# Advanced Microbubbles as a Multifunctional Platform Combining Imaging and Therapy
## 2 Advanced Microbubbles for Imaging and Therapy  
### Tumor Detection  
Although MBs can improve ultrasound image resolution, there is no specific targeting capability for them to differentiate between normal and cancerous tissues. Thus, MBs are always constructed with the attachment to specific binding ligands such as peptides, affibodies, antibodies, etc. to be able to actively target the tumor areas. With the targeted MBs anchoring at the cancerous regions, ultrasonic echo signals can be significantly enhanced which leads to early diagnosis of cancers. In the process of tumor angiogenesis, the integrin family is a main factor of mediating cell and extracellular matrix adhesion. The relationship between integrin \(\alpha\
u\beta 3\) and tumor angiogenesis is the most attractive to researchers and has become a hot topic. Integrin \(\alpha\
u\beta 3\) receptor is overexpressed on the surface of a variety of malignant tumors and endothelial cells of tumor tissue angiogenesis, otherwise not expressed in mature vascular endothelial cells and the vast majority of normal organ systems. Its overexpression provides molecular targets as imaging probes and could be used in ultrasound to detect angiogenesis. [PERSON] developed iRGD (internalizing arginine-glycine-aspartate)-lipopeptide-based \(\alpha\
u\beta 3\) integrin-targeted MBs (1.36 \(\pm\) 0.89, 1.24 \(\pm\) 0.74, 1.09 \(\pm\) 0.61 \(\upmu\)m) as non-invasive molecular imaging probes to tumor angiogenesis [33]. The binding specificity for endothelial cells was significantly stronger (\(p\) < 0.01) under in vitro static and dynamic conditions. US images revealed strong contrast enhancement within the mice bearing 4T1 breast tumors compared with the control MBs. In addition to integrins, vascular endothelial growth factor receptors (VEGFRs) are regarded as another kind of specific biomarker of neoangiogenesis. [PERSON] designed VEGFR2-targeted MBs in combination with 40 MHz endoluminal ultrasound biomicroscopy (eUBM) to detect colon tumors [34]. [PERSON] developed VEGFR2-targeted MBs in an orthotopic model of renal cell carcinoma (RCC) to image angiogenesis. Compared with MBs conjugated to an isotype control antibody (MBC) or naked microbubbles (MBN), the duration of contrast enhancement of MBs conjugated to anti-VEGFR2 antibody (MBV) demonstrated prolonged contrast intensity within the tumor. Furthermore, the contrast intensity for MBV was significantly higher in the tumors than in normal parenchyma (\(p\) < 0.01) [35]. [PERSON] purified phospholipid-PEG2000-peptide which has nanomolar affinity for VEGFR2, and incorporated it into gas-filled microbubbles. In vivo experiments showed the VEGFR2-targeted microbubbles (BR55) greatly provided imaging of angiogenesis [36] (Figure 2). In a further study, [PERSON] established triple-targeted MBs with antibodies against mouse \(\alpha\
u\beta 3\) integrin, P-selectin and VEGFR2 for evaluating the early response to antiangiogenic therapy. Tumor-bearing mice were injected with the multi-targeted microbubbles for contrast-enhanced US imaging and microbubble accumulation was calculated by destruction-replenishment techniques and time-intensity curve analysis. It was found that the vascularity of breast cancer decreased within Day 1 of bevacizumab injection by real-time monitoring of molecular US imaging, and further reduction of vascularity was detected by day 3 [37]. [PERSON] found that vascular expression of B7H3 was selectively and significantly higher in breast cancer tissues through an IHC analysis of 248 human breast specimens. Then a kind of B7H3-targeted MB was prepared to identify that the US imaging signal increased significantly in breast cancer tissues in vivo. These findings concluded that the use of B7H3-targeted MBs can be a useful tool to improve the diagnostic accuracy of breast cancer detection in patients [38]. US imaging can also be applied to monitor the tumor angiogenesis during tumor development [39]. [PERSON] et al. conjugated RGD peptides to the MB shell via biotin-avidin linkage.