**2. LNP fundamentals**

Structural lipids such as cholesterol and glycerophospholipids are the major components of biological membranes. Without doubt, one of the great triumphs of nature is the manner in which lipid molecules such as these are able to form into cellular membranes sufficient to compartmentalize volumes within cells and between cells. In short, lipids are able to form the vast macromolecular assemblies that come to make up cellular membranes and other barriers

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in nature precisely because they have an unrivalled capability for self-association, driven by weak short range forces and the hydrophobic effect (Figure 1) [1]. This capacity for selfassociation can be exploited in the laboratory in order to create self-assembly LNPs (approx 100nm in diameter) (Figure 2) that are proving to be a powerful platform to enable the functional delivery of active pharmaceutical ingredients (APIs) to target cells *in vivo*. Suitable APIs might include biopharmaceutical agents (e.g. DNA, RNA interference effectors), selected small molecule drugs (e.g. anticancer cytotoxic drugs), and/or imaging agents (e.g. magnetic resonance imaging contrast metals – gadolinium (III) (Gd3+), radiometals, and/or fluorescent probes). The number of lipid variations is almost limitless, so too the number and variety of possible LNPs that may be produced for use in biological situations. Therefore, LNPs offer the potential opportunity for tailor-made preparation and production leading in the future even to the possibility of LNP-mediated personalized medicine, no less [2, 3].

**Figure 1.** Principles of lipid structure and self-assembly.

Structural lipids of all types consist broadly of a non-polar, hydrophobic "chain" or "tail" region attached to a "polar" or "head" region (a); biological membranes primarily adopt a normal topology lamellar LαI fluid mesophase (bilayer) structure (b); under certain circum‐ stances biological membranes adopt other mesophases in particular the inverse topology hexagonal HII fluid mesophase (c), where hydrophobic chain regions face outwards and hydrophilic polar regions face inwards to form aqueous channels (the darker circles). (Dia‐ grams reproduced from [1])

in nature precisely because they have an unrivalled capability for self-association, driven by weak short range forces and the hydrophobic effect (Figure 1) [1]. This capacity for selfassociation can be exploited in the laboratory in order to create self-assembly LNPs (approx 100nm in diameter) (Figure 2) that are proving to be a powerful platform to enable the functional delivery of active pharmaceutical ingredients (APIs) to target cells *in vivo*. Suitable APIs might include biopharmaceutical agents (e.g. DNA, RNA interference effectors), selected small molecule drugs (e.g. anticancer cytotoxic drugs), and/or imaging agents (e.g. magnetic resonance imaging contrast metals – gadolinium (III) (Gd3+), radiometals, and/or fluorescent probes). The number of lipid variations is almost limitless, so too the number and variety of possible LNPs that may be produced for use in biological situations. Therefore, LNPs offer the potential opportunity for tailor-made preparation and production leading in the future even

to the possibility of LNP-mediated personalized medicine, no less [2, 3].

80 Advances in Bioengineering

**Figure 1.** Principles of lipid structure and self-assembly.

However, and there always is a however, LNPs that have seen service *in vivo* are turning out to have one fundamental design weakness which can be summarized by saying that those chemical modifications to LNP surfaces that are necessary for such nanoparticles to be stable to storage and in biological fluids, plus minimally visible to a host immune system, now turn out to limit the efficiency of functional delivery once LNPs reach their target cells. Accordingly, one of the primary ways to overcome this problem in recent years has been to introduce the concept of nanoparticle triggerability to LNP design [2, 4]. Nanoparticles possess triggerability (or are said to be triggerable) when designed for stability in biological fluids (from a desired point of administration to disease-target cells) then become triggered for the controlled release of associated APIs at target cells either through local changes in local endogenous (intrinsic) conditions, or through the application of an exogenous (extrinsic) stimulus trained onto target cell regions where nanoparticles are also located. What we are now learning is that MBs used in combination with ultrasound can be used in with LNPs, to provide a potent way to introduce LNP triggerability. Moreover, the introduction of ultrasound critically introduces an oppor‐ tunity for real-time, diagnostic imaging of LNP mediated delivery of APIs to target cells *in vivo*. Nanoparticles that combine functional API delivery *in vivo* with real-time, diagnostic imaging are known as theranostic nanoparticles (TNPs) [5]. Most importantly, TNPs offer the opportunity for true image-guided therapy. As such this may well be a primary future of the MBs-ultrasound-LNP combination as the rest of this chapter will now aim to demonstrate.

**Figure 2.** Functional lipid-based nanoparticle delivery systems.

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