In this study, the lipid targeted nanobubble carrying the A10-3.2 aptamer against prostate specific membrane antigen was fabricated, and its effect in the ultrasound imaging of prostate cancer was investigated. Materials including 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, carboxyl-modified 1,2-distearoyl-sn-glycero3-phosphoethanolamine, and polyethyleneglycol-2000 were for mechanical oscillation, and nanobubbles were obtained through the centrifugal flotation method. After mice were injected with nanobubbles, abdominal color Doppler blood flow imaging significantly improved. Through left ventricular perfusion with normal saline to empty the circulating nanobubbles, nanobubbles still existed in tumor tissue sections, which demonstrated that nanobubbles could enter tissue spaces via the permeability and retention effect. Fluorinated A10-3.2 aptamers obtained by chemical synthesis had good specificity for PSMA-positive cells, and were linked with carboxyl-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine lipid molecules from the outer shell of nanobubbles via amide reaction to construct targeted nanobubbles. Gel electrophoresis and immunofluorescence confirmed that targeted nanobubbles were fabricated successfully. Next, targeted nanobubbles could bind with PSMA-positive cells (C4-2 cells), while not with PSMA-negative cells (PC-3 cells), using in vitro binding experiments and flow cytometry at the cellular level. Finally, C4-2 and PC-3 xenografts in mice were used to observe changes in parameters of targeted and non-targeted nanobubbles in the contrast-enhanced ultrasound mode, and the distribution of Cy5.5-labeled targeted nanobubbles in fluorescent imaging of live small animals. Comparison of ultrasound indicators between targeted and non-targeted nanobubbles in C4-2 xenografts showed that they had similar peak times (P.0.05), while the peak intensity, half time of peak intensity, and area under the curve of ½ peak intensity were significantly different (P,0.05). In PC-3 xenografts, there were no differences in these four indicators. Fluorescent imaging indicated that targeted nanobubbles had an aggregation ability in C4-2 xenograft tumors. In conclusion, targeted nanobubbles carrying the anti-PSMA A10-3.2 aptamer have a targeted imaging effect in prostate cancer.
With the in-depth understanding of ultrasound contrast agents (UCAs) and the development of material science, the outer shell of UCAs can be constructed of materials such as lipid components, denatured albumin (ALB), and polymers based on different needs.1 Of all, the lipid outer shell is the most classic and most appropriate component of diagnostic contrast agents, due to the common features of excellent elasticity, easy modification of lipid molecules, and non-toxicity to humans.2 Based on these traits, groups in lipid molecules are easily linked with ligands specific to molecules of interest, such as compounds with small molecular weight and antibodies. In this way, outer shell components of targeted lipid UCAs were fabricated, which is also a key part in ultrasound molecular imaging.3 However, current studies about ultrasound molecular imaging are mainly applied in targeted ultrasound diagnosis of thrombosis, inflammation, and tumors.4–7 The newly emerging lipid ultrasound nanobubble (NB) is a UCA with a particle size smaller than 1,000 nm, which can overcome the limitations of traditional ultrasound microbubbles exclusively in blood vessels.8 Nano-scale UCAs in tumor tissues can utilize the permeability and retention effects of tumors, such as larger endothelial gaps, lack of a basement membrane, and poor lymph drainage in tumors, to enter extravascular spaces to achieve imaging of tumor parenchyma.9,10
In this study, we combined our previous findings with the advantages of the small molecular weight and excellent specificity of aptamers, and then used the safe amide reaction connection method to construct targeted NBs carrying the anti-PSMA aptamer A10-3.2. These were safe and had excellent penetration ability, strong specificity, and potential for clinical translation.39,40 This study can not only provide a basis for ultrasound molecular imaging of prostate cancer, but can also provide methods for constructing targeted ultrasound probes carrying aptamers. In addition, because the aptamer is a gene fragment in itself, it can be intercalated with anthracyclines or can be connected to other gene fragments, which can lay the foundation for future construction of targeted NBs carrying drugs (or genes) in targeted therapy under ultrasound irradiation.41–44