FITC) was purchased from Nanosoft Polymers (Winston-Salem, USA). All other chemicals were of analytical purity and were used as received.

2.2 Fabrication of LBs

Firstly, the lipid formulation composed of DSPC and DSPE- PEG2K was added into hydration liquid, which consisted of glycerol (V/V, 10%), saline (V/V, 90%) and ethanol (4 wt.%), and then dispersed by sonication for 5 min. Secondly, the lipid dispersion was heated to 60 °C and mixed with free bubbles generated by previously developed repeated compression method [20]. Briefly, 2 mL of dispersion was transferred into 3-mL vials sealed with plastic caps, and the ceiling air in the vials was replaced with SF6 gas and additional 3 mL of SF6 was retained in a 5-mL syringe. The SF6 gas was injected into lipid suspension through a needle to reach the maximum pressure of 0.3 MPa. Then, the syringe plunger was pulled up to induce small volume (1 mL) changes, causing sufficient mixing of gas and liquid by the pressure difference between 0.2 and 0.3 MPa. Further decompression to atmospheric pressure resulted in the formation of free bubbles in lipid suspension. The process from compression to decompression was repeated 5 times. Lastly, the

suspension was cooled down to 20 °C and the LBs dispersion was obtained. Different formulations with DSPC and different concentrations of DSPE-PEG2K were further investigated.

2.3 Physicochemical characterization of LBs

The size distribution of LBs was obtained by dynamic light scattering (DLS) measurement using a Malvern NanoSizer (Zeta-Sizer, Malvern Instrument, British). The concentration of LBs was measured by a Multisizer (Multi4e, Beckman, USA). The morphology of LBs was examined using optical microscopy (Ti2-U, Nikon, Japan) and scanning electron microscopy (SEM, Ultra Plus, Carl Zeiss, Germany). The freshly prepared LBs dispersion was dropped onto a clean glass slide and covered with a coverslip for microscopic imaging. For SEM analysis, a drop of sample solution was dispersed onto a 1 cm × 1 cm silicon wafer with sequential drying at room temperature.

2.4 Mechanism study on self-assembly of lipids at

free bubbles interface

2.4.1 Transition of lipids aggregates in solution

Size distribution of lipid self-assembled aggregates at different temperature was measured by DLS, before which membrane filtration (0.22 μm) was conducted to remove the interference of large vesicles. The aggregate structure was observed by trans-mission electronic microscopy (TEM, JEM-2100, JEOL, Japan) after negative stain using 2% aqueous phosphotungstic acid.

2.4.2 Interaction of lipids and free bubbles

Lipids were labeled by DiI and their assembly at free bubbles interface was imaged by fluorescence microscopy (Ti2-U, Nikon, Japan). To further investigate the distribution of DSPE-PEG2k on the lipid shell, DSPE-PEG2k was replaced with green fluorescent DSPE-PEG2000-FITC for fluorescence imaging. To simulate the behavior of the adsorbed lipid monolayer at the gas–water interface of free bubbles, surface pressure versus surface area (π/A) isotherm was measured on a Langmuir- Blodgett Troughs (KSV NIMA LB Troughs, Biolin Scientific, Sweden) at 25 °C. Lipid formulation in chloroform (1 mg/mL) was spread on the surface of water, and the solvent was evaporated for 10 min. The surface pressure was measured using the Wilhelmy plate arrangement attached to a microbalance.

2.4.3 Stabilization of the LBs induced by cooling

The temperature–time curve of the lipid dispersion after assembly at 60 °C was measured by optical fiber spectrometer (FISO UMI 4, Canada). Lipids dispersed at 60 °C and LBs cooled to 20 °C were freeze-dried by a lyophilizer (VirTis, SP Scientific, USA) for the measurements of differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared spectra (ATR−FTIR) and rheological properties. DSC measure-ment of LBs was carried out at 2.5 °C/min and at temperatures ranging from 0 to 120 °C using a differential scanning calorimeter (DCS 214 Nevio, NETZSCH, Germany). ATR−FTIR measurement was conducted by an attenuated total reflection spectrometer (IRAffinity-1, Shimadzu, Japan) with a horizontal ATR plate containing ZnSe crystal. The infrared spectra were recorded with a resolution of 0.5 cm−1, and a total of 20 scans were measured in the range of 600−3,800 cm−1 at 298 K. The rheological properties were measured with a rotational rheometer (MCR302, Anton Paar, Austria). Parallel-plate configurations with a diameter of 20 mm at a gap of 1 mm were set for measurements at pre-determined temperatures (30, 40, 50, 55, 60 °C).

2.5 In vitro ultrasound imaging

For acoustic imaging of the LBs in vitro, a homemade agar

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phantom was used, which consisted of 3% agar, 86% distilled degassed water, and 11% glycerol. The sample loading wells in the gel phantom were prepared by using a mold of 1.5-mL centrifuge tubes.

Ultrasound imaging was conducted by using microimaging systems (VisualSonics Vevo 2100, USA) with an MS-250 transducer. The imaging settings for the ultrasound imaging system were center frequency of 18 MHz, intensity power of 4%, and contrast gain of 35 dB. No parameters were changed throughout all imaging acquisition steps. Degassed water was scanned before sampling to confirm a clear background signal. Then, freshly prepared LBs dispersion was injected into the gel phantom to be imaged. The mean power intensities under contrast-mode were analyzed in the region of interest (ROI).

2.6 In vivo ultrasound imaging

Before ultrasound imaging, mice were anesthetized by injection of 1% pentobarbital sodium solution. 200 μL samples of different sized LBs dispersions were administered by tail vein injection. The liver region was imaged with the microimaging system using both B-mode and contrast-mode. The imaging settings for the ultrasound imaging system were center frequency of 18 MHz, intensity power of 4%, and contrast gain of 35 dB. The mean power intensity in the ROIs of the liver was analyzed and normalized to the intensity at the time of contrast agent injection (time = 0). Time-dependent ultrasound imaging was monitored after sample injection. The animal study protocol was approved by the Institutional Animal Care and Use Committee at Southeast University.

3 Results and discussion

3.1 Fabrication and characterization of LBs

As shown in Fig. 1(a), the fabrication of LBs included the following three steps: (1) dispersion of the lipids powder in hydration liquid (0.1 mg/mL DSPC and 0.1 mg/mL DSPE- PEG2K); (2) heating the lipid dispersion to 60 °C and mixing it with free bubbles generated by the repeated compression method; (3) cooling the dispersion to 20 °C to obtain LBs. The lipid dispersion became transparent when heated to 60 °C, and became milky white after mixed with free bubbles, indicating the generation of LBs (Fig. S1 in the Electronic Supplementary Material (ESM)). As shown in Fig. 1(b), lots of encapsulated

microbubbles were successfully prepared. The size distribution of the LBs is shown in Fig. 1(c). The average size of the LBs was 2.18 ± 0.22 μm, which was suitable as an ultrasound contrast agent. Based on the microscopy image and size distribution results, it indicated that the size of the particles was not so homogeneous. In the future, in order to improve the homogeneity of the LBs, the formulation of the lipid mixture, the ratio of lipid and free bubbles, and the temperature profiles during cooling can be further optimized. Besides, some posttreatments such as centrifugation and filtration can be performed. The zeta potential of the LBs was −7.9 ± 0.3 mV. The measured concentration of LBs was approximately (2.06 ± 0.9) × 109/mL, which was higher than that of commercial SonoVue (1 × 108– 5 × 108/mL) [21]. The LBs were stable for 5 h with a slight size decrease (approximately 2.07 ± 0.26 μm after 5 h) at room temperature, which could meet clinical ultrasound imaging requirements (Fig. S2 in the ESM).

To prove the feasibility and universality of this method, we also conducted a series of control experiments (Fig. S3 in the ESM). For DSPC (Tm, 55 °C), LBs could be obtained when heated above its Tm (at 60 and 80 °C) and mixed with free bubbles, while lipid droplets were generated when mixed with free bubbles at 20 and 40 °C (Fig. S3(a) in the ESM). More amount of LBs could be generated by increasing the concentration of DSPC, and the optimized DSPC concentration was 0.10 mg/mL (Fig. S3(b) in the ESM). When increased to 0.20 mg/mL, lipid droplets were generated due to the excess of DSPC. Besides, stable LBs could also be obtained when the DSPC was placed by DMPC (Tm, 23 °C) or DPPC (Tm, 41 °C), while the assembled bubbles would dissolve again for ePC (Tm, −8 °C), which had a Tm below the final cooling temperature (Fig. S3(c) in the ESM). These results indicate two key factors to obtain stable LBs: Lipids were prone to self-assembly at the free bubble interface when above the Tm; and the assembled LBs could only be stable below the Tm. The assembly mechanism was further investigated as follows.

3.2 Temperature-regulated self-assembly mechanism

of lipids to form LBs

3.2.1 Heating-induced vesicle to micelle transition of lipids

aggregate in solution

Size distributions of the mixed DSPC/DSPE-PEG2K aggregate in solution at different temperatures are shown in Fig. 2(a).

 Figure 1 Fabrication and characterization of LBs. Schematic of fabrication of LBs (a). Microscopy image of LBs (b). Size distribution of LBs (c).

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DLS plot showed a peak around 98 nm at 20 °C. Similar result was obtained at 40 °C. However, when the temperature increased to 60 °C, the peak of size distribution remarkably decreased to 13 nm, which was indicative of the size of micelles. As the temperature further increased to 80 °C, the peak was slightly decreased at 10 nm. Similar size decrease was also observed in the system of DMPC/DSPE-PEG2K and DPPC/DSPE-PEG2K when heated above their Tm (Fig. S4 in the ESM).

TEM images of DSPC/DSPE-PEG2K aggregates formed at 20 and 60 °C are shown in Figs. 2(b) and 2(c). Vesicles with a diameter of 50–100 nm were observed in the sample dispersed at 20 °C. However, for the sample dispersed at 60 °C, micelles with a diameter of 10–20 nm were observed, which coincided with the DLS results. It indicates that micelles are the major aggregates in the system when heated above Tm.

Amphiphilic lipid molecules can self-assemble into different aggregates such as micelles and vesicles in aqueous solution, and they can be transformed by the change of various environmental factors. Temperature often plays an important role in such transitions. It’s reported that transition from a higher ordered state to a lower ordered one usually occurs when increasing the temperature [9, 10]. Here, DLS and TEM results confirm that heating above Tm induces the transition from vesicles to micelles (Fig. 2(d)). Compared to vesicles, the lipid molecules in the form of micelles are more active to be adsorbed onto the gas–liquid interface of free bubbles.

3.2.2 Self-assembly of lipids at the free bubbles interface

The lipids were labeled with DiI, and the evolution of bubbles fluorescence image over time after mixing with free bubbles is shown in Fig. 3(a). Bubbles coated with lipid shell were observed and then the bubbles size decreased until to a balanced size. Magnified images are shown in Fig. 3(b). As previously reported, free bubbles with size above 10 μm were firstly generated by repeated compression method [20]. It showed that the lipid molecules were rapidly adsorbed to the free bubble interface and formed a fluorescent shell with relatively uniform fluorescence intensity. With the shrinkage of the bubbles due to the Laplace pressure, the lipid shell was compressed, and the shell exhibited heterogeneous fluorescence distribution. The inhomogeneity of the shell was more obvious and domains with high fluores-cence intensity were observed with further shrinkage. The bubbles eventually shrank to a balanced size of 2–3 μm with heterogeneous shells.

For lipid monolayer at the free bubble interface, the area of a single lipid molecule (A) can be calculated as Eq. (1)

2π /A D N= (1) where D is the mean diameter of the bubble and N is the average lipid molecule number on one bubble. The total lipid molar amount in the whole dispersion (2 mL) is 0.25 μmol, and the average total number of bubbles is approximately 4.12 × 109; thus, the calculated N is 3.65 × 107 molecules/bubble. As shown in Fig. 3(b), the bubble diameters are approximately 10, 6, 4, and 2.5 μm, and the calculated corresponding A is 860, 310, 138, and 54 Å2, respectively.

To simulate the self-assembly of lipid molecules at the bubble interface, we characterized the pressure–area isotherm of the DSPC/DSPE-PEG2K as used on a flat Langmuir trough, which is shown in Fig. 3(c). As the monolayer was compressed, the lipid underwent several phase changes, from a “gaseous-like” (G) state to a “liquid-expanded” (LE) state, to “liquid-condensed” (LC) and “solid” (S) states, characterized by the orientation of the hydrophobic chain of the lipid with respect to its neighbors [22]. The measured critical area (Ac) from LE to LC was approximately 63 Å2.

According to Figs. 3(b) and 3(c), the lipid monolayer assembled at the free bubble interface was initially in a G state. It indicates that the fresh free bubbles provide enough air– liquid interface to adsorb lipid with spread of hydrophobic chains. With the shrink of the bubble under Laplace pressure, the lipid monolayer was compressed and phase separation occurred, which is confirmed by fluorescence image in Fig. 3(b). When the bubble shrank to a stable size, the calculated single lipid molecule (A4) was smaller than Ac, demonstrating the LC state of the lipid shell. The lipid molecules were arranged tightly and the surface pressure was significantly increased if further compressed, leading to more stable bubbles at LC state compared to G state. As shown in Fig. 3(d), the lipid monolayer was compressed from disordered G state to an ordered LC state, conferring a beneficial size of the lipid encapsulated bubbles. If it maintained at 60 °C, LBs could deform due to collapses or buckles out of plane of the monolayer with further compression.

3.2.3 Cooling-induced phase transition of the lipid shell and

bubbles stabilization

Further cooling below Tm is important to stabilize the final LBs. The temperature–time curve of the lipid dispersion after mixed with free bubbles is shown in Fig. 4(a). The temperature was cooled below the Tm of DSPC (55 °C) after 56 s, and remained around 20 °C after 40 min. Figure 4(b) is the DSC

 Figure 2 Heating-induced vesicle to micelle transition. Size distributions of self-assembled lipids aggregates at different temperatures (a). TEM images of self-assembled lipids aggregates at 20 °C (b) and 60 °C (c). Schematic of heating-induced vesicle to micelle transition (d).

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heating curve of the LBs. A broad peak was seen from appro-ximately 15 °C, and the peak temperature was roughly 55 °C. It has been reported that DSC profiles of the lipid melting process are broadened for curved membranes [23, 24], which is consistent with the lipid shell around the bubbles. Besides, the broad feature of the DSC peak suggested the noncooperative way in the phase transition of the lipid shell.

Figure 4(c) shows the FTIR spectra of the lipids dispersed at 60 °C and LBs cooled to 20 °C. Peaks at 2,919 and 2,880 cm−1

were assigned to asymmetric and symmetric stretching bands of CH2 in the hydrophobic tail region; peaks around 1,072 and 1,215 cm−1 were attributed to the stretching vibrations of the PO2

− group in the hydrophilic head region; the peak centered at 1,091 cm−1 was assigned to the C–O–C stretching vibration originating from the PEG part of DSPE-PEG2K [25, 26]. From Fig. 4(c), changes mainly occurred in the hydrophobic tail region: The peak position of CH2 shifted toward a low wavenumber. This property has been used frequently to follow the conformational

 Figure 3 Self-assembly of lipids at free bubbles interfaces. Evolution of LBs after mix of lipid dispersion and free bubbles (a), scale bar: 20 μm. Magnified fluorescence microscopy images of LBs and their calculated mean single molecule areas at the bubble shell (b), scale bar: 3 μm. Isotherms of a DSPC monolayer at 25 °C (c). Schematic of self-assembly of lipids at free bubble interface (d).

Figure 4 Cooling-induced phase transition of the lipids shell. Temperature–time curve of the lipid dispersion(a). DSC heating curve of LBs (b). FTIR spectra (c) and steady flow curves as a function of temperature ((d), (e)) of the samples for lipids dispersed at 60 °C and LBs cooled to 20 °C. Schematic of cooling-induced phase transition of lipids shell (f).

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order of lipid methylene chains and the trans-gauche isomeriza-tion of CH2 groups in lipid tail regions [27]. FTIR results revealed that lipid acyl tails had evident conformational rearrangements on lipid shell after cooling.

The rheological responses for lipid dispersed at 60 °C and LBs cooled to 20 °C are shown in Figs. 4(d) and 4(e). The steady flow curves of lipid dispersed at 60 °C revealed the Newtonian nature of the system from 30 to 60 °C (Fig. 4(d)), and thus indicated the existence of symmetric aggregates. It was consistent with the presence of mixed DSPC/DSPE-PEG2K micelles. However, the flow curve for LBs revealed remarkable shear- thinning feature at 30 °C and above 55 °C. It has been reported that the transition temperature for DSPE-PEG2K is approximately 20 °C [28]. We attributed this rheological response to the phase transition of asymmetric DSPE-PEG2K and DSPC domains on the lipid shell. It indicated that the DSPE-PEG2K and DSPC components had segregated and formed asymmetric domains on the lipid shell after cooling.

The above-mentioned results indicate that cooling induced the phase transition from fluid state to glassy state of the lipid shell (Fig. 4(f)), and the segregated DSPE-PEG2K and DSPC components formed into domains. Phase transition of the lipid shell can confine the monolayer from collapsing or buckling out of plane, which can further stabilize the LBs. Besides, it has been reported that the polycrystalline microstructure formed on the lipid shell and exhibited a significantly enhanced resistance to shear deformation after cooling [25, 29, 30].

3.2.4 The formation mechanism of LBs through self-assembly of

lipids at free bubbles interface

On the basis of the above experimental results, we proposed the following mechanism for temperature-regulated self-assembly of lipids at free bubbles interface to obtain LBs (Fig. 5). Usually, lipids self-assembled into vesicles in solution at room temperature. Heating above Tm improved the fluidity of acyl tails in lipids, and induced the transition from vesicles to mixed micelles. The micelles were unstable and active to be absorbed at gas− liquid interfaces. After mixed with free bubbles, lipid molecules spread on the free bubble interface and self-assembled into monolayer around the bubble. With the shrinkage of the bubble, the lipid monolayer changed from an unstable G state to a more ordered LC state, which confers the microsize of the LBs from big bubbles. After cooling below Tm, the lipid shell underwent phase transition and formed asymmetric domains on the lipid shell, which further confined the lipids on the shell and stabled the bubbles.

Due to their hydrophobic tails in the molecules, it is difficult to directly disperse lipids in water. An organic solvent such as chloroform is often used to first dissolve the lipids and then mix them with aqueous solution [4–6]. In this method,

 Figure 5 Schematic of temperature-regulated self-assembly of lipids at free bubbles interfaces to prepare LBs.

heating above Tm and the existence of gas–water interface promotes the lipids dispersion in the form of micelles and subsequently assembly into bubble shells, which avoids the use of organic solvent. On the other hand, the self-assembled vesicles in solution hinder direct adsorption of lipid molecules to the gas−liquid interface [6, 31], and shear forces are subsequently needed through homogenization or sonication with gas in traditional preparation methods (such as film emulsification) [32, 33]. The self-assembly of lipid molecules to the pre-existing bubble interfaces may reduce energy consumption to mix lipids and gas and avoids complicated preparation processes.

3.3 DSPE-PEG2K content-dependent LB size regulation

More importantly, it is noticed that increasing the content of DSPE-PEG2K results in a further decrease of the LBs size. We fixed the concentration of DSPC and increased the amounts of DSPE-PEG2K: LBs-1 (DSPE-PEG2K: DSPC=1.96 mol/mol), LBs-2 (2.8 mol/mol) and LBs-3 (4.2 mol/mol). DLS measurements showed that the average sizes of LBs-1, LBs-2 and LBs-3 were 1.68 ± 0.11 μm, 704 ±7 nm and 208 ± 6 nm, respectively (Fig. S5 in the ESM). Their structures were further investigated by optical microscopy and SEM shown in Fig. 6. Bubble structure with gas core could be observed for LBs-1 (Fig. 6(a)) and LBs-2 (Fig. 6(b)), and LBs-3 showed as dots assigned to relatively large bubbles under optical microscopy (Fig. 6(c)). In the SEM micrographs, LBs-1 and LBs-2 appeared as unaggregated hollows, which was due to the vacuum during measurement (Figs. 6(d) and 6(e)). Importantly, LBs-3 revealed similar hollows with much smaller size, which confirmed their gas core in bubbles rather than solid spheres as liposomes (Fig. 6(f)). The sizes of LBs shown in Figs. 6(d)–6(f) were around 1.5 μm, 700 nm and 200 nm, respectively, which was consistent with the DLS results. In addition, as shown in Fig. S6 in the ESM, the size stability of LBs-3 could be maintained within 8 h, which was more potential for clinical applications.

The lipid membrane probe DiI was added and DSPE- PEG2K was replaced by DSPE-PEG2000-FITC to show their spatial distribution on the LBs shell. Figure 7(a) shows bright field and fluorescent images of LBs with increasing DSPE- PEG2K content, which is the relatively larger bubble in the sample due to the resolution limit of optical microscopy. Heterogeneous fluorescence distribution was observed on the shell. The co-localization of the DSPE-PEG2K and membrane probe was not complete, indicating the different enriched regions of the two lipid species. With the increase of DSPE-PEG2K content, the phase separation was more obvious, and the ratio of domains with high fluorescence intensity was increased. We proposed an interpretation for the size control of LBs by the addition of DSPE-PEG2K as shown in Fig. 7(b). It has been reported that DSPE-PEG2K preferentially enriches the parts

 Figure 6 Microscopy images, scale bar: 10 μm ((a)–(c)), and SEM char-acterization, scale bar: 1 μm ((d)–(f)) for different sized LBs fabricated with different formulations where the DSPC was fixed and the amount of DSPE- PEG2K were increased.

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 Figure 7 Effect of DSPE-PEG2K content on LBs size. Bright field and fluorescent images of LBs with increased DSPE-PEG2K content (a), scale bar: 3 μm. Schematic representation of the domain in bubble shell with different DSPE-PEG2K content (b).

of the membrane with higher curvature, which has been explained by the hydrophilic polymers. The large head groups endow a higher steric demand than conventional lipids with nonpolymeric head groups [34–36]. At a low concentration, DSPE-PEG2K was scattered in the monolayer shell composed of DSPC and had little effect on it. Increasing the DSPE-PEG2K content induced a greater degree of phase separation on the shell, and more DSPE-PEG2K-enriched domains formed, which further drove the bending of the shell. The increase of shell curvature led to the decrease of bubble size. Through this bottom-up regulation, nanobubbles could be obtained without postseparation steps or other surfactant addition, which may affect the bubble yield or generate material waste [37].

3.4 In vitro ultrasound imaging

In vitro ultrasound imaging was performed using these three types of LBs with different sizes. In order to maintain the similar imaging enhancement at the initial timepoint, the concentration of LBs-1, LBs-2, and LBs-3 sample was around 4.12 × 107, 2.06 × 108, and 2.06 × 109/mL, respectively. The

ultrasound images of the samples at different time intervals are presented in Fig. 8(a). Ultrasound imaging enhancement could be acquired by contrast-mode for all of the samples, indicating the good echogenic properties of the bubbles. The brightness of LBs-1 declined sharply within 2.5 min. For LBs-2, with a decreased diameter, the enhanced duration was increased. Signi-ficant imaging enhancement could be observed within 30 min of observation time for LBs-3. The contrast mean power was quantitatively calculated from the average grayscale values of the images at different time points, and the plotted curves are shown in Fig. 8(b). After 2.5 min, the gray value declined, with a decrease of  61% of its initial value for LBs-1, but only a 17% decrease for LBs-2. No significant decrease was observed for LBs-3 at this timepoint. In particular, the gray value of LBs-3 was maintained at only a 49% decrease from its initial value for  20 min. These results indicate that the duration of ultrasound imaging enhancement was significantly prolonged with the decrease in the bubble size.

3.5 In vivo ultrasound imaging

In vivo ultrasound imaging was performed using mice, and ultrasound images of liver were acquired. Images in B-mode and contrast-mode for the three different sizes of LBs are shown in Fig. 9(a). After the LBs were intravenously injected, enhanced acoustic contrast was observed in the liver region for all three types of LBs. However, the enhanced imaging areas and duration time were different. For LBs-1, significant enhancement was first observed in the vessel region in the liver (shown as ROI 1), but not in the other main liver tissue region marked as ROI 2. Additionally, the ultrasonic imaging signal decreased significantly after 1 min. For LBs-2, several bright spots became visible in ROI 2, and the ultrasound imaging time lasted longer than that of LBs-1. Notably, the whole liver tissue area in both ROI 1 and 2 presented significant contrast enhancement for LBs-3. The enhancement remained obvious after 5 min and could last more than 8 min. These results indicate that the nanosized bubbles could further enter

 Figure 8 In vitro ultrasound imaging. Ultrasound images of different sized LBs at different time intervals (a). Normalized contrast mean powers as a function of time (b).

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the microvasculature in the liver and enhance ultrasound echoes of the whole liver for a longer time.

To perform quantitative analysis on the ultrasound imaging for these LBs, the contrast mean power time-courses in the ROI 1 and 2 are shown in Fig. 9(b). After LBs-1 injection, the contrast mean power was increased up to approximately 10 times than the initial value in ROI 1 at 10 s but remained at baseline intensity levels without enhancement in ROI 2. For LBs-2, ultrasound enhancement was weakened in ROI 1 (approximately 2 times the initial value), but the contrast mean power in ROI 2 was significantly increased. The image enhan-cements for ROI 1 and ROI 2 were synchronized after LBs-3 injection, and the contrast mean power in ROI 2 increased up to approximately 3 times compared with the initial value. Furthermore, the ultrasound imaging time of LBs-3 lasted longer than both LBs-1 and LBs-2. Therefore, the excellent per-formance of the LBs proves their great potential as ultrasound contrast agents. LBs-3, with their approximate 200 nm size, also maintained good ultrasound imaging capacity and were able to enter small blood vessels in the tissue due to their smaller size, which has great application potential in molecular imaging and ultrasound-mediated drug delivery in deep tissue.

4 Conclusions In this work, the self-assembly of lipids was regulated by temperature in the presence of free bubbles to fabricate micro/ nanobubbles. When heated above the Tm, the directly dispersed lipids aggregated into micelles transformed from vesicles, which were active to be adsorbed to the gas–liquid interface. After reaction with free bubbles, lipids self-assembled to the bubble interface from initial G state to LC state, resulting in the formation of lipid shelled microbubbles. Cooling below the Tm induced the transition from fluid to glassy state of the lipid shell, which further stabilized the LBs. Microbubbles with concentration of (2.06 ± 0.9) × 109/mL were obtained through this bottom-up assembly. Furthermore, by adjusting the content of DSPE-PEG2K, the shell curvature could be

manipulated, and the LBs size could be further decreased. The LBs with diameters of approximately 1.68 ± 0.11 μm, 704 ± 7 nm and 208 ± 6 nm could be successfully prepared. The in vitro and in vivo ultrasound imaging experiments demonstrated the ultrasound imaging capacities of the different-sized LBs. With the decrease of size, LBs presented longer imaging duration times and more detailed tissue contrast imaging enhancement capacity. Therefore, the generality of this approach would be suitable for drug or gene loading to have potential applications in micro/nanobubble-mediated ultrasound-assisted diagnosis and therapy in the future.

Acknowledgements This investigation was financially funded by the National Key Research and Development Program of China (Nos. 2017YFA0104302 and 2018YFA0704103), and the National Natural Science Foundation of China (Nos. 61821002 and 51832001). Funding also partially comes from the Natural Science Foundation of Jiangsu Province (No. BK20191266) and Zhong Ying Young Scholar of Southeast University. The authors also would like to thank the support from the Fundamental Research Funds for the Central Universities.

Electronic Supplementary Material: Supplementary material (experimental diagram of fabrication process, size stability of the LBs, microscopy images for control experiments, size dis-tribution change of self-assembled aggregates in the system of DMPC/DSPE-PEG2K and DPPC/DSPE-PEG2K, size distribution of LBs-1, LBs-2 and LBs-3, size stability of the LBs-3) is available in the online version of this article at https://doi.org/10.1007/ s12274-020-2732-x.

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Figure 9 In vivo ultrasound imaging. B-mode and contrast-mode ultrasound images of mouse liver after intravenous injection of different sized LBs (a).Normalized contrast mean powers in ROI 1 and ROI 2 in liver as a function of time (b).

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