Construction of ultrasonic nanobubbles carrying CAIX polypeptides to target carcinoma cells derived from various organs

Abstract

Background: Ultrasound molecular imaging is a novel diagnostic approach for tumors, whose key link is the construction of targeted ultrasound contrast agents. However, available targeted ultrasound contrast agents for molecular imaging of tumors are only achieving imaging in blood pool or one type tumor. No targeted ultrasound contrast agents have realized targeted ultrasound molecular imaging of tumor parenchymal cells in a variety of solid tumors so far. Carbonic anhydrase IX (CAIX) is highly expressed on cell membranes of various malignant solid tumors, so it's a good target for ultrasound molecular imaging. Here, targeted nanobubbles carrying CAIX polypeptides for targeted binding to a variety of malignant tumors were constructed, and targeted binding ability and ultrasound imaging effect in different types of tumors were evaluated.

Results: The mean diameter of lipid targeted nanobubbles was (503.7 ± 78.47) nm, and the polypeptides evenly distributed on the surfaces of targeted nanobubbles, which possessed the advantages of homogenous particle size, high stability, and good safety. Targeted nanobubbles could gather around CAIX-positive cells (786-O and Hela cells), while they cannot gather around CAIX-negative cells (BxPC-3 cells) in vitro, and the affinity of targeted nanobubbles to CAIX-positive cells were significantly higher than that to CAIX-negative cells (P < 0.05). Peak intensity and duration time of targeted nanobubbles and blank nanobubbles were different in CAIX-positive transplanted tumor tissues in vivo (P < 0.05). Moreover, targeted nanobubbles in CAIX-positive transplanted tumor tissues produced higher peak intensity and longer duration time than those in CAIX-negative transplanted tumor tissues (P < 0.05). Finally, immunofluorescence not only confirmed targeted nanobubbles could pass through blood vessels to enter in tumor tissue spaces, but also clarified imaging differences of targeted nanobubbles in different types of transplanted tumor tissues.

Conclusions: Targeted nanobubbles carrying CAIX polypeptides can specifically enhance ultrasound imaging in CAIX-positive transplanted tumor tissues and could potentially be used in early diagnosis of a variety of solid tumors derived from various organs.

Keywords: Carbonic anhydrase IX; Malignant tumors; Targeted nanobubbles; Ultrasound molecular imaging.

Fig. 1

Basic characteristics of targeted nanobubbles. a1 The particle size of targeted nanobubbles. a2 The particle size of blank nanobubbles. b The distribution of targeted nanobubbles under an optical microscope. c The morphology of targeted nanobubbles under a transmission electron microscope. d MTT assay of targeted and blank nanobubbles. *P < 0.05

Fig. 2

Dual-fluorescence validates targeted nanobubbles. a DiI-labeled targeted nanobubbles. b Nanobubbles carrying the FITC-modified polypeptides. c Merged image confirms that biotin–streptavidin system successfully linked the polypeptides to the surfaces of targeted nanobubbles

Fig. 3

Stability of targeted nanobubbles. a Histogram of the concentration of targeted nanobubbles changes over time. b Histogram of the particle size of targeted nanobubbles changes over time. *P < 0.05

Fig. 4

In vitro targeting experiments of targeted and blank nanobubbles. a–c The binding of nanobubbles (black arrows) to 786-O cells. d–f The binding of nanobubbles (black arrows) to Hela cells. g–i The binding of nanobubbles to BxPC-3 cells. j The number of nanobubbles binding to the three types of tumor cells. *P < 0.05

Fig. 5

Affinity of targeted and blank nanobubbles to three types of cells. a The affinity of nanobubbles to 786-O cells. b The affinity of nanobubbles to HeLa cells. c The affinity of nanobubbles to BxPC-3 cells. d Quantification of the affinity of the two types of nanobubbles to the three types of cells. *P < 0.05

Fig. 6

In vitro imaging of targeted and blank nanobubbles. a1 Ultrasound images of nanobubbles at different concentrations in vitro. a2 Quantification of imaging intensity of nanobubbles in vitro. b1 The decay rate of targeted nanobubbles in vitro. b2 The decay rate of blank nanobubbles in vitro. c1 Ultrasound images of targeted nanobubbles before and after destruction. c2 Quantification of imaging intensity of targeted nanobubbles before and after destruction. *P < 0.05

Fig. 7

Imaging characteristics of targeted and blank nanobubbles in three types of transplanted tumor tissues. a Ultrasound images of nanobubbles in the three types of transplanted tumor tissues. Red circles indicate the regions of transplanted tumor tissues. b1–b3 Time-intensity curves of nanobubbles in the three types of transplanted tumor tissues. c Area under the curve of nanobubbles in the three types of transplanted tumor tissues. *P < 0.05

Fig. 8

Localization of targeted nanobubbles in nude mice body. a–d The distribution of targeted nanobubbles in 786-O transplanted tumor tissues. e–h The distribution of targeted nanobubbles in Hela transplanted tumor tissues. i–l The distribution of targeted nanobubbles in BxPC-3 transplanted tumor tissues. m–p The distribution of targeted nanobubbles in the right thigh muscle tissues

Fig. 9

Blood vessels in three types of transplanted tumor tissues. a Blood vessels in 786-O transplanted tumor tissues. b Blood vessels in Hela transplanted tumor tissues. c Blood vessels in BxPC-3 transplanted tumor tissues. d MVDs in the three types of transplanted tumor tissues were assessed. Red arrows indicate tumor blood vessels

Fig. 10

Expression of CAIX in three types of transplanted tumor tissues. a Immunohistochemistry on 786-O transplanted tumor tissues. b Immunohistochemistry on Hela transplanted tumor tissues. c Immunohistochemistry on BxPC-3 transplanted tumor tissues. The yellow–brown areas indicate positive CAIX expression, black arrows indicate tumor stroma