Lipid-shelled vehicles: engineering for ultrasound molecular imaging and drug delivery

Abstract

Ultrasound pressure waves can map the location of lipid-stabilized gas micro-bubbles after their intravenous administration in the body, facilitating an estimate of vascular density and microvascular flow rate. Microbubbles are currently approved by the Food and Drug Administration as ultrasound contrast agents for visualizing opacification of the left ventricle in echocardiography. However, the interaction of ultrasound waves with intravenously-injected lipid-shelled particles, including both liposomes and microbubbles, is a far richer field. Particles can be designed for molecular imaging and loaded with drugs or genes; the mechanical and thermal properties of ultrasound can then effect localized drug release. In this Account, we provide an overview of the engineering of lipid-shelled microbubbles (typical diameter 1000-10 000 nm) and liposomes (typical diameter 65-120 nm) for ultrasound-based applications in molecular imaging and drug delivery. The chemistries of the shell and core can be optimized to enhance stability, circulation persistence, drug loading and release, targeting to and fusion with the cell membrane, and therapeutic biological effects. To assess the biodistribution and pharmacokinetics of these particles, we incorporated positron emission tomography (PET) radioisotopes on the shell. The radionuclide (18)F (half-life approximately 2 h) was covalently coupled to a dipalmitoyl lipid, followed by integration of the labeled lipid into the shell, facilitating short-term analysis of particle pharmacokinetics and metabolism of the lipid molecule. Alternately, labeling a formed particle with (64)Cu (half-life 12.7 h), after prior covalent incorporation of a copper-chelating moiety onto the lipid shell, permits pharmacokinetic study of particles over several days. Stability and persistence in circulation of both liposomes and microbubbles are enhanced by long acyl chains and a poly(ethylene glycol) coating. Vascular targeting has been demonstrated with both nano- and microdiameter particles. Targeting affinity of the microbubble can be modulated by burying the ligand within a polymer brush layer; the application of ultrasound then reveals the ligand, enabling specific targeting of only the insonified region. Microbubbles and liposomes require different strategies for both drug loading and release. Microbubble loading is inhibited by the gas core and enhanced by layer-by-layer construction or conjugation of drug-entrapped particles to the surface. Liposome loading is typically internal and is enhanced by drug-specific loading techniques. Drug release from a microbubble results from the oscillation of the gas core diameter produced by the sound wave, whereas that from a liposome is enhanced by heat produced from the local absorption of acoustic energy within the tissue microenvironment. Biological effects induced by ultrasound, such as changes in cell membrane and vascular permeability, can enhance drug delivery. In particular, as microbubbles oscillate near a vessel wall, shock waves or liquid jets enhance drug transport. Mild heating induced by ultrasound, either before or after injection of the drug, facilitates the transport of liposomes from blood vessels to the tissue interstitium, thus increasing drug accumulation in the target region. Lipid-shelled vehicles offer many opportunities for chemists and engineers; ultrasound-based applications beyond the few currently in common use will undoubtedly soon multiply as molecular construction techniques are further refined.

Figure 1

Overview of vehicles, lipid components and biodistribution. (a) phase transition temperature as a function of acyl chain length for four lipid head groups- phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphotidylethanolamine (PE). (b)–(d) Cartoon representation (not drawn to scale) of microbubbles (b), temperature-sensitive liposomes below their transition temperature (c), temperature-sensitive liposomes above their transition temperature (d). Typical microbubble and liposome diameters are 1–10 µm and 65–120 nm. Phase transition temperatures near 40°C are typically chosen for temperature-sensitive vehicles.

Figure 2

Synthesis and stability of microbubbles. Mechanical agitation of liposomes in an aqueous environment results in lipid-coated microbubbles. If the microbubble shell is cooled slowly domains of lipid components form. On left, various microstructures are shown with permission from (23), including (a) large domains after slow cooling, (b) snowflake domains after rapid cooling and (c) network domains (scale bars are 20 µm). On right, microbubbles with varying acyl chain length are shown before and after a sequence of ultrasound pulses, microbubbles expel a small volume of gas and lipid with each pulse, shown with permission from (24). After insonation, C₁₂ decreases in diameter but exhibits no optically-discernable shed particles, C₁₈ sheds a small bud, and C₂₂ accumulates lipid that remains attached to the monolayer shell (scale bars are 5 µm). For C₂₂, increasing the acyl chain length increases the cohesiveness of the microbubble shell and as a result excess lipid remains bound to the smaller bubble after insonation.

Figure 3

(a)–(c) Positron emission tomography (PET) 90-minute maximum intensity projection images of (a) long-circulating liposomes in a rat model, where the image is dominated by the blood pool; (b) short linear heart-targeted peptide (Cys-Arg-Pro-Pro-Arg) coated liposomes in a mouse model, where the image is dominated by the heart; and (b) microbubbles in a rat model, where the image is dominated by the spleen. (a) and (c) are reproduced with permission from (16), and (b) is reproduced with permission from (17). Particles were radiolabeled by incorporating ([¹⁸F]FDP into the particle shell.

Figure 4

Insonation and imaging of microbubbles. (a) Image of a line through the center of a microbubble over time as the sound waves produce oscillations in microbubble diameter. Two-dimensional images of the microbubble are overlaid at the corresponding time points. (b) Grey scale ultrasound image of a tumor acquired at a center frequency of 14 MHz. (c) Corresponding image of microbubble density within the tumor created with Contrast Pulse Sequence (CPS) from Siemens Medical Solutions (MountainView CA). (d) Parametric map of the time required for microbubbles to fill each voxel after a high intensity pulse ruptures the bubbles, where color indicates time from 0.5 (yellow) to 10 seconds (pink). A 7-MHz pulse sequence consisting of low amplitude imaging pulses interleaved with high amplitude disruptive pulses was used to acquire the data. Such maps provide an assessment of local vascular functionality.

Figure 5

Vascular targeting. (a)–(b) Ultrasound images of microbubbles bound to angiogenic vasculature in a fibroblast-growth-factor stimulated Matrigel plug where (a) is control microbubble and is (b) Echistatin-targeted microbubble. (a)–(b) are reproduced with permission from (26). (c) Prior to the application of ultrasound radiation force, microbubbles are uniformly distributed within the blood vessel. (d) After the application of radiation force, microbubbles are deflected to the vessel wall, resulting in enhanced targeting. Cartoons (c–d) are reproduced with permission from (28).

Figure 6

Enhancing drug and gene loading. (a) Layer-by-layer loading of DNA on the surface of a particle. (a) and (c) are reproduced with permission from (32). (b) Hybrid vehicle composed of microbubbles and liposomes or nanoparticles, reproduced with permission from (38). (c) Confocal fluorescence image of a microbubble showing heterogeneous distribution and co-localization of DNA (red) and fluorescein-isothiocyanate-poly(L-lysine). Scale bar is 5 um.

Figure 7

Enhanced drug and gene delivery achieved with the combination of particles and ultrasound. Insonation of circulating microbubbles in the chorioallantoic membrane model results in small vascular defects through which a fluorescent dye is transported, reproduced with permission from (27). (a)–(f) are a sequence of images acquired from 1.00-MHz center frequency insonation at a peak negative pressure of 1.3 MPa. (a) was acquired before insonation, (b)–(f) were acquired 0.06, 0.12, 1.24, 2.24 and 3.24 seconds after insonation began, respectively, demonstrating the transport of the fluorescent probe from the vasculature to the tissue interstitium.

Scheme 1

(a) Structure of [¹⁸F]fluorodipalmitin ([¹⁸F]FDP), (b) integration of [¹⁸F]FDP within particles.

Scheme 2

(a) Structure of BAT-PEG-lipid, (b) integration of BAT-PEG-lipid within particles and radiolabeling with ⁶⁴Cu.

Scheme 3

Scheme employed in peptide-PEG-lipid conjugate synthesis.