Skip to content
BY 4.0 license Open Access Published by De Gruyter May 19, 2021

Magnetic black phosphorus microbubbles for targeted tumor theranostics

  • Yao Zhu , Yingying Liu , Zhongjian Xie , Tianzhen He , Lili Su , Fengjuan Guo , Gulzira Arkin , XiaoShu Lai , Jinfeng Xu EMAIL logo and Han Zhang ORCID logo EMAIL logo
From the journal Nanophotonics

Abstract

Black phosphorus (BP) is attracting more and more interest for the biomedical application. The absorption in a wide spectral range and high photothermal conversion efficiency make BP suitable for photothermal therapy. However, BP alone is hard to realize the targeted therapy, which limits the precision and efficiency of the therapy. Magnetic microbubbles (MBs) are favored drug carriers because they can resist the sheer force of blood flow in a magnetic field, which improves the efficiency of MBs adhesion to the vascular wall for targeted ultrasound diagnosis and therapy. This study first optimized the magnetic MBs configurations through controlling the connecting polyethylene glycol (PEG) chain length. The magnetic MBs with PEG2000 have been chosen for targeted BP nanosheets delivery due to the better stability and magnetic responsiveness. The magnetic black phosphorus microbubbles (MBBPM) can realize the targeted tumor theranostics in vitro and in vivo. They could be applied for the targeted ultrasound imaging with an enhanced echogenicity by three times when accumulated at the target site where the magnetic field is applied. As the NIR laser irradiation was applied on the accumulated MBBPM, they dynamited and the temperature increased rapidly. It improved the cell membrane permeability, thus accelerating and enhancing a precision photothermal killing effect to the breast cancer cells, compared to BP alone.

1 Introduction

Cancer has become one of the major diseases that seriously endanger human health, thus many researchers have devoted to studying its mechanism, diagnosis, and therapy [1], [2], [3], [4]. BP has a layered structure. It possesses a direct adjustable band gap from 0.3 to 2.0 eV as the thickness changes [5]. It has unique thermal, mechanical, and semiconductor properties, which has attracted wide attention of researchers for the application of thermoelectricity, energy storage, flexible electronics, and quantum information technology [6], [7], [8], [9]. BP possesses ideal biodegradability and its potential in biomedical applications has been studied in drug delivery, photothermal therapy, photodynamic therapy, sonodynamic therapy, and photoacoustic imaging of diseases [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. The BP alone cannot realize the targeted therapy and it is taken up by tumor cells mainly via endocytosis pathway, which takes about 4 h and limits the therapy efficiency [10, 21, 22]. Thus, the design of carriers for the targeted delivery of BP is important for its biomedical applications.

Ultrasound is considered a safe excitation source for various biomedical applications since it is noninvasive, nontoxic, and low-cost. Most importantly, ultrasound can achieve deeper tissue penetration than light [23], [24], [25], [26], [27], [28], [29], [30]. In addition to its use in traditional ultrasonography applications, ultrasound is attracting increasing attention for disease therapy [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], similar to light-triggered therapy [45], [46], [47], [48]. The resolution, sensitivity, and specificity of ultrasound diagnosis are significantly improved by ultrasound contrast agents. In recent decades, a diverse range of ultrasound contrast agents—including microbubbles (MBs), liposomes, and cerasomes—have been developed [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62]. Among them, gas filled MBs have been approved by the US Food and Drug Administration (FDA) [63], [64]. Recently, MBs have been applied for targeted drug/gene delivery and controlled release based on ultrasound-targeted microbubble destruction (UTMD) [39], [40]. However, MBs have low binding ability, short retention time, and lack specificity and sensitivity, which limit their application in ultrasound theranostics [4951, 54].

To achieve more efficient and specific binding ability, targeted MBs are created via adding targeting ligands such as folate, RGD peptide, and glycoprotein transferrin [65], [66], [67], [68], [69], [70], [71] to the microbubble shell surface. However, it only works at target areas with low blood flow speed, like microvasculature [72]. Comparatively, magnetic MBs can resist the sheer force of blood flow and change their distribution in the blood vessel under a magnetic field. It can make the MBs closer to the endothelial cells of the vascular wall, thus to increase their contact probability with the target area and greatly improve the targeted binding efficiency. Thus, magnetic MBs possess great potential in the contrast-enhanced ultrasound imaging, targeted drug delivery and site directed vascular gene delivery [72], [73], [74], [75], [76]. If it is optimized, it will have broad clinical application prospects, such as improving sensitivity of visualizable treatment of cardiovascular diseases, enhancing the efficiency of diagnosis and therapies of tumors, and as imaging probes for immune adherence and other special biological processes. Different methods have been used to combine MBs and magnetic nanoparticles (NPs) to form magnetic MBs [73, 7787]. The magnetic NPs have been embedded in the inner layer or shell of the MBs, and have also been attached to the shell surface of the MBs through certain chelating agents, biotin–avidin bridging, or electrostatic coupling. It affects the properties of the as synthesized magnetic MBs. Magnetic NPs embedded in the MB shell stiffen the surface and thus make the ultrasound image quality poorer [84], [85]. In contrast, coupling the magnetic NPs to the MB shell surface with polymer spacer arms, such as polyethylene glycol (PEG), increases the binding rate with minor effect on the surface properties [71]. The effects of PEG chain length and concentration on the mechanical property, dynamical properties, and stability of MBs have been investigated [72], [88]. However, the effects of the PEG chain length on the stability, the magnetic responsiveness, and the ultrasound image quality of the magnetic MBs have not been studied comprehensively.

In this study, different PEG chain lengths have been used to connect the magnetic NPs and the MBs, in order to find the chain length with these properties optimized. Then, the magnetic MBs with PEG2000 were applied for targeted BP nanosheets delivery, due to the better stability, magnetic responsiveness, and echogenicity. Compared with the BP alone, the magnetic black phosphorus MBs (MBBPM) show accelerated and enhanced photothermal therapy efficiency. Moreover, it can achieve the integration of targeted disease diagnosis and treatment. The schematic diagram of the synthesis process of the MBBPM and their application in the targeted theranostics are illustrated in Schematic 1. This work helps to design and optimize targeted theranostic magnetic MBs for enhanced diagnosis and therapy efficiency.

Schematic 1: 
The schematic diagram of (A) the synthesis process of the MBBPM and (B) its application for the targeted theranostics.
Schematic 1:

The schematic diagram of (A) the synthesis process of the MBBPM and (B) its application for the targeted theranostics.

2 Results and discussion

2.1 Characterization of the BP nanosheets

The BP nanosheets were prepared by a liquid exfoliation method as previously reported [17]. Briefly, bulk BPs were firstly ground and then underwent probe sonication and bath sonication. From the transmission electron microscopy (TEM) image in Figure 1A, it can be seen that the BP nanosheets have a lateral size from 100 to 200 nm. The atomic force microscopy (AFM) in Figure 1B demonstrates that the BP nanosheets have a thickness of 3–3.5 nm, corresponding to 5–6 layers. The chemical element in the obtained BP nanosheets was investigated by X-ray photoelectron spectroscopy (XPS). Three XPS peaks were observed in Figure 1C, which located at 129.8, 130.6, and 133.9 eV, respectively. Among them, the peaks at 129.8 and 130.6 eV are assigned to P 2p 3/2 and P 2p 1/2 in BP while the peak at 133.9 eV corresponds to P in oxidized state (P ox) [89]. P ox peak is commonly observed in BP, because it is easily oxidized in open air [90]. Raman spectroscopy was applied to study the crystallinity of the bulk BPs and the exfoliated BP nanosheets (Figure 1D). In bulk BPs, three Raman peaks can be detected at about 362, 437, and 465 cm−1, respectively. They are correspondingly consistent with the typical out-of-plane phonon mode Ag 1 and the in-plane phonon modes B2g and Ag 2 [91]. The three phonon modes can also be seen in exfoliated BP nanosheets. It demonstrates that the obtained BP nanosheets are well crystallized but their Raman peak intensities dramatically decrease, which is attributed to the reduced number of layers [91]. Moreover, their Ag 2 vibration mode blue shifts compared to that in bulk BPs, also confirming the reduced thickness [92], [93]. The absorption of the exfoliated BP nanosheets was also measured. As shown in Figure 1E, the BP nanosheets have a broad absorption band covering the ultraviolet to infrared spectral range.

Figure 1: 
(A)–(C) The transmission electron microscope (TEM) image, atomic force microscope (AFM) image, and X-ray photoelectron spectroscopy (XPS) spectrum of the exfoliated black phosphorus (BP) nanosheets, respectively. The scale bar in (a) is 0.2 μm. (B2) The height profile along the white line in (B1). (D) The Raman spectra of bulk BPs andBP nanosheets. (E) The absorption spectrum of the exfoliated BP nanosheets.
Figure 1:

(A)–(C) The transmission electron microscope (TEM) image, atomic force microscope (AFM) image, and X-ray photoelectron spectroscopy (XPS) spectrum of the exfoliated black phosphorus (BP) nanosheets, respectively. The scale bar in (a) is 0.2 μm. (B2) The height profile along the white line in (B1). (D) The Raman spectra of bulk BPs andBP nanosheets. (E) The absorption spectrum of the exfoliated BP nanosheets.

2.2 Properties of magnetic MBs controlled by PEG chain length

To optimize the magnetic MBs configuration for targeted delivery of the BP nanosheets, five types of magnetic MBs were synthesized by mixing the streptavidin-coated superparamagnetic Fe3O4 NPs with the biotinylated MBs with different connecting PEG chain lengths (none, PEG400, PEG1000, PEG2000, and PEG3400), as illustrated in Figure 2A. The concentrations of the as prepared biotinylated MBs are ∼1.3 × 109 MBs/mL. To verify the formation of the magnetic MBs, the magnet was used to see if they were magnetically responsive. It can be seen in the optical images (Figure 2B), that for both biotinylated and magnetic MBs, there is a cake layer in the upper portion of the solution but with different colors. It illustrates the existence of both MBs. When the magnet was placed near the MBs, biotinylated MBs showed no changes but magnetic MBs accumulated at the side where the magnet is placed (Figure 2C). It confirms the magnetic MB formation. The morphology of the biotinylated MBs and magnetic MBs have been analyzed using scanning electron microscope (SEM) and transmission electron microscope (TEM) micrographs, respectively. As can be seen from Figure 2D, the MBs are spherical but they are collapsed under the electron beam with high energy, because they are filled with gas and the MBs shell are soft. The TEM micrograph of the magnetic MBs in Figure 2E further corroborates the attachment of the Fe3O4 NPs to the MB’s surface.

Figure 2: 
(A) Illustration of the synthesized five types of biotinylated microbubbles (MBs) with different connecting polyethylene glycol (PEG) chain lengths (none, PEG400, PEG1000, PEG2000, and PEG3400) to combine with magnetic NPs for magnetic MBs formation. (B) Optical images of the as prepared biotinylated MBs and magnetic MBs without a magnet and (C) with a magnet placed near them. (D) The scanning electron microscope (SEM) micrograph of biotinylated MBs and (E) the TEM micrographs of magnetic MBs. The scale bar is 2 μm.
Figure 2:

(A) Illustration of the synthesized five types of biotinylated microbubbles (MBs) with different connecting polyethylene glycol (PEG) chain lengths (none, PEG400, PEG1000, PEG2000, and PEG3400) to combine with magnetic NPs for magnetic MBs formation. (B) Optical images of the as prepared biotinylated MBs and magnetic MBs without a magnet and (C) with a magnet placed near them. (D) The scanning electron microscope (SEM) micrograph of biotinylated MBs and (E) the TEM micrographs of magnetic MBs. The scale bar is 2 μm.

An inverted fluorescence microscope was also used to study the morphology of the synthesized biotinylated MBs and magnetic MBs. The bright-field images of the five types of biotinylated MBs (no PEG spacer arm, MBb; with PEG400, MBb400; with PEG1000, MBb1k; with PEG2000, MBb2k and with PEG3400, MBb3400) and the corresponding magnetic MBs (MBM, MBM400, MBM1k, MBM2k, and MBM3400) are shown in Figure 3A1–A5 and B1–B5, respectively. They are circle-shaped, which illustrates the MBs formation. The fluorescent images of the FITC-labeled magnetic MBs (Figure 3C1–C5) show green fluorescence along the hollow circle edge, which also confirms the coupling of the magnetic NPs to the MBs’ shell surface.

Figure 3: 
(A1–A5) The bright-field images of biotinylated MBs with different PEG chain lengths (no PEG spacer arm, MBb; with PEG400, MBb400; with PEG1000, MBb1k; with PEG2000, MBb2k and with PEG3400, MBb3400) and (B1–B5) the corresponding magnetic MBs (MBM, MBM400, MBM1k, MBM2k and MBM3400). (C1–C5) The fluorescent images of FITC-labeled magnetic MBs with different PEG chain lengths. The scale bar is 5 μm.
Figure 3:

(A1–A5) The bright-field images of biotinylated MBs with different PEG chain lengths (no PEG spacer arm, MBb; with PEG400, MBb400; with PEG1000, MBb1k; with PEG2000, MBb2k and with PEG3400, MBb3400) and (B1–B5) the corresponding magnetic MBs (MBM, MBM400, MBM1k, MBM2k and MBM3400). (C1–C5) The fluorescent images of FITC-labeled magnetic MBs with different PEG chain lengths. The scale bar is 5 μm.

To further comprehensively study the effect of the PEG chain length on the properties of the MBs, the particle size distribution and zeta potential of the biotinylated and magnetic MBs were measured. The binding capacity of the biotinylated MBs with the streptavidin-coated Fe3O4 NPs was then tested by measuring the percentage of magnetic MBs obtained.

For biotinylated MBs, their particle size distributions are 1.7 ± 0.2 μm (Figure 4A) and their zeta potential are near −20 mV (Figure 4B). The PEG chain length shows negligible effects on them. After coupling the streptavidin-coated Fe3O4 NPs to the biotinylated MB shell surfaces, the average particle sizes of the MBM, MBM400, and MBM1k were not different while the particle size distributions of MBM2k and MBM3400 were changed to 2.7 ± 0.3 and 3.1 ± 0.3 μm, respectively (Figure 4A). The zeta potential of all the magnetic MBs remained negative but the absolute values decreased, compared with the biotinylated MBs (Figure 4B). The concentrations of the obtained magnetic MBs were also measured. The concentrations of MBM and MBM400 were about 2.4 × 108 MBs/mL and that of MBM1k was 6.4 × 108 MBs/mL. The concentrations of MBM2k and MBM3400 were 1.2 × 109 and 1.1 × 109 MBs/mL, respectively. It was found that, MBb2k and MBb3400 had significantly better binding capacity with streptavidin-coated Fe3O4 NPs than MBb, MBb400, and MBb1k. It resulted in the markedly higher percentages of MBM2k and MBM3400 (∼90%) than those of MBM, MBM400, and MBM1k (∼20–50%) in the original concentrations of the biotinylated MBs (1.3 × 109 MBs/mL) (Figure 4C).

Figure 4: 
(A) The average particle size and (B) zeta potential of biotinylated MBs and the generated magnetic MBs with different PEG chain lengths; the black and red bars represent biotinylated MBs and magnetic MBs, respectively. (C) The percentage of magnetic MBs obtained in the total MB population. The remaining proportion of (D) biotinylated MBs and (E) magnetic MBs in the total MBs measured immediately after preparation and after storage in PBS under ambient conditions for 20, 40, and 60 min. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4:

(A) The average particle size and (B) zeta potential of biotinylated MBs and the generated magnetic MBs with different PEG chain lengths; the black and red bars represent biotinylated MBs and magnetic MBs, respectively. (C) The percentage of magnetic MBs obtained in the total MB population. The remaining proportion of (D) biotinylated MBs and (E) magnetic MBs in the total MBs measured immediately after preparation and after storage in PBS under ambient conditions for 20, 40, and 60 min. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

The biotin linked to the MB’s shell surface with shorter PEG chain length than PEG2000 may make it buried within the PEG2000 spacer arms added for long circulation in vivo. Thus, the streptavidin-coated Fe3O4 NPs are hindered in binding with the biotin [68, 9496], which leads to the much lower magnetic MBs percentage. The coupled magnetic NPs to these MBs are shielded [95], [96], which results in the average particle sizes of MBM, MBM400, and MBM1k similar to those of MBb, MBb400, and MBb1k. In contrary, the biotin linked to the MB’s shell surface with PEG2000 and PEG3400 is exposed, which can well connect with the streptavidin-coated Fe3O4 NPs and thus leads to much higher binding rate and the increased average particle sizes.

2.3 Stability of magnetic MBs controlled by PEG chain length

Owing to the intended practical application of the MBs, their stability was also tested (Figure 4D and E). A total of 20, 40, and 60 min after the storage of the biotinylated MBs and magnetic MBs in PBS (with the same initial concentration of 1 × 108 MBs/mL) in the ambient conditions, their concentrations were measured and compared with the as-prepared samples to evaluate the stability of each type of MBs. All of the biotinylated MBs spontaneously disassembled when stored in PBS in ambient conditions. MBb, MBb400, and MBb1k disassembled gradually, with the remaining percentage being less than 80% after 60 min while MBb2k and MBb3400 remained stable after 40 min with the remaining percentage being more than 80% after 60 min (Figure 4D). For the magnetic MBs, all of them decreased significantly with time when stored in PBS under ambient conditions (Figure 4E). Only ∼30% of MBM and MBM400 in the total MBs are left after 60 min. The percentage of MBM1k is decreased to 60%. In comparison, the percentages of MBM2k and MBM3400 were significantly higher than 70% even after 60 min. In general, both biotinylated MBs and magnetic MBs with longer PEG chain lengths are more stable than those with shorter PEG chain lengths [84], [85].

2.4 Magnetic responsiveness of magnetic MBs controlled by PEG chain length

As the magnetic MBs are intended for efficient targeted theranostic applications, their magnetic responsiveness was measured with a home-made parallel plate flow chamber assay under sheer forces from 5 to 45 dyn/cm2, as shown in Figure 5A. The magnetic responsiveness of biotinylated MBs was measured as a control. Since the magnetic responsiveness of the biotinylated MBs with different PEG chain lengths show no significant difference (Figure 5B), only that of MBb was shown in Figure 5C for a control.

Figure 5: 
(A) Schematic of the home-made parallel plate flow chamber assay. The captured percentage of (B) biotinylated MBs and (C) magnetic MBs by an applied magnetic field (B ≈ 1.2 T) under sheer forces from 5 to 45 dyn/cm2. The biotinylated MBs were used as a control. Data represent mean ± SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5:

(A) Schematic of the home-made parallel plate flow chamber assay. The captured percentage of (B) biotinylated MBs and (C) magnetic MBs by an applied magnetic field (B ≈ 1.2 T) under sheer forces from 5 to 45 dyn/cm2. The biotinylated MBs were used as a control. Data represent mean ± SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

It was shown in Figure 5C, as the sheer force increased the number of captured magnetic MBs by the magnetic field decreased for all types of magnetic MBs studied. Compared with the biotinylated MBs, all of the magnetic MBs showed a higher captured percentage even under high sheer forces of 45 dyn/cm2. It demonstrated that the magnetic MBs showed potential for targeted theranostic applications, even at target areas with a high blood flow speed, such as in the aorta. Among the magnetic MBs, MBM2k, and MBM3400 with longer PEG spacer arm length showed better magnetic responsiveness than MBM, MBM400, and MBM1k. It may be because the higher magnetic NPs binding capacity to them compared to the MBs with shorter PEG length [95], [96].

2.5 Echogenicity of magnetic MBs controlled by PEG chain length

Achieving contrast-enhanced ultrasound images is one of the important intended functions of the MBs, therefore the effect of the PEG chain length on it was also studied. The locations of the ultrasound probe and the magnet placed near the agar plates are shown in Figure 6A. It showed no US signal for PBS solution (Figure 6B). In contrast, the ultrasound signal in B-mode and contrast-mode using different types of biotinylated and magnetic MBs are significantly enhanced in Figure 6C.

Figure 6: 
(A) Schematic diagram of the setup for acquiring ultrasonograms whilst applying a magnet. (B) The ultrasonogram of PBS solution. (C) B-mode and contrast-mode ultrasonograms of biotinylated and magnetic MBs suspensions in PBS without and with magnet application (5 × 106/mL, pH 7.4, B ≈ 1.2 T). (D) In vitro echogenicity at the region of interest (ROI) of MBs measured as signal enhancement normalized to the PBS solution signal. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6:

(A) Schematic diagram of the setup for acquiring ultrasonograms whilst applying a magnet. (B) The ultrasonogram of PBS solution. (C) B-mode and contrast-mode ultrasonograms of biotinylated and magnetic MBs suspensions in PBS without and with magnet application (5 × 106/mL, pH 7.4, B ≈ 1.2 T). (D) In vitro echogenicity at the region of interest (ROI) of MBs measured as signal enhancement normalized to the PBS solution signal. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

For all of the biotinylated MBs, the ultrasound images showed no notable difference with and without magnet application. Although the MBM, MBM400, and MBM1k can be attracted by the magnet in the parallel plate flow chamber assay, they showed no marked differences with and without a magnet under the ultrasound. It may be due to their poor magnetic responsiveness and stability. Moreover, in the parallel plate flow chamber, the distance between the magnetic MBs and the magnet is 0.13–0.17 mm, while it is about 1 cm, i.e. 80 times further in the echogenicity measurements. The magnetic field strength decreases with the distance, so the magnetic responsiveness of MBM, MBM400, and MBM1k in the echogenicity measurements may be too low to accumulate at the target site. In contrast, when the magnet was applied, MBM2k and MBM3400 accumulated at the side where the magnet was placed and the ultrasound image signal was significantly enhanced. For quantitative analysis, the image signal in the contrast-mode at the accumulation region (region of interest [ROI]) was recorded and the enhancement was normalized to the PBS solution signal (Figure 6B). It can be seen from Figure 6D, that for the biotinylated MBs and magnetic MBs with no magnet, the signal enhancement first increased and then decreased as the PEG chain length linking the biotin to the MB shell surface increased. MBb1k and MBM1k (no magnet) showed the highest echogenicity. Meanwhile, it was found that all of the magnetic MBs with no magnet showed a lower echogenicity compared with the biotinylated MBs. However, it was encouraging to find that the MBM2k and MBM3400 accumulated to the magnet side, leading to the echogenicity increased to three times and twice, respectively, which is much higher than those for all of the biotinylated MBs.

In both parallel plate flow chamber assay and echogenicity measurements, the magnetic responsiveness of magnetic MBs can be observed under magnetic field. It shows promising potential for the targeted diagnosis and drug delivery. The longer PEG chains linking the biotin to the MB shell surface and subsequently combined with the magnetic NPs were beneficial for improving the ultrasonogram quality as they accumulated under the magnetic field. In conclusion, the magnetic MBs with longer PEG spacer arms lengths (PEG2000 and PEG3400) showed increased average particle sizes compared with the corresponding biotinylated MBs. They also possessed better stability, magnetic responsiveness, and echogenicity than the magnetic MBs with shorter PEG chain lengths (none, PEG400, and PEG1000). These effects are thought to be resulted from their different configurations. The biotin linked to the MBs’ shell surface with PEG2000 and PEG3400 are well exposed and so is the coupled streptavidin-coated Fe3O4 NPs, which leads to the increased average particle size and good magnetic responsiveness. Contrarily, the biotin linked to the MBs’ shell surface with no PEG, PEG400, or PEG1000 is buried [68, 9496]. It is hard to couple with the streptavidin-coated Fe3O4 NPs, which make the magnetic MBs’ average particle size with minor variation and the magnetic responsiveness worse. Thus, in the further study, the magnetic MBs with PEG2000 linking their surface with the magnetic NPs are chosen for the targeted delivery of BP nanosheets for the photothermal therapy.

2.6 Characterization of MBBPM

To load the BP nanosheets on the surface of the magnetic MBs electrostatically, stearic-PEI 600 has been added in the MBs synthesis formula to achieve cationic MBs (MBc). Its initial concentration was about 1.1 × 109 MBs/mL. BP nanosheets solution was added to the MBc solution and they were incubated for 15 min. Then the magnetic NP dispersion was added to the mixture and incubated for further 15 min. The synthesized BP loaded magnetic MBs solution has a concentration of around 8.1 × 108 MBs/mL. The schematic of the finally obtained MBBPM was shown in Figure 7A. As shown in the photographs in Figure 7B, the white MBc become black after adding BP nanosheets (i.e. MBBP) and subsequently magnetic NPs (i.e. MBBPM). The obtained MBBPM can be accumulated to the side at which the magnetic field is applied. It illustrated the successful adsorption of BP nanosheets and magnetic NPs on the surface of the MBs. From the optical images in Figure 7C, MBBP and MBBPM have a black layer on the MBs surface. Their zeta potential has also been measured and they were presented in Figure 7D. The exfoliated BP nanosheets have an average zeta potential of −18.4 mV and the MBc has a surface charge of 20.5 mV. After mixing them, the zeta potential of MBBP is negative. It further confirms the attachment of the BP nanosheets on the MBc. The obtained MBBPM possessed a surface charge of about −12.6 mV. The particle size distribution of bare MBc is 2.7 ± 0.2 μm. The respective particle size distributions of MBBP and MBBPM are 3.6 ± 0.1 and 3.7 ± 0.3 μm, with the average particle sizes increased (Figure 7E).

Figure 7: 
(A) The schematic of the finally obtained magnetic black phosphorus microbubbles (MBBPM). (B) The photographs and (C) the optical images of the cationic MBs (MBc), black phosphorus microbubbles (MBBP), and MBBPM. The scale bars are all 20 μm (D) The zeta potential and (E) the average particle sizes of BP nanosheets, MBc, MBBP, and MBBPM. (F) The remaining percentage of MBBP and MBBPM with time. (G) The absorption spectra of the subnatant in the MBBP solution with time. (H) B-mode and contrast-mode ultrasonograms of the MBBPM suspension in PBS without and with magnet application (5 × 106/mL, pH 7.4, B ≈ 1.2 T) and the ratio of the signal intensity at the target region (ROI 1) to that at the nontarget region (ROI 2). Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7:

(A) The schematic of the finally obtained magnetic black phosphorus microbubbles (MBBPM). (B) The photographs and (C) the optical images of the cationic MBs (MBc), black phosphorus microbubbles (MBBP), and MBBPM. The scale bars are all 20 μm (D) The zeta potential and (E) the average particle sizes of BP nanosheets, MBc, MBBP, and MBBPM. (F) The remaining percentage of MBBP and MBBPM with time. (G) The absorption spectra of the subnatant in the MBBP solution with time. (H) B-mode and contrast-mode ultrasonograms of the MBBPM suspension in PBS without and with magnet application (5 × 106/mL, pH 7.4, B ≈ 1.2 T) and the ratio of the signal intensity at the target region (ROI 1) to that at the nontarget region (ROI 2). Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

To study the stability of the BP nanosheets loaded magnetic MBs, the remaining percentages of MBBP and MBBPM have been analyzed from as prepared to 1 h after the storage in the ambient condition. It can be observed in Figure 7F, MBBP is stable without obvious reduction. Moreover, through measuring the absorption of the subnatant in the MBBP solution, it shows no detected BP nanosheets desorption from MBBP (Figure 7G). It illustrates the BP nanosheets are stably electrostatically adsorbed on the MBs surface. MBBPM has decreased with time, which is consistent with the previous study on the magnetic MBs but its percentage still remains more than 80% after 1 h.

The ultrasonograms of the final BP-incorporated magnetic MB without and with the magnet application have been measured, which is shown in Figure 7H. To verify its targeting effect, the signal intensity of the target region where the magnet is placed (ROI 1) is divided by that of nontarget region (ROI 2). It is enhanced significantly from about 1 as no magnet is applied to about 4 and 7 as the magnet is applied for B-mode and contrast mode, respectively. It demonstrates a good targeting effect of the MBBPM.

2.7 Photothermal effect of MBBPM

The BP nanosheets have a wide absorption spectrum from ultraviolet to infrared spectral range (Figure 1E), which shows a promising potential in photothermal therapy under near infrared (NIR) light. To study the photothermal performance of the synthesized MBBPM, the MBBPM dispersion underwent magnetic field for 3 min to attract the MBBPM to the bottom of the eppendorf (EP) tube and then it was irradiated with an 808 nm laser at 2.0 W/cm2 for 10 min. The measurement schematic is shown in Figure 8A. For comparison, the photothermal performance of BP nanosheets and the PBS were also measured in the same condition. As can be seen in Figure 8B–D, without NIR laser irradiation, the temperature almost remains at about 28 °C for all of the PBS, BP nanosheets, and MBBPM solutions. Under NIR laser irradiation for 10 min, the temperature of the BP nanosheets and MBBPM dispersion could increase to more than 40 °C, but the different regions could reach different temperatures. For BP nanosheets dispersion, the temperature gradually increased from the top (region 2) to the bottom (region 1) with the irradiation time increasing (Figure 8D). The temperature in region 2 reached about 44 °C while that in region 1 reached just about 36 °C. On the contrary, the MBBPM was attracted at the bottom (region 1), the temperature increased rapidly at the accumulation site to about 42 °C, while that in the upper region 2 is lower and slower. The rate of the temperature increase and the finally reached temperature after 10 min of NIR irradiation are different in different regions for BP nanosheets alone and MBBPM suspension under the magnetic field application. Figure 8A show the measurement schematic on the photothermal performance of the MBBPM. Under the magnetic field, the MBBPM accumulate at the bottom region 1, resulting in the local concentration of BP nanosheets in the region 1 high while that in the upper region 2 much lower. Therefore, the temperature in region 1 increases quickly to more than 40 °C. Contrarily, the temperature increases much slower and the final temperature is lower in the region 2 for the MBBPM. However, for BP nanosheets alone, they are still uniformly dispersed, which is not affected by the magnetic field and not like the case of MBBPM in Figure 8A. Since the upper region 2 is closer to the NIR laser than the bottom region 1, the temperature gradually increases from the region 2 to the region 1 with the irradiation time increasing.

Figure 8: 
(A) The measurement schematic on the photothermal performance of the MBBPM under a magnetic field. (B) The infrared (IR) thermal images of PBS, BP nanosheets, and MBBPM without and with 808 nm laser irradiation (2.0 W/cm2) for 10 min. (C) and (D) The corresponding photothermal heating curves of the PBS, BP nanosheets, and MBBPM without and with the 808 nm laser irradiation (2.0 W/cm2) for 10 min, respectively.
Figure 8:

(A) The measurement schematic on the photothermal performance of the MBBPM under a magnetic field. (B) The infrared (IR) thermal images of PBS, BP nanosheets, and MBBPM without and with 808 nm laser irradiation (2.0 W/cm2) for 10 min. (C) and (D) The corresponding photothermal heating curves of the PBS, BP nanosheets, and MBBPM without and with the 808 nm laser irradiation (2.0 W/cm2) for 10 min, respectively.

2.8 In vitro photothermal therapy with MBBPM

To study the targeted photothermal therapy effect of the MBBPM in vitro, MBBPM and MBBP were incubated with the MCF-7 breast cancer cells under the magnetic field (about 1.2 T) for 3 min and then the 808 nm laser irradiation (2.0 W/cm2) was applied for 10 min. It can be seen from Figure 9A, there was no obvious MBBP (optical image of the MCF-7 breast cancer cells incubated with MBBP) but numerous MBBPM (optical image of the MCF-7 breast cancer cells incubated with MBBPM before NIR irradiation) can be observed on the MCF-7 cells. It illustrated the capacity of the magnetic MBs for targeted delivery of the BP nanosheets. The circle-shaped MBBPM can be seen before the NIR irradiation (optical image of the MCF-7 breast cancer cells incubated with MBBPM before NIR irradiation), but they diminish after the NIR irradiation (optical image of the MCF-7 breast cancer cells incubated with MBBPM after NIR irradiation), which are dynamited. After the treatments with different concentrations of MBBPM (from 1 × 105 to 1 × 107 MBBPM/mL, 5×10−5 μg BP nanosheets/MB), the cells were further cultured for 12 h. Afterwards, the MCF-7 cells were rinsed with PBS three times and then costained with Calcein AM and PI for 30 min. After rinsing with PBS, they were observed by the inverted fluorescence microscope and the results were shown in Figure 9B. The number of live and dead cells were counted by ImageJ software and the cell viability was calculated (Figure 9C). The corresponding relative cell viability decreased from about 80% to no more than 10% as the MBBPM concentration increased to 1 × 107 MBBPM/mL. The TEM analysis for the cellular uptakes of the MBBPM (1 × 107 MBBPM/mL) in MCF-7 cells was also performed, in order to assess the interactions of the MBBPM with the cell membrane and the influence of the photothermal therapy on the cells. It can be observed from Figure 9D, as the red arrows indicate, a huge amount of nanomaterials including BP nanosheets and some iron oxide NPs have been uptaken inside the cells only after 3 min of incubation with the MCF-7 cells under the magnetic field and 10 min of treatment under the NIR irradiation. It demonstrates that the cell membrane permeability is improved, thus shortening the uptake time of the BP nanosheets inside the cells greatly. Moreover, it also can be seen in the TEM image of the MCF-7 cells after the photothermal therapy, the mitochondrion has already collapsed or its inner membrane ridges are irregularly arranged, as the green and blue arrows indicate. These illustrate the photothermal therapy with the MBBPM lead to the apoptosis of the MCF-7 cells and the generation of the autophagolysosome, as the yellow arrows indicate.

Figure 9: 
(A) The optical images of MCF-7 cancer cells incubated with MBBP and MBBPM under the magnetic field (B ≈ 1.2 T) for 3 min and then under the NIR laser irradiation. (B) The fluorescence images of the treated MCF-7 cancer cells stained with calcein AM (green) and PI (red) for live and dead cells, respectively. (C) The corresponding relative viability of the MCF-7 cancer cells treated with different concentration of MBBPM under the 808 nm laser irradiation (2.0 W/cm2) for 10 min. (D) The TEM image of the MCF-7 cancer cells incubated with MBBPM (1 × 107 MBBPM/mL) under the magnetic field (B ≈ 1.2 T) for 3 min and then treated under the 808 nm laser irradiation (2.0 W/cm2) for 10 min. 1: the nanomaterials uptaken inside the MCF-7 cells indicated by the red arrows; 2 and 3: the mitochondrion already collapsed and with the inner membrane ridges irregularly arranged indicated by the green and blue arrows, respectively. 4: Autophagolysosome containing nanomaterials and some subcellular indicated by the yellow arrows. The scale bar in the optical images of MCF-7 cancer cells incubation with MBBP and MBBPM before NIR irradiation in (A) is 400 μm. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 9:

(A) The optical images of MCF-7 cancer cells incubated with MBBP and MBBPM under the magnetic field (B ≈ 1.2 T) for 3 min and then under the NIR laser irradiation. (B) The fluorescence images of the treated MCF-7 cancer cells stained with calcein AM (green) and PI (red) for live and dead cells, respectively. (C) The corresponding relative viability of the MCF-7 cancer cells treated with different concentration of MBBPM under the 808 nm laser irradiation (2.0 W/cm2) for 10 min. (D) The TEM image of the MCF-7 cancer cells incubated with MBBPM (1 × 107 MBBPM/mL) under the magnetic field (B ≈ 1.2 T) for 3 min and then treated under the 808 nm laser irradiation (2.0 W/cm2) for 10 min. 1: the nanomaterials uptaken inside the MCF-7 cells indicated by the red arrows; 2 and 3: the mitochondrion already collapsed and with the inner membrane ridges irregularly arranged indicated by the green and blue arrows, respectively. 4: Autophagolysosome containing nanomaterials and some subcellular indicated by the yellow arrows. The scale bar in the optical images of MCF-7 cancer cells incubation with MBBP and MBBPM before NIR irradiation in (A) is 400 μm. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

The optimized concentration, i.e. 1 × 107 MBBPM/mL was used in the further study of the in vitro photothermal therapy effect comparison between the MBBPM and the BP nanosheets (Figure 10). It was found that, BP nanosheets and MBBPM addition without NIR irradiation or only NIR irradiation did not kill the MCF-7 cells (Figure 10A). With the magnetic field applied, incubation of BP nanosheets with the MCF-7 cells for 3 min and then applying 808 nm laser of 2.0 W/cm2 for 10 min did not lead to obvious killing effect. Extending the incubation time to 3 h, the viability decreased to less than 50% (Figure 10B). It may be due to that the BP nanomaterials taken up by tumor cells via endocytosis pathway takes about 4 h [10, 21, 22]. In contrast, incubation of MBBPM with the MCF-7 cells under the magnetic field for 3 min and then applying the NIR laser irradiation almost kill the MCF-7 cells completely. Extending the incubation time to 3 h showed no obvious difference. To further extend the incubation to 5 h, the cell viabilities of the treated MCF-7 cells show no obvious difference after the treatment with both the BP nanosheets alone and the MBBPM. It is proposed that the accelerated and enhanced photothermal killing effect was attributed to: 1. the targeted effect of the MBBPM, resulting in the large amount of MBBPM accumulated at the breast cancer cells and the temperature rapidly increase locally; 2. the dynamited MBBPM under the NIR laser irradiation (Figure 9A) is beneficial for increasing the cell membrane permeability and thus the BP nanosheets could be delivered into the MCF-7 cells quickly [97]; 3. The local mild temperature increase also improved the cell membrane permeability [98]. Thus, MBBPM accompanied with the magnetic field and NIR laser irradiation can accelerate and enhance the photothermal therapy effect.

Figure 10: 
(A) The fluorescence images of the MCF-7 cancer cells treated with BP nanosheets, MBBPM, or none of them (blank) without or with NIR laser irradiation after incubation for 3 min, 3 h, or 5 h and then stained with calcein AM (green) and PI (red) for live and dead cells, respectively. (B) The corresponding relative viability of the treated MCF-7 cancer cells. The scale bar is 400 μm. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 10:

(A) The fluorescence images of the MCF-7 cancer cells treated with BP nanosheets, MBBPM, or none of them (blank) without or with NIR laser irradiation after incubation for 3 min, 3 h, or 5 h and then stained with calcein AM (green) and PI (red) for live and dead cells, respectively. (B) The corresponding relative viability of the treated MCF-7 cancer cells. The scale bar is 400 μm. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

2.9 In vivo ultrasound imaging with MBBPM

To show the potential of the MBBPM for the targeted diagnosis, the ultrasound images of the tumors before injection, after injection of 20 min and after the burst of MBc and MBBPM have been obtained (Figure 11A). It can be seen that, after the injection of both MBc and MBBPM, the ultrasound signals at the tumors were enhanced, but which is higher for MBBPM. For a quantitative analysis, the echo–power–time profile of the tumor regions (ROI) 15 s before and after the burst of MBc and MBBPM after 20 min of their injection for their accumulation under the magnetic field. As shown in Figure 11B, the US signal intensity is higher for MBBPM before their burst, which illustrated its better magnetic responsiveness than MBc. Since the US signal before the burst of the MBs comes from both the adherent MBs attracted by the magnetic field and the circulating MBs and the US signal after the burst of the MBs comes only from the circulating MBs [99], the signal intensity before the burst minus that after the burst (i.e. the differential targeted enhancement parameter, dTE) was used to evaluate their targeting efficiency. In Figure 11C, the dTE parameter was shown for MBc and MBBPM and it was much bigger of MBBPM (12.1 ± 1.2 a.u.) than that of MBc (3.8 ± 0.6 a.u.), which demonstrated the much higher targeting efficiency.

Figure 11: 
(A) Ultrasound images of the tumors before injection, after injection for 20 min and after the burst of MBc and MBBPM. (B) The echo–power–time profile of the tumor regions (ROI) 15 s before and after the burst of MBc (blue dots) and MBBPM (grey dots) and their average values (MBc: orange dots and MBBPM: purple dots). (C) The differential targeted enhancement (dTE) parameter for the MBc and MBBPM. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 11:

(A) Ultrasound images of the tumors before injection, after injection for 20 min and after the burst of MBc and MBBPM. (B) The echo–power–time profile of the tumor regions (ROI) 15 s before and after the burst of MBc (blue dots) and MBBPM (grey dots) and their average values (MBc: orange dots and MBBPM: purple dots). (C) The differential targeted enhancement (dTE) parameter for the MBc and MBBPM. Data represent mean with SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001.

2.10 In vivo photothermal therapy with MBBPM

To show the targeted PTT efficiency of the MBBPM, the tumor-bearing mice were injected intravenously the PBS, BP nanosheets, and MBBPM, with the magnet placed near the tumor sites. After 10 min of the injection, the tumors were irradiated by the 808 nm laser (2.0 W/cm2, 10 min). The treatment was conducted every three days for three times. The photographs of the tumor-bearing mice and the isolated tumors on the 19th day after the photothermal therapy have been captured. As shown in Figure 12A, the tumor volume was the biggest after the PTT with PBS. Compared with that, the tumor volume was smaller after the PTT with BP nanosheets and that was the smallest after the PTT with MBBPM. From the H&E staining of the tumor tissue slices with the PTT in the three groups, it can be observed that the tumor cells mostly maintained their normal morphology while those were destroyed and became necrotic for the BP nanosheets and MBBPM groups. Moreover, the tumor cells were more severely destroyed. It demonstrated that the MBBPM has higher in vivo PTT efficiency. The tumor volumes and body weights of the tumor-bearing mice received the PTT with PBS, BP nanosheets, and MBBPM were also measured every other day. It illustrated that the tumor grew fast during 19 days after the PTT with PBS (Figure 12B). In comparison, the tumor grew slower after the PTT with BP nanosheets while the tumor growth was almost completely suppressed from the 7th day, confirming the higher efficient PTT of MBBPM. The weight of the treated mice was nearly not affected by the treatments (Figure 12C). It was also found that the no significant histological abnormalities were seen from the H&E staining of the major organs after the PTT with MBBPM (Figure 12D), which demonstrated its good biosafety.

Figure 12:
Figure 12:

3 Conclusions

The few-layer BP nanosheets of 100–200 nm have been synthesized via liquid phase exfoliation. It possesses a wide absorption band even in the NIR range. To deliver them into cancer cells for targeted theranostics, different configurations of magnetic MBs were achieved via the combination of streptavidin-coated Fe3O4 NPs and biotinylated MBs, with different connecting PEG chain lengths (no PEG spacer arm, PEG400, PEG1000, PEG2000, and PEG3400). As the PEG chain length increased, the stability was improved. For the shorter connecting PEG chain lengths, the biotin is shielded and thus the streptavidin-coated Fe3O4 NPs is hindered to couple with it. It results in the low yield of MBM, MBM400, and MBM1k (lower than 50%) and poor magnetic responsiveness. In contrast, for the magnetic MBs with longer connecting PEG chain lengths—MBM2k and MBM3400—the percentages were near 100%. Moreover, MBM2k and MBM3400 showed better stability, magnetic responsiveness, and echogenicity under magnetic field than the other magnetic MBs. Then, a precision delivery strategy by magnetic MBs with PEG2000 was adopted for targeted BP nanosheets delivery. The synthesized MBBPM are stable and can achieve targeted theranostics. They accumulate at the site with magnetic field application, increasing the US imaging signal intensity significantly both in vitro and in vivo and also leading to the local temperature increases rapidly to a saturation. Moreover, the in vitro and in vivo phtotothermal therapy also demonstrated that the MBBPM can dramatically improve the photothermal therapy efficiency with a significantly shortened incubation time needed for BP nanosheets delivered into the cancer cells under the magnetic field and NIR laser irradiation.

4 Methods

4.1 Materials

The main component materials of the lipid microbubbles—1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)—were purchased from Avanti Polar Lipids (Alabaster, USA). The coupling agent with different PEG chain lengths, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-x)] (DSPE-PEGx-biotin) with x = 0, 400, 1000, 2000 and 3400, were obtained from Ruixi Biological Technology Co. Ltd (Xi’an, China). The streptavidin-coated superparamagnetic Fe3O4 NPs (5 mg/mL, ∼130 nm) were purchased from Zhongkeleiming Technology Co. Ltd (Beijing, China). The bulk BP was obtained from the supplier of Smart-Elements (Austria) and anhydrous N-methyl-2-pyrrolidone (NMP) (99.5%) was bought from Aladdin Bio-Chem Technology Co. LTD (Shanghai, China). Calcein AM and PI were obtained from KeyGEN BioTECH (Nanjing, China). All of the materials were used as received without further modification.

4.2 Preparation of magnetic MBs

First, the biotinylated MBs were synthesized. DSPC, DSPE-PEG2000, and DSPE-PEGx-Biotin (x = 0, 400, 1000, 2000, and 3400) were dissolved in a mixture of 18 mL of chloroform and 2 mL of methanol with a molar ratio of 82:9:9, as previously reported [79], [81]. The mixture was then magnetically stirred for 30 min. After rotary evaporation, vacuum drying, hydration and ultrasound treatment, the obtained solution was evacuated and octafluoropropane (C3F8) was introduced. Finally, the MBs were achieved with shaking for 30 s. MBs were purified by two centrifugation (400 g)/redispersion cycles. The as synthesized biotinylated MBs with different PEG chain lengths were denoted MBb, MBb400, MBb1k, MBb2k, and MBb3400, for x = 0, 400, 1000, 2000, and 3400, respectively.

To prepare the magnetic MBs, a suspension of streptavidin-coated superparamagnetic Fe3O4 NPs was added to the five as synthesized biotinylated MB solutions for 15 min. To reduce the possibility that one streptavidin binds multiple biotins, and thus avoid the aggregation of the magnetic MBs, the streptavidin-coated superparamagnetic Fe3O4 NPs firstly undergo ultrasound treatment and its ratio to biotinylated MBs was set to 4:1. Then the obtained magnetic MB solutions were subjected to two centrifugation (400 g)/redispersion cycles to remove the magnetic NPs not coupled to the MB shell surface. Finally, a magnet (25 × 10 × 5 mm, ∼1.2 T) was used to attract the magnetic MBs while the nonmagnetic MBs in the solutions were collected for measuring the nonmagnetic MBs. The magnet was then removed and the purified magnetic MBs were redispersed in PBS for further characterization. The obtained magnetic MBs were denoted MBM, MBM400, MBM1k, MBM2k, and MBM3400, for x = 0, 400, 1000, 2000, and 3400, respectively. To check the magnetic NPs connected on the MBs surface, the Labeling Check Reagent-FITC (MACS Miltenyi Biotec, Germany) was added in the magnetic MBs suspension for 15 min at 4 °C and the excess Labeling Check Reagent-FITC was removed by two centrifugation (400g)/redispersion cycles.

4.3 Preparation of MBBPM

The BP nanosheets were synthesized via liquid exfoliation as previously reported [89]. An amount of 20 mg of the bulk BP powder dispersed in 2 mL of NMP (10 mL) was ground. Then 18 mL additional NMP was added to the dispersion and the mixture underwent probe sonication for 8 h (duty cycle of 50%) using a power of 260 W. Afterwards, the mixture underwent sonication overnight with a power of 300 W. To avoid the overheat, they were performed in an ice bath. The dispersion was centrifuged at 7000 rpm for 20 min and the collected supernatant was further centrifuged at 15,000 rpm for 5 min. Some of the obtained precipitate was dispersed in PBS for the measurement and the others was dispersed in NMP and stored at 4 °C.

For the electrostatic adsorption of BP nanosheets on the MBs surface, the stearic-PEI600 was added in the MBs formulation to synthesize MBc with the molar ratio of DSPC, DSPE-PEG2000, DSPE-PEG2000-biotin, and stearic-PEI600 to be 46:9:9:36. Then the BP nanosheets dispersion was mixed with the MBc. After their incubation for 15 min, the magnetic NP dispersion was then added to the mixture and incubated for further 15 min. To mix them well, the mixture was shaken gently. Finally, the mixture was centrifuged at 1400 rpm for 4 min and the unadsorbed BP and nonconnected magnetic NPs in the suspension was removed.

4.4 Property characterization

The particle size distribution and zeta potential of all of the biotinylated, magnetic MBs, and MBBPM were analyzed by dynamic light scattering (DLS) with a Zetasizer Nano ZSE (Malvern, United Kingdom). Since the zeta potential of samples was influenced by PBS, it was measured in deionized water. The concentration of the as prepared MBs was measured using accusizer (Particle Sizing Systems, PSS A7000AD, USA). The binding rate of the magnetic NPs to the MBs was evaluated from the percentage of magnetic MBs in the entire MB population, which could be calculated using the formula: (entire MBs concentration − nonmagnetic MBs concentration)/entire MBs concentration. Their morphologies were observed with an inverted fluorescence microscope (Leica DMi8, Germany), scanning electron microscopy (SEM, ZEISS SUPRA55, Germany) and transmission electron microscopy (TEM, Hitachi 7500, Japan). The thickness of BP nanosheets was measured using Atomic Force Microscope (AFM, Bruker, Germany). XPS was achieved via a ULVAC PHI 5000 Versa Probe II (Japan). Raman spectra was obtained by Witec Alpha 300R using a 532 nm continuous-wave laser (Witec, Germany). Absorption spectra were acquired through a Tecan Spark multifunctional microplate reader (Switzerland).

4.5 Stability

To test the stability of the biotinylated MBs, magnetic MBs, BP MBBP, and MBBPM, they were dispersed in PBS solution and then their remaining percentage were measured as prepared and after storage in ambient conditions for 20, 40, and 60 min.

4.6 Magnetic responsiveness

Analysis of the magnetic responsiveness was conducted with a home-made parallel plate flow chamber assay. A total of 1 mL of magnetic MBs (with the same initial concentration of 1 × 108 MBs/mL) was injected into a vacuum parallel plate flow chamber (Glycotech 31-001, USA) by a stepping motor (Yuhui, China) through capillary tubing (r = 0.15 cm) at sheer forces of 5–45 dyn/cm2. A magnet (25 × 10 × 5 mm, ∼1.2 T) was placed on the chamber throughout the experiments to capture the magnetic MBs, and then they were redispersed in 1 mL of PBS. The magnetic responsiveness was evaluated from the ratio of captured MBs to MBs originally injected and was plotted as a function of the sheer force.

4.7 In vitro echogenicity

To obtain the in vitro ultrasound imaging, the Vevo 2100 ultrasound imaging platform (VisualSonics, Canada) was used. Agarose gel powder and microcentrifuge tube were used to prepare agar plates with holes, and MBs of the same concentration (5 × 106 MBs/mL, 400 μL) were added to the agar plates. Their ultrasound images and the signal intensities in the region of interest (ROI) were recorded (B-mode and contrast mode, frequency of 18 MHz, power of 2%, gain of 35 dB) under the magnet for 20 min. The signal enhancement was normalized by that of PBS solution of the same volume to evaluate the in vitro echogenicity.

4.8 Photothermal performance

0.5 mL of samples in EP tubes were irradiated by a fiber-coupled continuous laser of 808 nm (Yuanming Laser Technology, China) with a power density of 2.0 W/cm2 for 10 min. Real-time thermal imaging was captured and the temperature was recorded by the infrared thermal imaging camera (Fluke Ti27, USA).

4.9 In vitro photothermal therapy study

MCF-7 cells (human breast cancer cells) were seeded in a 96-well plate for 12 h with 200 μL of DMEM (HyClone) supplemented with 10% (volume ratio) of fetal bovine serum and kept in an incubator consisting of 5% CO2 at 37 °C. Then they were incubated with MBBPM or BP nanosheets for 3 min or 3 h under magnetic field and then were irradiated by 808 nm laser with the power of 2.0 W/cm2 for 10 min. For each sample, three multiple holes were set. The height of laser tip to 96-well plates was adjusted to cover one well. After the treatments, the cells were further cultured for 12 h. Afterwards, the MCF-7 cells were rinsed with PBS three times and then costained with Calcein AM and PI for 30 min. After rinsing with PBS, they were observed by the inverted fluorescence microscope. The relative cell viability was calculated with ImageJ software depending on the fluorescence images. After the treatment with MBBPM, the cells were washed with PBS to remove the materials. Then they were detached, centrifuged, fixed, and dehydrated. The cell pellets were infiltrated in a mixture of epoxy resin in 100% ethanol and leave it polymerized to make ultrathin slices. Finally, their TEM images were captured using the 120 kV transmission electron microscope (Tecnai G2 Spirit BioTWIN, FEI, USA).

4.10 Establishment of subcutaneous tumor model

The Female BALB/c nude mice aged six weeks old with the weight about 20 g were bought from the Huafukang Biotechnology Co., Ltd. (Beijing, China). To establish the subcutaneous tumor model, MCF-7 cells (5 × 107/mL, 100 μL) were injected subcutaneously into their right forelimb armpit. When the tumor diameters reached approximately 5–7 mm, the nude mice were used for the in vivo ultrasound imaging and the in vivo photothermal therapy.

4.11 In vivo ultrasound imaging

The tumor-bearing nude mice were first anesthetized with 0.5% pentobarbital (30 mg/kg mouse weight). The Vevo 2100 ultrasound imaging platform (VisualSonics, Canada) was used to obtain the contrast-enhanced ultrasound imaging of the tumors before the MBs injection. Then they were administrated intravenously the MBc and the MBBPM (n = 5 for each group, 1 × 107 MBs/mL, 150 μL), with the magnet placed next to the tumors. After 20 min of their injection for the MBs accumulation, the US imaging was captured for 15 s, followed by the burst of the MBs and the magnet removed. The US imaging is captured for 15 s further to allow freely circulating MBs to replenish in tumors. The differential targeted enhancement (dTE) parameter was analyzed with the built-in Vevo CQ software to evaluate the targeting effect of the MBBPM.

4.12 In vivo photothermal therapy study

To study the PTT efficacy of the MBBPM in vivo, the tumor-mice were administrated intravenously the PBS, MBBP, and the MBBPM (n = 5 for each group, 150 μL) with the magnet placed next to the tumors. After 10 min of the injection, the tumors were irradiated by the 808 nm laser (2.0 W/cm2, 10 min). The treatment was conducted every three days for three times and the changes in the tumor volume and body weight were recorded for 19 days. The tumor size was measured using a vernier caliper, and the volumes were calculated by the equation (volume = length × width2/2). At the 19th day, all of the mice were executed cervical dislocation and the tumors of each group were isolated for H&E staining. To assess the biosafety of the MBBPM, the organs of heart, liver, spleen, lung, and kidney of the MBBPM group were also isolated for H&E staining.

4.13 Statistical analysis

Comparisons between two groups of data were made using the unpaired t-test. The means of more than two groups were compared with the one-way ANOVA or two-way ANOVA, followed by post Tukey’s pairwise comparisons. Probability values p < 0.05 were considered statistically significant. Statistical analyses were carried out with Prism software packages (version 5.01).


Corresponding authors: Jinfeng Xu, Department of Ultrasonography, Shenzhen Medical Ultrasound Engineering Center, Shenzhen People’s Hospital, Second Clinical Medical College of Jinan University, First Clinical Medical College of Southern University of Science and Technology, Shenzhen 518020, China; and Han Zhang, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, and Otolaryngology Department and Biobank of the First Affiliated Hospital, Shenzhen Second People’s Hospital, Health Science Center, Shenzhen University, Shenzhen 518060, P.R. China, E-mail: , .
Yao Zhu and Yingying Liu contributed equally to this work.

Award Identifier / Grant number: 81771841

Funding source: Guangdong Basic and Applied Basic Research Foundation

Award Identifier / Grant number: 2019A1515111132

Award Identifier / Grant number: 2019B1515120043

Funding source: Science and Technology Project of Shenzhen

Award Identifier / Grant number: JCYJ20180508152903208

Award Identifier / Grant number: 2020A151501612

Funding source: Longhua District Science and Innovation Commission Project Grants of Shenzhen

Award Identifier / Grant number: JCYJ201904

  1. Author contributions: Y.Z. performed all the characterizations and wrote the manuscript. Y.-Y.L. designed the experiments. Z.-J.X. edited and revised the manuscript. T.-Z.H. synthesized microbubbles and L.-L.S. conducted echogenicity measurements. F.-J.G. prepared magnetic microbubbles. G.A. cultured cells and performed photothermal measurements. X.-S.L. performed TEM measurements. J.-F.X. and H. Z. conceived and supervised the study. All authors approved the manuscript.

  2. Research funding: The authors acknowledge financially supported by the National Natural Science Foundation of China (No. 81771841). Yao Zhu also acknowledges the Guangdong Basic and Applied Basic Research Foundation (Nos. 2019A1515111132 and 2019B1515120043) and the Research Subsidy for post-doc staying in Shenzhen from Shenzhen Municipal Bureau of Human Resources and Social Affairs. The Science and Technology Project of Shenzhen (JCYJ20180508152903208), Natural Science Foundation of Guangdong Province (Grant No. 2020A151501612) and Longhua District Science and Innovation Commission Project Grants of Shenzhen (JCYJ201904) are also grateful acknowledged.

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

References

[1] X. Sun, X. He, Y. Zhang, et al., “Inflammatory cell-derived CXCL3 promotes pancreatic cancer metastasis through a novel myofibroblast-hijacked cancer escape mechanism,” Gut, 2021, Epub ahead of print, https://doi.org/10.1136/gutjnl-2020-322744.Search in Google Scholar PubMed

[2] Q. Liu, Z. Xie, M. Qiu, et al.., “Prodrug-loaded zirconium carbide nanosheets as a novel biophotonic nanoplatform for effective treatment of cancer,” Adv. Sci., vol. 7, p. 2001191, 2020, https://doi.org/10.1002/advs.202001191.Search in Google Scholar PubMed PubMed Central

[3] X. He, X. Yin, J. Wu, et al.., “Visualization of human T lymphocyte-mediated eradication of cancer cells in vivo,” Proc. Natl. Acad. Sci. USA, vol. 117, pp. 22910–22919, 2020, https://doi.org/10.1073/pnas.2009092117.Search in Google Scholar PubMed PubMed Central

[4] Y. Cao, “Adipocyte and lipid metabolism in cancer drug resistance,” J. Clin. Invest., vol. 129, pp. 3006–3017, 2019, https://doi.org/10.1172/jci127201.Search in Google Scholar PubMed PubMed Central

[5] S. Wu, K. S. Hui, and K. N. Hui, “2D black phosphorus: from preparation to applications for electrochemical energy storage,” Adv. Sci., vol. 5, p. 1700491, 2018, https://doi.org/10.1002/advs.201700491.Search in Google Scholar PubMed PubMed Central

[6] M. Liu, S. Feng, Y. Hou, et al.., “High yield growth and doping of black phosphorus with tunable electronic properties,” Mater. Today, vol. 36, pp. 91–101, 2020, https://doi.org/10.1016/j.mattod.2019.12.027.Search in Google Scholar

[7] J. Pang, A. Bachmatiuk, Y. Yin, et al.., “Applications of phosphorene and black phosphorus in energy conversion and storage devices,” Adv. Energy Mater., vol. 8, pp. 1–43, 2018, https://doi.org/10.1002/aenm.201702093.Search in Google Scholar

[8] D. K. Sang, H. Wang, Z. Guo, N. Xie, and H. Zhang, “Recent developments in stability and passivation techniques of phosphorene toward next-generation device applications,” Adv. Funct. Mater., vol. 29, pp. 1–22, 2019, https://doi.org/10.1002/adfm.201903419.Search in Google Scholar

[9] Z. Liu, Y. Sun, H. Cao, et al.., “Unzipping of black phosphorus to form zigzag-phosphorene nanobelts,” Nat. Commun., vol. 11, pp. 1–10, 2020, https://doi.org/10.1038/s41467-020-17622-6.Search in Google Scholar PubMed PubMed Central

[10] W. Zhou, H. Cui, L. Ying, and X. F. Yu, “Enhanced cytosolic delivery and release of CRISPR/cas9 by black phosphorus nanosheets for genome editing,” Angew. Chem. Int. Ed., vol. 57, pp. 10268–10272, 2018, https://doi.org/10.1002/anie.201806941.Search in Google Scholar PubMed

[11] J. Shao, H. Xie, H. Huang, et al.., “Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy,” Nat. Commun., vol. 7, pp. 1–13, 2016, https://doi.org/10.1038/ncomms12967.Search in Google Scholar PubMed PubMed Central

[12] W. Zhou, T. Pan, H. Cui, Z. Zhao, P. K. Chu, and X. Yu, “Black phosphorus: bioactive nanomaterials with inherent and selective chemotherapeutic effects,” Angew. Chem., vol. 58, pp. 769–774, 2018, https://doi.org/10.1002/anie.201810878.Search in Google Scholar PubMed

[13] J. Peng, Y. Lai, Y. Chen, J. Xu, L. Sun, and J. Weng, “Sensitive detection of carcinoembryonic antigen using stability-limited few-layer black phosphorus as an electron donor and a reservoir,” Small, vol. 13, pp. 1–11, 2017, https://doi.org/10.1002/smll.201603589.Search in Google Scholar PubMed

[14] Z. Li, T. Zhang, F. Fan, F. Gao, H. Ji, and L. Yang, “Piezoelectric materials as sonodynamic sensitizers to safely ablate tumors: a case study using black phosphorus,” J. Phys. Chem. Lett., vol. 11, pp. 1228–1238, 2020, https://doi.org/10.1021/acs.jpclett.9b03769.Search in Google Scholar PubMed

[15] J. Ouyang, L. Deng, W. Chen, et al.., “Two dimensional semiconductors for ultrasound-mediated cancer therapy: the case of black phosphorus nanosheets,” Chem. Commun., vol. 54, pp. 2874–2877, 2018, https://doi.org/10.1039/c8cc00392k.Search in Google Scholar PubMed

[16] W. Chen, J. Ouyang, H. Liu, et al.., “Black phosphorus nanosheet-based drug delivery system for synergistic photodynamic/photothermal/chemotherapy of cancer,” Adv. Mater., vol. 29, pp. 1–7, 2017, https://doi.org/10.1002/adma.201603864.Search in Google Scholar PubMed

[17] Z. Xie, M. Peng, R. Lu, et al.., “Black phosphorus-based photothermal therapy with aCD47-mediated immune checkpoint blockade for enhanced cancer immunotherapy,” Light Sci. Appl., vol. 9, p. 161, 2020, https://doi.org/10.1038/s41377-020-00388-3.Search in Google Scholar PubMed PubMed Central

[18] S. Xiong, Z. Li, Y. Liu, et al.., “Brain-targeted delivery shuttled by black phosphorus nanostructure to treat Parkinson’s disease,” Biomaterials, vol. 260, p. 120339, 2020, https://doi.org/10.1016/j.biomaterials.2020.120339.Search in Google Scholar PubMed

[19] Z. Xie, T. Fan, J. An, et al.., “Emerging combination strategies with phototherapy in cancer nanomedicine,” Chem. Soc. Rev., vol. 49, pp. 8065–8087, 2020, https://doi.org/10.1039/d0cs00215a.Search in Google Scholar PubMed

[20] J. Ouyang, X. Ji, X. Zhang, et al.., “In situ sprayed NIR-responsive, analgesic black phosphorus-based gel for diabetic ulcer treatment,” Proc. Natl. Acad. Sci. USA, vol. 117, pp. 28667–28677, 2020, https://doi.org/10.1073/pnas.2016268117.Search in Google Scholar PubMed PubMed Central

[21] W. Tao, X. Zhu, X. Yu, et al.., “Black phosphorus nanosheets as a robust delivery platform for cancer theranostics,” Adv. Mater., vol. 29, pp. 1–9, 2017, https://doi.org/10.1002/adma.201770002.Search in Google Scholar

[22] S. Zong, L. Wang, Z. Yang, H. Wang, Z. Wang, and Y. Cui, “Black phosphorus-based drug nanocarrier for targeted and synergetic chemophotothermal therapy of acute lymphoblastic leukemia,” ACS Appl. Mater. Interfaces, vol. 11, pp. 5896–5902, 2019, https://doi.org/10.1021/acsami.8b22563.Search in Google Scholar PubMed

[23] Z. Xie, C. Xing, W. Huang, et al.., “Ultrathin 2D nonlayered tellurium nanosheets: facile liquid phase exfoliation, characterization, and photoresponse with high performance and enhanced stability,” Adv. Funct. Mater., vol. 28, pp. 1705833–1705844, 2018, https://doi.org/10.1002/adfm.201705833.Search in Google Scholar

[24] C. Xing, Z. Xie, Z. Liang, et al.., “Selenium nanosheets: 2D nonlayered selenium nanosheets: facile synthesis, photoluminescence, and ultrafast photonics,” Adv. Opt. Mater., vol. 5, pp. 1700884–1700894, 2017, https://doi.org/10.1002/adom.201700884.Search in Google Scholar

[25] T. J. Fan, Z. J. Xie, W. C. Huang, Z. J. Li, and H. Zhang, “Two-Dimensional non-layered selenium nanoflakes: facile fabrications and applications for self-powered photo-detector,” Nanotechnology, vol. 30, p. 114002, 2019, https://doi.org/10.1088/1361-6528/aafc0f.Search in Google Scholar PubMed

[26] L. Wu, Z. Xie, L. Lu, et al.., “Few-layer tin sulfide: a promising black-phosphorus-analogue 2D material with exceptionally large nonlinear optical response, high stability, and applications in all-optical switching and wavelength conversion,” Adv. Opt. Mater., vol. 6, pp. 1–10, 2018, https://doi.org/10.1002/adom.201700985.Search in Google Scholar

[27] Z. Xie, F. Zhang, Z. Liang, T. Fan, and H. Zhang, “Revealing of the ultrafast third-order nonlinear optical response and enabled photonic application in two-dimensional tin sulfide,” Photonics Res., vol. 7, pp. 494–502, 2019, https://doi.org/10.1364/prj.7.000494.Search in Google Scholar

[28] W. Huang, Z. Xie, T. Fan, et al.., “Black-phosphorus-analogue tin monosulfide: an emerging optoelectronic two-dimensional material for high-performance photodetection with improved stability under ambient/harsh conditions,” J. Mater. Chem. C, vol. 6, pp. 9582–9593, 2018, https://doi.org/10.1039/c8tc03284j.Search in Google Scholar

[29] C. Xing, W. Huang, Z. Xie, et al.., “Ultra-small bismuth quantum dots: facile liquid-phase exfoliation, characterization, and application in high-performance UV–Vis photo-detector,” ACS Photonics, vol. 5, pp. 621–629, 2017, https://doi.org/10.1021/acsphotonics.7b01211.Search in Google Scholar

[30] Z. Xie, Y. P. Peng, L. Yu, et al.., “Solar-inspired water purification based on emerging two-dimensional materials: status and challenges,” Sol. RRL, vol. 4, p. 1900400, 2020, https://doi.org/10.1002/solr.201900400.Search in Google Scholar

[31] T. Yoshida, T. Kondo, R. Ogawa, et al.., “Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma,” Canc. Chemother. Pharmacol., vol. 61, pp. 559–567, 2008, https://doi.org/10.1007/s00280-007-0503-y.Search in Google Scholar PubMed

[32] A. P. Sviridov, V. G. Andreev, E. M. Ivanova, et al.., “Porous silicon nanoparticles as sensitizers for ultrasonic hyperthermia,” Appl. Phys. Lett., vol. 103, p. 193110, 2013, https://doi.org/10.1063/1.4829148.Search in Google Scholar

[33] X. Lin, J. Song, and X. Chen, “Ultrasound activated sensitizers and applications,” Angew. Chem., vol. 59, pp. 14212–14233, 2019, https://doi.org/10.1002/anie.201912768.Search in Google Scholar PubMed

[34] B. Yang, Y. Chen, and J. Shi, “Reactive oxygen species (ROS)-based nanomedicine,” Chem. Rev., vol. 119, pp. 4881–4985, 2019, https://doi.org/10.1021/acs.chemrev.8b00626.Search in Google Scholar PubMed

[35] M. Ma, H. Xu, H. Chen, X. Jia, K. Zhang, and Q. Wang, “A drug – perfluorocarbon nanoemulsion with an ultrathin silica coating for the synergistic effect of chemotherapy and ablation by high-intensity focused ultrasound,” Adv. Mater., vol. 26, pp. 7378–7385, 2014, https://doi.org/10.1002/adma.201402969.Search in Google Scholar PubMed

[36] W. Tsai, H. Lai, J. Lee, C. Lo, and W. Chen, “Enhancement of the cytotoxicity and selectivity of doxorubicin to hepatoma cells by synergistic combination of galactose-decorated γ-poly (glutamic acid) nanoparticles and low-intensity ultrasound,” Langmuir, vol. 30, pp. 5510–5517, 2014, https://doi.org/10.1021/la500352g.Search in Google Scholar PubMed

[37] S. Rizzitelli, P. Giustetto, C. Boffa, et al.., “Nanomedicine Nanotechnology, in vivo MRI visualization of release from liposomes triggered by local application of pulsed low-intensity non-focused ultrasound,” Biol. Med., vol. 10, pp. 901–904, 2014, https://doi.org/10.1016/j.nano.2014.03.012.Search in Google Scholar PubMed

[38] K. Zhang, H. Xu, H. Chen, et al.., “Theranostics CO2 bubbling-based ‘nanobomb’ system for targetedly suppressing panc-1 pancreatic tumor via low intensity ultrasound-activated inertial cavitation,” Theranostics, vol. 5, pp. 1291–1302, 2015, https://doi.org/10.7150/thno.12691.Search in Google Scholar PubMed PubMed Central

[39] S. Tinkov, C. Coester, S. Serba, et al.., “New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: in-vivo characterization,” J. Contr. Release, vol. 148, pp. 368–72, 2010, https://doi.org/10.1016/j.jconrel.2010.09.004.Search in Google Scholar PubMed

[40] F. Yan, L. Li, Z. T. Deng, et al.., “Paclitaxel-liposome–microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers,” J. Contr. Release, vol. 166, pp. 246–255, 2013, https://doi.org/10.1016/j.jconrel.2012.12.025.Search in Google Scholar PubMed

[41] Y. Chen, T. Liu, P. Chang, et al.., “A theranostic nrGO@MSN-ION nanocarrier developed to enhance the combination effect of sonodynamic therapy and ultrasound hyperthermia for treating tumor,” Nanoscale, vol. 8, pp. 12648–12657, 2016, https://doi.org/10.1039/c5nr07782f.Search in Google Scholar PubMed

[42] X. Qian, Y. Zheng, and Y. Chen, “Micro/nanoparticle-augmented sonodynamic therapy (SDT): breaking the depth shallow of photoactivation,” Adv. Mater., vol. 28, pp. 8097–8129, 2016, https://doi.org/10.1002/adma.201602012.Search in Google Scholar PubMed

[43] T. Liu, N. Zhang, Z. Wang, et al.., “Endogenous catalytic generation of O2 bubbles for in situ ultrasound-guided high intensity focused ultrasound ablation,” ACS Nano, vol. 11, pp. 9093–9102, 2017, https://doi.org/10.1021/acsnano.7b03772.Search in Google Scholar PubMed

[44] K. Kaczmarek, T. Hornowski, and M. Kubovcíkova, “Biological and medical applications of materials and interfaces heating induced by therapeutic ultrasound in the presence of magnetic nanoparticles heating induced by therapeutic ultrasound in the presence of magnetic nanoparticles,” ACS Appl. Mater. Interfaces, vol. 72, pp. 28–48, 2018.Search in Google Scholar

[45] Z. Xie, D. Wang, T. Fan, et al.., “Black phosphorus analogue tin sulfide nanosheets: synthesis and application as near-infrared photothermal agents and drug delivery platforms for cancer therapy,” J. Mater. Chem. B, vol. 6, pp. 4747–4755, 2018, https://doi.org/10.1039/c8tb00729b.Search in Google Scholar PubMed

[46] Z. Xie, S. Chen, Y. Duo, et al.., “Biocompatible two-dimensional titanium nanosheets for multimodal imaging-guided cancer theranostics,” ACS Appl. Mater. Interfaces, vol. 11, pp. 22129–22140, 2019, https://doi.org/10.1021/acsami.9b04628.Search in Google Scholar PubMed

[47] C. Xing, S. Chen, M. Qiu, et al.., “Conceptually novel black phosphorus/cellulose hydrogels as promising photothermal agents for effective cancer therapy,” Adv. Healthc. Mater., vol. 7, pp. 1–11, 2018, https://doi.org/10.1002/adhm.201870030.Search in Google Scholar

[48] X. Liang, X. Ye, C. Wang, et al.., “Photothermal cancer immunotherapy by erythrocyte membrane-coated black phosphorus formulation,” J. Contr. Release, vol. 296, pp. 150–161, 2019, https://doi.org/10.1016/j.jconrel.2019.01.027.Search in Google Scholar PubMed

[49] C. Fan, C. Ting, H. Lin, et al.., “Biomaterials SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumor drug delivery,” Biomaterials, vol. 34, pp. 3706–3715, 2013, https://doi.org/10.1016/j.biomaterials.2013.01.099.Search in Google Scholar PubMed

[50] C. Fan, C. Ting, H. Liu, et al.., “Biomaterials antiangiogenic-targeting drug-loaded microbubbles combined with focused ultrasound for glioma treatment,” Biomaterials, vol. 34, pp. 2142–2155, 2013, https://doi.org/10.1016/j.biomaterials.2012.11.048.Search in Google Scholar PubMed

[51] N. Zhang, F. Yan, X. Liang, et al.., “Theranostics Localized delivery of curcumin into brain with polysorbate 80-modified cerasomes by ultrasound-targeted microbubble destruction for improved Parkinson’s disease therapy,” Theranostics, vol. 8, pp. 2264–2277, 2018, https://doi.org/10.7150/thno.23734.Search in Google Scholar PubMed PubMed Central

[52] E. E. Paoli, E. S. Ingham, H. Zhang, et al.., “Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes,” J. Contr. Release, vol. 178, pp. 108–117, 2015, https://doi.org/10.1016/j.jconrel.2014.01.005.Search in Google Scholar PubMed PubMed Central

[53] S. Shen, D. Huang, J. Cao, et al.., “Magnetic liposomes for light-sensitive drug delivery and combined photothermal-chemotherapy of tumors,” J. Mater. Chem. B, vol. 7, pp. 1096–1106, 2019, https://doi.org/10.1039/c8tb02684j.Search in Google Scholar PubMed

[54] K. Kooiman, H. J. Vos, M. Versluis, and N. De Jong, “Acoustic behavior of microbubbles and implications for drug delivery,” Adv. Drug Deliv. Rev., vol. 72, pp. 28–48, 2014, https://doi.org/10.1016/j.addr.2014.03.003.Search in Google Scholar PubMed

[55] M. Chen, X. Liang, C. Gao, et al.., “Ultrasound triggered conversion of porphyrin/camptothecin-fluoroxyuridine triad microbubbles into nanoparticles overcomes multidrug resistance in colorectal cancer,” ACS Nano, vol. 12, pp. 7312–7326, 2018, https://doi.org/10.1021/acsnano.8b03674.Search in Google Scholar PubMed

[56] J. R. Lindner, J. Song, A. R. Jayaweera, J. Sklenar, and S. Kaul, “Microvascular rheology of definity microbubbles after intra-arterial and intravenous administration,” J. Am. Soc. Echocardiogr., vol. 15, pp. 396–403, 2002, https://doi.org/10.1067/mje.2002.117290.Search in Google Scholar PubMed

[57] K. W. Ferrara, M. A. Borden, and H. Zhang, “Lipid-shelled vehicles: engineering for ultrasound molecular imaging and drug delivery,” Acc. Chem. Res., vol. 42, pp. 881–892, 2009, https://doi.org/10.1021/ar8002442.Search in Google Scholar PubMed PubMed Central

[58] Y. Du, X. Liang, Y. Li, et al.., “Liposomal nanohybrid cerasomes targeted to PD-L1 enable dual-modality imaging and improve antitumor treatments,” Canc. Lett., vol. 414, pp. 230–238, 2017, https://doi.org/10.1016/j.canlet.2017.11.019.Search in Google Scholar PubMed

[59] X. Liang, J. Gao, L. Jiang, et al.., “Nanohybrid liposomal cerasomes with good physiological stability and rapid temperature responsiveness for high intensity nanohybrid liposomal cerasomes with good physiological stability and rapid temperature responsiveness for high intensity focused ultrasound,” ACS Nano, vol. 9, pp. 1280–1293, 2015, https://doi.org/10.1021/nn507482w.Search in Google Scholar PubMed

[60] K. Matsui, S. Sando, T. Sera, et al.., “Cerasome as an infusible, cell-friendly, and serum-compatible transfection agent in a viral size,” J. Am. Chem. Soc., vol. 128, pp. 3114–3115, 2006, https://doi.org/10.1021/ja058016i.Search in Google Scholar PubMed

[61] Y. Sasaki, K. Matsui, Y. Aoyama, and J. Kikuchi, “Cerasome as an infusible and cell-friendly gene carrier: synthesis of cerasome-forming lipids and transfection using cerasome,” Nat. Protoc., vol. 1, pp. 1227–1234, 2006, https://doi.org/10.1038/nprot.2006.182.Search in Google Scholar PubMed

[62] Z. Tang, N. Kong, X. Zhang, et al.., “A materials-science perspective on tackling COVID-19,” Nat. Rev. Mater., vol. 5, pp. 1–14, 2020, https://doi.org/10.1038/s41578-020-00247-y.Search in Google Scholar PubMed PubMed Central

[63] M. C. Olson, T. D. Atwell, and J. M. Knudsen, “Anaphylactic reaction to an ultrasound contrast agent (Lumason) in a patient with systemic mastocytosis,” J. Clin. Ultrasound, vol. 46, pp. 533–535, 2018, https://doi.org/10.1002/jcu.22585.Search in Google Scholar PubMed

[64] A. Q. X. Nio, A. Faraci, Kirsten. Christensen-Jeffries, et al.., “Optimal control of SonoVue microbubbles to estimate hydrostatic pressure,” IEEE Trans. Ultrason. Ferroelectrics Freq. Contr., vol. 67, pp. 557–567, 2020, https://doi.org/10.1109/tuffc.2019.2948759.Search in Google Scholar

[65] Y. Lv, Y. Cao, P. Li, et al.., “Ultrasound-triggered destruction of folate-functionalized mesoporous silica nanoparticle-loaded microbubble for targeted tumor therapy,” Adv. Healthc. Mater., vol. 6, p. 1700354, 2017, https://doi.org/10.1002/adhm.201700354.Search in Google Scholar PubMed

[66] C. H. Fan, E. L. Chang, C. Y. Ting, Y. C. Lin, and C. K. Yeh, “Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery,” Biomaterials, vol. 106, pp. 46–57, 2016, https://doi.org/10.1016/j.biomaterials.2016.08.017.Search in Google Scholar PubMed

[67] W. Luo, G. Wen, L. Yang, et al.., “Dual-targeted and pH-sensitive doxorubicin prodrug-microbubble complex with ultrasound for tumor treatment,” Theranostics, vol. 7, pp. 452–465, 2017, https://doi.org/10.7150/thno.16677.Search in Google Scholar PubMed PubMed Central

[68] M. A. Borden, H. Zhang, R. J. Gillies, P. A. Dayton, and K. W. Ferrara, “A stimulus-responsive contrast agent for ultrasound molecular imaging,” Biomaterials, vol. 29, pp. 597–606, 2008, https://doi.org/10.1016/j.biomaterials.2007.10.011.Search in Google Scholar PubMed PubMed Central

[69] L. Oddo, B. Cerroni, F. Domenici, et al.., “Next generation ultrasound platforms for theranostics,” J. Colloid Interface Sci., vol. 491, pp. 151–160, 2017, https://doi.org/10.1016/j.jcis.2016.12.030.Search in Google Scholar PubMed

[70] L. Duan, F. Yang, W. He, et al.., “A multi‐gradient targeting drug delivery system based on RGD‐l‐TRAIL‐labeled magnetic microbubbles for cancer theranostics,” Adv. Funct. Mater., vol. 26, pp. 8313–8324, 2016, https://doi.org/10.1002/adfm.201603637.Search in Google Scholar

[71] C. R. Anderson, X. Hu, H. Zhang, et al.., “Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent,” Invest. Radiol., vol. 46, pp. 215–224, 2011, https://doi.org/10.1097/rli.0b013e3182034fed.Search in Google Scholar

[72] B. Chertok and R. Langer, “Circulating magnetic microbubbles for localized real-time control of drug delivery by ultrasonography-guided magnetic targeting and ultrasound,” Theranostics, vol. 8, pp. 341–357, 2018, https://doi.org/10.7150/thno.20781.Search in Google Scholar PubMed PubMed Central

[73] H. Mannell, J. Pircher, F. Fochler, et al.., “Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles,” Nanomed. Nanotechnol. Biol. Med., vol. 8, pp. 1309–1318, 2012, https://doi.org/10.1016/j.nano.2012.03.007.Search in Google Scholar PubMed

[74] M. D. Saint Victor, D. Carugo, L. C. Barnsley, J. Owen, C. Coussios, and E. Stride, “Magnetic targeting to enhance microbubble delivery in an occluded microarterial bifurcation Magnetic targeting to enhance microbubble delivery in an occluded microarterial bifurcation,” Phys. Med. Biol., vol. 62, pp. 7451–7470, 2017.10.1088/1361-6560/aa858fSearch in Google Scholar PubMed

[75] E. Beguin, M. D. Gray, K. A. Logan, et al.., “Magnetic microbubbles mediated chemo-sonodynamic therapy using a combined magnetic-acoustic device,” J. Contr. Release, vol. 317, pp. 23–33, 2020, https://doi.org/10.1016/j.jconrel.2019.11.013.Search in Google Scholar PubMed

[76] L. Yan, W. Miao, and D. Li, “Ultrasound imaging based on magnetic lipid microbubble contrast agent with Fe3O4 nanoparticles,” J. Nanosci. Nanotechnol., vol. 20, pp. 6087–6093, 2020, https://doi.org/10.1166/jnn.2020.18519.Search in Google Scholar PubMed

[77] C. Crake, J. Owen, S. Smart, et al.., “Enhancement and passive acoustic mapping of cavitation from fluorescently tagged magnetic resonance-visible magnetic microbubbles in vivo,” Ultrasound Med. Biol., vol. 42, pp. 3022–3036, 2016, https://doi.org/10.1016/j.ultrasmedbio.2016.08.002.Search in Google Scholar PubMed

[78] F. Yang, Y. Li, Z. Chen, Y. Zhang, J. Wu, and N. Gu, “Superparamagnetic iron oxide nanoparticle-embedded encapsulated microbubbles as dual contrast agents of magnetic resonance and ultrasound imaging,” Biomaterials, vol. 30, pp. 3882–3890, 2009, https://doi.org/10.1016/j.biomaterials.2009.03.051.Search in Google Scholar PubMed

[79] C. H. Fan, C. Yu-Hang, T. Chien-Yu, et al.., “Ultrasound/magnetic targeting with SPIO-DOX-Microbubble complex for image-guided drug delivery in brain tumors,” Theranostics, vol. 6, pp. 1542–1556, 2016, https://doi.org/10.7150/thno.15297.Search in Google Scholar PubMed PubMed Central

[80] Y. Liu, F. Yang, C. Yuan, et al.., “Magnetic nanoliposomes as in situ microbubble bombers for multimodality image-guided cancer theranostics,” ACS Nano, vol. 11, pp. 1509–1519, 2017, https://doi.org/10.1021/acsnano.6b06815.Search in Google Scholar PubMed

[81] Z. Liu, T. Lammers, J. Ehling, et al.., “Iron oxide nanoparticle-containing microbubble composites as contrast agents for MR and ultrasound dual-modality imaging,” Biomaterials, vol. 32, pp. 6155–6163, 2011, https://doi.org/10.1016/j.biomaterials.2011.05.019.Search in Google Scholar PubMed

[82] J. Wu, H. Leong-Poi, J. Bin, et al.., “Efficacy of contrast-enhanced US and magnetic microbubbles targeted to vascular cell adhesion molecule-1 for molecular imaging of atherosclerosis,” Radiology, vol. 260, pp. 463–471, 2011, https://doi.org/10.1148/radiol.11102251.Search in Google Scholar PubMed

[83] A. Barrefelt, T. B. Brismar, G. Egri, et al.., “Multimodality imaging using SPECT/CT and MRI and ligand functionalized 99mTc-labeled magnetic microbubbles,” EJNMMI Res., vol. 3, pp. 1–14, 2013, https://doi.org/10.1186/2191-219x-3-12.Search in Google Scholar PubMed PubMed Central

[84] C. Sciallero, L. Balbi, G. Paradossi, and A. Trucco, “Magnetic resonance and ultrasound contrast imaging of polymer-shelled microbubbles loaded with iron oxide nanoparticles,” R. Soc. Open Sci., vol. 3, p. 160063, 2016, https://doi.org/10.1098/rsos.160063.Search in Google Scholar PubMed PubMed Central

[85] D. Vlaskou, O. Mykhaylyk, F. Krötz, et al.., “Magnetic and acoustically active lipospheres for magnetically targeted nucleic acid delivery,” Adv. Funct. Mater., vol. 20, pp. 3881–3894, 2010, https://doi.org/10.1002/adfm.200902388.Search in Google Scholar

[86] X. Cai, F. Yang, and N. Gu, “Applications of magnetic microbubbles for theranostics,” Theranostics, vol. 2, pp. 103–112, 2012, https://doi.org/10.7150/thno.3464.Search in Google Scholar PubMed PubMed Central

[87] J. Owen, Q. Pankhurst, and E. Stride, “Magnetic targeting and ultrasound mediated drug delivery: benefits, limitations and combination,” Int. J. Hyperther., vol. 28, pp. 362–373, 2012, https://doi.org/10.3109/02656736.2012.668639.Search in Google Scholar PubMed

[88] M. A. Borden, M. R. Sarantos, S. M. Stieger, S. I. Simon, K. W. Ferrara, and P. A. Dayton, “Ultrasound radiation force modulates ligand availability on targeted contrast agents,” Mol. Imaging Off. J. Soc. Mol. Imaging, vol. 5, pp. 139–147, 2006, https://doi.org/10.2310/7290.2006.00016.Search in Google Scholar

[89] A. Ambrosi, Z. Sofer, and M. Pumera, “Electrochemical exfoliation of layered black phosphorus into phosphorene,” Angew. Chem. Int. Ed., vol. 56, pp. 10443–10445, 2017, https://doi.org/10.1002/anie.201705071.Search in Google Scholar PubMed

[90] A. Favron, E. GaufrHs, F. Fossard, et al., “Photooxidation and quantum confinement effects in exfoliated black phosphorus,” Nat. Mater., vol. 14, pp. 826–832, 2015. https://doi.org/10.1038/nmat4299.Search in Google Scholar PubMed

[91] P. Ares, J. J. Palacios, G. Abellán, J. Gómez-Herrero, and F. Zamora, “Recent progress on antimonene: a new bidimensional material,” Adv. Mater., vol. 30, pp. 1–27, 2018, https://doi.org/10.1002/adma.201703771.Search in Google Scholar PubMed

[92] Z. Guo, H. Zhang, S. Lu, Z. Wang, and P. K. Chu, “From black phosphorus to phosphorene: basic solvent exfoliation, evolution of Raman scattering, and applications to ultrafast photonics,” Adv. Funct. Mater., vol. 25, pp. 6996–7002, 2016.10.1002/adfm.201502902Search in Google Scholar

[93] Y. Feng, J. Zhou, Y. Du, et al.., “Raman spectra of few-layer phosphorene studied from first-principles calculations,” J. Phys. Condens. Matter, vol. 27, p. 185302, 2015, https://doi.org/10.1088/0953-8984/27/18/185302.Search in Google Scholar PubMed

[94] C. C. Chen and M. A. Borden, “Ligand conjugation to bimodal PEG brush layers on microbubbles,” Langmuir, vol. 26, pp. 13183–13194, 2010, https://doi.org/10.1021/la101796p.Search in Google Scholar PubMed PubMed Central

[95] D. H. Kim, A. L. Klibanov, and D. Needham, “The influence of tiered layers of surface-grafted poly(ethylene glycol) on ReceptorLigand-mediated adhesion between phospholipid monolayer-stabilized microbubbles and coated glass beads,” Langmuir, vol. 16, pp. 2808–2817, 2000, https://doi.org/10.1021/la990749r.Search in Google Scholar

[96] M. A. Borden, M. R. Sarantos, S. M. Stieger, S. I. Simon, K. W. Ferrara, and P. A. Dayton, “Ultrasound radiation force modulates ligand availability on targeted contrast agents,” Mol. Imag., vol. 5, pp. 139–147, 2006, https://doi.org/10.2310/7290.2006.00016.Search in Google Scholar

[97] Y. Zhou, X. Y. Zhou, Z. G. Wang, Y. F. Zhu, and P. Li, “Elevation of plasma membrane permeability upon laser irradiation of extracellular microbubbles,” Laser Med. Sci., vol. 25, pp. 587–594, 2010, https://doi.org/10.1007/s10103-010-0773-1.Search in Google Scholar PubMed

[98] M. Terakawa, M. Ogura, S. Sato, et al.., “Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave,” Opt. Lett., vol. 29, pp. 1227–1229, 2004, https://doi.org/10.1364/ol.29.001227.Search in Google Scholar PubMed

[99] J. Willmann, R. Paulmurugan, K. Chen, et al.., “US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice,” Radiology, vol. 246, pp. 508–518, 2008, https://doi.org/10.1148/radiol.2462070536.Search in Google Scholar PubMed PubMed Central

Received: 2021-03-01
Accepted: 2021-05-03
Published Online: 2021-05-19

© 2021 Yao Zhu et al., published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

Downloaded on 19.3.2024 from https://www.degruyter.com/document/doi/10.1515/nanoph-2021-0085/html
Scroll to top button