*3.3. Extravascular Targeting (NB)*

Since MB are trapped in the bloodstream due to their size, extravascular imaging is not possible. This is not necessarily a drawback, since many promising targets are still accessible. Furthermore, the intravascular distribution minimizes the unspecific MB accumulation in tissues leading to a low background. However, if extravascular targeting is desired one can adapt the UCA size to the size of the vascular fenestrations. In leaky tumor vessels fenestrations are known to be around 400–800 nm large [147]. Therefore, bubbles in the nanoscale were introduced, which can leak from the vasculature into the tumor tissue [148–150]. Due to their accumulation via the enhanced permeability and retention effect (EPR), they show prolonged persistence at tumor site compared to MB. By targeting over-expressed tumor markers, the retention of the NB in the tumor tissue can be enhanced [7,151]. An overview of targeted NB is provided in Table 3.

**Table 3.** Summary of extravascular targets investigated for molecular US imaging.


Due to their small size, NB are expected to backscatter less and, thus, show less signal during US imaging [159]. Furthermore, for smaller bubbles higher frequencies are needed, which can hamper imaging, since high frequencies do not reach deep into tissue [160]. Moreover, US devices used in the clinics usually do not have the transducers for high frequencies.

Surprisingly, some studies report that NB show similar echogenicity as MB (Figure 9) [148,161]. There are no clear explanations for this observation. However, one group discusses that there might be an underestimation of NB concentrations in the phantoms because NB are difficult to count [161].

Furthermore, highly flexible shells of some NB may increase the echogenicity significantly, even at lower frequencies than their resonance frequency [159–162]. For instance, Perera and colleagues designed a new shell, which was inspired by nature, where shells with several layers exist. The layers differ in their elastic properties such as in bacterial cell envelopes [163]. Perera adapted this concept and designed a shell consisting of two layers differing in elasticity, one compliant layer, another stiff adlayer with PEG on top. This composition provides high shell stability, which reduces air loss during oscillation and improves better persistence in the blood circulation. Together, these properties promise an extended effective visualization of the tumor.

**Figure 9.** Contrast harmonic images of NB and commercially available Definity® MB (DEF) at different concentrations and frequencies. Reproduced with permission from [161]. Copyright Elsevier, 2013.

By targeting NB a longer tumor persistence but not necessarily a big difference in US signals was observed [152,157]. This is in line with results on other nanoparticles, were targeting only improved retention but not accumulation, the latter being mostly mediated via the EPR effect [164]. Thus, obtaining exact information about extravascular molecular marker expression with particles of this size will remain difficult. However, the exploration of the diagnostic benefits resulting from NB accumulation is just beginning and needs further research and reasonable conceptual considerations.
