**5. Microbubbles (MBs)**

There are a number of pathways by which LNPs could enter mammalian cells, namely by phagocytosis, pinocytosis, clathrin and calveolin independent endocytosis [9, 17]. Of these, phagocytosis is a process of engulfing and internalisation of large particles (1 um) by cells. The majority of phagocytic cells of the immune system, including macrophages, neutrophils, monocytes, and microglia, are able to internalise foreign bodies via phagocytosis. Pinocytosis is a method of particle internalisation by non-phagocytic cells but this is a very non-specific mechanism of internalisation into cells [9]. On the other hand, clathrin-mediated endocytosis (receptor-mediated) and caveolin-mediated endocytosis are much the more specific means of

In original preclinical laboratory experiments, Gadonano-F LNPs were prepared and used to image xenograft tumours (IGROV-1) in mice. At just 2h post-injection the Gadonano-F LNPs mediated a similar signal enhancement to that attained at 24h post-injection by control (Gadonano LNPs, lacking folate ligand) (Figure 6). Specifically, Gadonano LNPs accumulated in tumour by the EPR mechanism ("passive" targeting) [12, 13], and labelled cells for MRI over a period of 24h. These imaging LNPs were seen to access the entire hyperpermeable, living volume of the tumour while being excluded from the necrotic regions. By contrast, the FRtargeted Gadonano-F LNPs accumulated in the tumour and labelled cells for MRI from 2h onwards, owing to EPR mediated tumour uptake from the blood pool followed by rapid FRmediated cell entry probably involving clathrin-mediated endocytosis. FR-targeted imaging

Gadonano-F LNPs accumulated in the tumour and labelled cells for MRI from 2h onwards, owing to EPR mediated tumour uptake from the blood pool followed by rapid FR-mediated cell entry probably involving clathrin-mediated endocytosis. FR-targeted

On the left 24 h post imaging LNP administration, (a) surface IGROV-1 tumor slices, Gadonano LNPs, (b) midtumor IGROV-1 slices, Gadonano LNPs, (c) surface IGROV-1 tumor slices, Gadonano-F LNPs, and (d) IGROV-1 midtumor slices, Gadonano-F LNPs (green bar represents 50 *μ*m). On the right, MRI contrast images of IGROV-1 tumour bearing Balb/C nude mice: images are from control mice injected with Gadonano LNPs (top); images are of mice injected with Gadonano-F LNPs (bottom) (white arrow

On the left 24 h post imaging LNP administration, (a) surface IGROV-1 tumor slices, Gadonano LNPs, (b) midtumor IGROV-1 slices, Gadonano LNPs, (c) surface IGROV-1 tumor slices, Gadonano-F LNPs, and (d) IGROV-1 midtumor slices, Gadonano-F LNPs (green bar repre‐ sents 50 *μ*m). On the right, MRI contrast images of IGROV-1 tumour bearing Balb/C nude mice:

Rapid and effective imaging of FR-bearing tumours by Gadonano-F LNPs in preclinical experiments, has since led to these LNPs being prepared for clinical trial by GlobalAcorn Ltd under the name GA101 NANO-GdF (Gadonano LNPs are also being developed for clinical trial by GlobalAcorn Ltd under the name GA100 NANO-Gd). Rapid and specific imaging of diseased tissues and tumours will be of paramount importance for preclinical research and also future diagnostic and theranostic applications in clinic [2,3].

Since the MBs were first proposed as suitable tools for ultrasound contrast agents more than three decades ago, there has been a remarkable progress in the field. In accordance with that, the research has been particularly concerned with the MB preparation techniques and their standardization, in order to ensure a steady composition and size distribution of MBs, both representing crucial features for the stability of MBs *in vivo* and thus providing a sufficient backscatter for ultrasound imaging. Although the very first MBs were generally considered to contribute to improvement of the imaging techniques, they were soon proved to serve as excellent drug and gene delivery systems both in diagnostics and therapy. Much has been discussed on ultrasound molecular imaging using MBs as contrast agents, including novel preparation techniques and their general design; however, the choice of a convenient system for drug and gene delivery and concept of MB functionalization is still an issue worth consideration. In this chapter, potential ways for application of MBs as contrast agents together with drug and gene delivery are shown, relating to the recently used functionalized

MBs are small gas-filled microspheres whose size typically ranges between 1 and 10 µm in diameter see (**Figure 7**). They comprise a gas-filled core, usually formed by inert, high molecular gases such as sulphur hexafluoride or perfluorocarbons, which due to their decreased solubility and low diffusion prolong the lifespan of MBs in circulation [19], by means of a stabilizing shell of lipid, polymer and/or protein [20, 21]. The composition of the shell determines the stiffness and therefore the stability of MBs in blood flow, where instability is caused by exposure of MBs to the rigors of systemic circulation [19, 22]. Limits on stability can effect the scope of ultrasound imaging utilization *in vivo* since the half-life of MBs depends on their stability. Generally, lipid shelled MBs are considered thinner and more flexible, whilst polymer and protein-shelled bubbles have a thicker and more rigid shell [23]. A thick permeable shell is beneficial to enhance the MB stability (also reducing the gas diffusion) and thus might be used for ultrasound drug delivery purposes [19, 24, 25]. However, the shell ought to only slightly limit the vibration of MBs in the ultrasound field generated

Another important aspect for any MB formulation is their size distribution that dictates the acoustic properties of MBs [22]. As the MBs smaller than 1 µm in diameter produce minimal acoustic contrast, the intent is to produce larger MBs [26-29]. On the other

LNPs accumulated in more perivascular and macrophage-rich locations [14,18].

*Figure 6. Fluorescence microscopy and MRI contrast imaging of 24 h post imaging LNP injection*.

**Figure 6.** Fluorescence microscopy and MRI contrast imaging of 24 h post imaging LNP injection.

shows location of tumour) (Adapted from [18])

by echo imaging systems (otherwise they do not provide sufficient contrast).

**Microbubbles (MBs)** 

86 Advances in Bioengineering

MBs in vitro and *in vivo*.

imaging LNPs accumulated in more perivascular and macrophage-rich locations [14,18].

cellular entry, of which the first is ideal for entry of LNPs to cells.

Since the MBs were first proposed as suitable tools for ultrasound contrast agents more than three decades ago, there has been a remarkable progress in the field. In accordance with that, the research has been particularly concerned with the MB preparation techniques and their standardization, in order to ensure a steady composition and size distribution of MBs, both representing crucial features for the stability of MBs *in vivo* and thus providing a sufficient backscatter for ultrasound imaging. Although the very first MBs were generally considered to contribute to improvement of the imaging techniques, they were soon proved to serve as excellent drug and gene delivery systems both in diagnostics and therapy. Much has been discussed on ultrasound molecular imaging using MBs as contrast agents, including novel preparation techniques and their general design; however, the choice of a convenient system for drug and gene delivery and concept of MB functionalization is still an issue worth consideration. In this chapter, potential ways for application of MBs as contrast agents together with drug and gene delivery are shown, relating to the recently used functionalized MBs in vitro and *in vivo*.

MBs are small gas-filled microspheres whose size typically ranges between 1 and 10 μm in diameter see (Figure 7). They comprise a gas-filled core, usually formed by inert, high molecular gases such as sulphur hexafluoride or perfluorocarbons, which due to their decreased solubility and low diffusion prolong the lifespan of MBs in circulation [19], by means of a stabilizing shell of lipid, polymer and/or protein [20, 21]. The composition of the shell determines the stiffness and therefore the stability of MBs in blood flow, where instability is caused by exposure of MBs to the rigors of systemic circulation [19, 22]. Limits on stability can affect the scope of ultrasound imaging utilization *in vivo* since the half-life of MBs depends on their stability. Generally, lipid shelled MBs are considered thinner and more flexible, whilst polymer and protein-shelled bubbles have a thicker and more rigid shell [23]. A thick perme‐ able shell is beneficial to enhance the MB stability (also reducing the gas diffusion) and thus might be used for ultrasound drug delivery purposes [19, 24, 25]. However, the shell ought to only slightly limit the vibration of MBs in the ultrasound field generated by echo imaging systems (otherwise they do not provide sufficient contrast).

Another important aspect for any MB formulation is their size distribution that dictates the acoustic properties of MBs [22]. As the MBs smaller than 1 μm in diameter produce minimal acoustic contrast, the intent is to produce larger MBs [26-29]. On the other hand, the MB size distribution should not exceed 10 μm of size in diameter; otherwise they get quickly filtered by the lungs and can pose a risk of embolus [22, 30]. Furthermore, the size distribution is also believed to affect the drug delivery [22, 31, 32].
