**4. Cellular imaging**

In functional lipid-based nanoparticles (LNPs), active pharmaceutical ingredients (APIs) (**A**) are condensed within functional concentric layers of chemical components designed for delivery into cells and intracellular trafficking (**B** components, primarily lipids and lipidrelated components), biological stability (**C** stealth/biocompatibility components–typically Polyethylene Glycol [PEG]) and biological targeting to target cells (**D** components, biological

Liposomes are the progenitors of all LNPs, and they are formulated directly from lipid constituents (Figure 3). Liposomes can be prepared by various techniques. The method based on hydration of lipid film represents the most exploited technique for preparation of lipo‐ somes. The lipids of known molarities are dissolved in organic solution which is subsequently slowly evaporated in vacuo to produce a thin film. The film is hydrated with a suitable aqueous buffer at temperature, which is generally above transition temperature of lipids used. Aqueous buffer contains also compound which are to be entrapped into liposomes. Size of liposomes can be reduced by freeze-thaw procedure followed by sonication, extrusion, microfluidisation or high pressure homogenisation. These procedures can tune size distribution of final liposo‐ mal preparation to desired values. The main parameters characterising liposomes are their size, spherical shape and degree of lamellarity. Liposomes may consist of one bilayer (unila‐ mellar), several bilayers (oligolamellar) or multiple bilayers (multilamellar). Membrane rigidity can be adjusted using appropriate lipids and membrane fluidity may be controlled

with the use of phospholipids with higher or lower transition temperature.

**Figure 3.** Various morphological types of liposomes and real appearance in cryoelectron microscopy.

concentric (MLV); Multivesicular vesicles (MVV)

Small unilamellar vesicles (SUV); Large unilamellar vesicles (LUV); Multilamellar vesicles –

receptor-specific targeting ligands) [2, 3].

**3. Liposomes to LNPs**

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Cellular imaging is "the visualisation of specific cells in an intact animal". This term also collectively denotes the visualisation of any type of the entire cell under different conditions [9]. While cellular imaging applies to the imaging of whole cells, molecular imaging deals with the visualisation of molecules and sub-cellular components within individual cells [10]. The exploitation of molecular probes or imaging agents is essential for the use of both cellular and molecular imaging strategies. Despite the fact that the method of cellular imaging of extrinsic fluorophores is highly efficient, the depth of tissue from which the signal emitted represents often a limiting factor for this technique. The possible use of positron emission tomography (PET) or single–photon computed tomography (SPECT) methods is also limited due to shortlived radioisotopes and poor spatial resolution. However, three-dimensional images of tissues containing water can be obtained by magnetic resonance imaging (MRI). This technique is characterised by high levels of spatial resolution and imaging of depth tissues is achieved.

A primary limitation of MRI is an inherent lack of sensitivity that can be overcome through the use of contrast agents that enhance signal sensitivity and hence image quality. MRI contrast agents consist of molecules that incorporate a paramagnetic metal ion, most commonly gadolinium (III) (Gd3+) or Iron (Fe3+/Fe2+). The improvement in image quality derives from the modulating effects of the coordinated metal ions on longitudinal (*T*1) or transverse (*T*2) relaxation times associated with proton resonance signals emanating from bulk water mole‐ cules surrounding the coordinated metal ions. Even though contrast agents incorporating Gd3 increase both 1//T1 and 1/T2 relaxivities, they are commonly used for T1-weighed (positive bright) contrast imaging, on condition that 1/T1 contribution is higher in tissue than the corresponding effect on 1/T2 enhancement. Due to the fact that more considerable increases in 1/T2 are induced by iron containing contrast agents, they are usually used in T2- weighed (negative dark) contrast imaging [11]. Despite the most frequently used MRI contrast agents are thermodynamically and kinetically stable low molecular weight Gd3+ complexes (that promote MRI contrast by non-specific enhancement of water proton relaxation rates within the blood pool), there has been a realization of the need for imaging LNPs suitable for the targeted delivery of imaging agents to sites of disease pathology such as cancerous lesions in order to obtain clear contrast images of their extent and location.

For cell populations to be imaged by MRI *in vivo*, these populations must be distinguishable from natural background noise signal. For this to happen, cells of interest should become labelled with appropriate agents, making use of controlling mechanisms such as enhanced permeability and retention (EPR) mechanism for imaging agents to reach target cells [12,13,14], followed by controlled target cell entry. According to Frangioni et al., the ideal cellular label should have the following features: a strong signalling effect, good biocompatibility, does not interfere with cellular genetics, remains retained only in the target cell population, and allows temporal imaging for long-lasting periods [15]. Controlled target cell entry is preferably by ligand-specific receptor-mediated cell entry processes, although alternative mechanisms of cell entry may also be acceptable (Figure 4) [16].

**Figure 4.** Cell entry mechanisms: the various uptake mechanisms of macromolecules and nanoparticles into cells are indicated according to their size (Adapted from [14]).

LNPs are an ideal platform to combine multimodal imaging (e.g. MRI contrast agent and fluorescent probes) with multiple functionalities in order to effect multimodal cellular labelling. Targeting of LNPs to various tissues and cells (e.g. tumour) can be accomplished by conjugation of LNP surfaces with biological receptor-specific targeting ligands (e.g. monoclo‐ nal antibodies, folate, RGD peptides) [2,3]. One of the best examples of such an imaging LNP recently described in the literature is shown (Figure 5). This imaging LNP system (Gadonano-F) is a novel Gd3+-containing, imaging LNP system that was designed to act as a folate receptor (FR)-targeted, positive contrast agent to enable magnetic resonance imaging (MRI) of FRpresenting cancerous lesions (primary and metastatic) including breast, lung, intestinal, renal, and ovarian cancers.

corresponding effect on 1/T2 enhancement. Due to the fact that more considerable increases in 1/T2 are induced by iron containing contrast agents, they are usually used in T2- weighed (negative dark) contrast imaging [11]. Despite the most frequently used MRI contrast agents are thermodynamically and kinetically stable low molecular weight Gd3+ complexes (that promote MRI contrast by non-specific enhancement of water proton relaxation rates within the blood pool), there has been a realization of the need for imaging LNPs suitable for the targeted delivery of imaging agents to sites of disease pathology such as cancerous lesions in

For cell populations to be imaged by MRI *in vivo*, these populations must be distinguishable from natural background noise signal. For this to happen, cells of interest should become labelled with appropriate agents, making use of controlling mechanisms such as enhanced permeability and retention (EPR) mechanism for imaging agents to reach target cells [12,13,14], followed by controlled target cell entry. According to Frangioni et al., the ideal cellular label should have the following features: a strong signalling effect, good biocompatibility, does not interfere with cellular genetics, remains retained only in the target cell population, and allows temporal imaging for long-lasting periods [15]. Controlled target cell entry is preferably by ligand-specific receptor-mediated cell entry processes, although alternative mechanisms of cell

**Figure 4.** Cell entry mechanisms: the various uptake mechanisms of macromolecules and nanoparticles into cells are

order to obtain clear contrast images of their extent and location.

entry may also be acceptable (Figure 4) [16].

84 Advances in Bioengineering

indicated according to their size (Adapted from [14]).

**Figure 5.** Multimodal imaging Gadonano-F LNPs for targeted delivery of MRI contrast agents to tumour cells.

Gadonano-F LNPs are prepared as above with the indicated lipid components. Double labelling is made possible using gadolinium metallochelating lipid (Gd.DOTA.DSA) for MRI positive contrast imaging and rhodamine fluorescent probe conjugated lipid (DOPE-Rhoda) for fluorescence imaging. Folate association with the Gadonano-F LNP surfaces is made possible using a polyethylene glycol (PEG) lipid (folate-PEG2000-DSPE), ensuring that Gado‐ nano-F LNPs are enabled for FR-specific targeting to tumour cells *in vivo*. The remaining PEG lipid (PEG2000-DSPE) ensures that the Gadonano-F LNPs possess stability in biological fluids (e.g. serum) (see [14]).

**Microbubbles (MBs)** 

MBs in vitro and *in vivo*.

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

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

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 LNPs accumulated in more perivascular and macrophage-rich locations [14,18]. 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 imaging 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*. On the left 24 h post imaging LNP administration, (a) surface IGROV-1 tumor slices, Gadonano LNPs, (b) midtumor IGROV-1 **Figure 6.** Fluorescence microscopy and MRI contrast imaging of 24 h post imaging LNP injection.

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

slices, Gadonano LNPs, (c) surface IGROV-1 tumor slices, Gadonano-F LNPs, and (d) IGROV-1 midtumor slices, Gadonano-F

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 images are from control mice injected with Gadonano LNPs (top); images are of mice injected with Gadonano-F LNPs (bottom) (white arrow shows location of tumour) (Adapted from [18])

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].
