Preparation Of Nanobubbles Modified With A Small-Molecule CXCR4 Antagonist For Targeted Drug Delivery To Tumors And Enhanced Ultrasound Molecular Imaging

Figures & data

Figure 1 Cell immunofluorescence assay. CXCR4 was expressed in MCF-7 cells and C33a cells but not in MDA-MB-468 cells. The nuclei are stained blue, and cell membranes expressing CXCR4 are shown in green.

Figure 2 Identification of DSPE-PEG2000-COOH. (A) The ¹H NMR spectrum of AMD070. (B) The ¹H NMR spectrum of DSPE-PEG2000-COOH. (C) The ¹H NMR spectrum of DSPE-PEG2000-AMD070.

Figure 3 Basic characteristics of the nanobubbles. (A) Observation of PTX-AMD070 NBs under a light microscope. (B) Observation of DiI-labeled PTX-AMD070 NBs under a fluorescence microscope. (C) Observation of PTX-AMD070 NBs under a transmission electron microscope. (D) Spectrum of PTX NB particle sizes. (E) Spectrum of PTX-AMD070 NB particle sizes. (F) An ultrasound image of PTX-AMD070 NBs in vitro. (G) Quantitative analysis of imaging intensity of PTX-AMD070 NBs in vitro. (H) Ultrasound images of PTX-AMD070 NBs in vitro before and after ultrasonic blast. (I) Quantitative analysis of imaging intensity of PTX-AMD070 NBs before and after ultrasonic blast (*P<0.05).

Figure 4 In vitro affinity of nanobubbles for cells. (A) Observation of DiI-labeled PTX-AMD070 NBs binding to MCF-7 cells, C33a cells, and MDA-MB-468 cells under a laser confocal microscope. The nuclei are stained blue; the cell membrane is stained with DiO (green); PTX-AMD070 NBs are stained with DiI (red). (B) Analysis of affinity between nanobubbles and MCF-7 cells determined by flow cytometry. (C) Analysis of affinity between nanobubbles and C33a cells determined by flow cytometry. (D) Analysis of affinity between nanobubbles and MDA-MB-46 cells determined by flow cytometry. (E) Quantitative analysis of affinity between the two types of nanobubbles and three types of cells. *There was significant difference between the cell binding ability of PTX-AMD070 NBs and PTX NBs (P<0.05).

Figure 5 Examination of tumor proliferation suppression ability in vitro using CCK-8 assays. (A) Tumor proliferation suppression ability of AMD070 and PTX at different concentrations. *The survival rate of MCF-7 cells was significantly different from that of cells without drug treatment (P<0.05). #The survival rate of C33a cells was significantly different from that of cells without drug treatment (P<0.05). ^▲The survival rate of MDA-MB-468 cells was significantly different from that cells without drug treatment (P<0.05). (B) Tumor proliferation suppression outcome in different treatment groups. *There was a significant difference in cell survival rate between the two groups (P<0.05).

Abbreviations: NBs, nanobubbles; PTX, paclitaxel; US, ultrasound.

Figure 6 Apoptosis of tumor cells detected by flow cytometry. (A) Apoptosis rates of MCF-7 cells, C33a cells, and MDA-MB-468 cells in the different treatment groups. (B–D) Quantitative comparison of apoptosis rates of MCF-7 cells, C33a cells, and MDA-MB-368 cells between different treatment groups; *There was significant difference in apoptosis rate between the two groups (P<0.05).

Abbreviations: EA, early apoptosis; LA, late apoptosis; TA, total apoptosis.

Figure 7 Ultrasound images of nanobubbles in xenografts. (A) Ultrasound images of nanobubbles in the three types of xenografts. The red dotted circle represents the xenograft area. (B–D) The time-intensity curves of the nanobubbles in the MCF-7, C33a, and MDA-MB-468 xenografts.

Table 1 Ultrasonic Imaging Parameters Of PTX-AMD070 NBs And PTX NBs In Xenograft Tumors

Tumor	Nanobubbles	Peak Time (Seconds)	Peak Intensity (dB)	AUC(dB·s)
MCF-7	PTX-AMD070 NBs	11.37±0.32	12.01±0.06*	6214.20±30.65*
Tumor	PTX NBs	11.27±0.31	11.28±0.01	5069.60±10.41
C33a	PTX-AMD070 NBs	11.41±0.39	12.08±0.15*	6301.00±39.69*
Tumor	PTX NBs	11.55±0.38	11.58±0.08	5201.20±58.07
MDA-MB-468	PTX-AMD070 NBs	11.44±0.47	11.52±0.12	5660.40±24.76
Tumor	PTX NBs	11.29±0.36	11.49±0.06	5625.80±24.14

Notes: *The same ultrasound imaging parameters of the same type of xenograft tumors differed significantly between PTX-AMD070 NBs and PTX NBs (P<0.05).

Abbreviation: AUC, area under the curve.

Figure 8 Distribution of PTX-AMD070 NBs in tissues. Distribution of PTX-AMD070 NBs in MCF-7 xenografts (upper panel), in C33a xenografts (middle and upper panel), in MDA-MB-468 xenografts (middle and lower panel), and in thigh muscle tissue (lower panel). The nuclei are stained blue; the tumor blood vessels are shown in green; the DiI-labeled PTX-AMD070 NBs are red.

Table 2 Tumor Mass And Tumor Inhibition Rate In Different Treatment Groups

Group	MCF-7 Tumor Weight (g)	Mean Tumor Inhibition Rate (%)	C33a Tumor Weight (g)	Mean Tumor Inhibition Rate (%)
Control	1.31±0.07	–	1.34±0.06	–
AMD070	1.20±0.09△	7.74±7.03	1.14±0.12△	14.88±8.98
PTX	0.92±0.02▲	29.38±3.45	0.80±0.03▲	40.63±2.17
PTX+AMD070	0.73±0.09#	44.15±8.88	0.74±0.09#	49.34±6.65
PTX-AMD070 NBs	0.59±0.05*	54.82±2.03	0.68±0.08*	58.10±4.35
PTX-AMD070 NBs+US	0.11±0.02	91.54±1.64	0.16±0.04	88.50±2.37

Notes: *There was a significant difference in the mass of xenografts between the PTX-AMD070 NBs+US group and PTX-AMD070 NBs group (P<0.05). #There was a significant difference in the mass of xenografts between the PTX-AMD070 NBs group and PTX+AMD070 group (P<0.05). ▲There was a significant difference in the mass of xenografts between the PTX group and PTX+AMD070 group (P<0.05). △There was a significant difference in the mass of xenografts between the AMD070 group and PTX+AMD070 group (P<0.05).

Figure 9 The curative effect in different treatment groups. (A) Xenografts in the tumor-bearing nude mice in different treatment groups. The red circles represent the xenograft areas. Scale, 1 cm. (B) Weight changes of nude mice bearing MCF-7 xenografts in different treatment groups. (C) Weight changes of nude mice bearing C33a xenografts in different treatment groups. (D) Tumor volume changes in nude mice bearing MCF-7 xenografts in different treatment groups. (E) Tumor volume changes in nude mice bearing C33a xenografts in different treatment groups.

Figure 10 H&E staining and TUNEL staining of xenograft tissues. Scale, 100 μm.