Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles.

It is known that Phosphatidyl choline-Phosphatidyl glycerol mixtures can be used for liposome formulations, making them less leaky than liposomes with only one lipid. We hypothesized that this might also be the case for bubbles, which can be used as ultrasound (US) contrast agents. Therefore, we have compared a series of mixed distearoyl phosphatidylcholine-distearoyl phosphatidylglycerol (DSPC-DPSG) bubbles and with bubbles containing either DSPC or DSPG (and distearoyl ethanolamine-polyethyleneglycol 2000, DSPE-PEG2k). Here, we describe the development, examination of stability in vitro and attenuation of broad frequency US pulses. Novel lipid-stabilized freeze-dried formulations for US applications, using the phospholipids DSPC, DSPG, and PEGylated DSPE-PEG2k and perfluoropropane gas were developed. It was found that the bubbles could effectively be preserved by freeze-drying and then re-constituted by addition of water. Average bubble sizes were around 2 µm for all bubbles after re-constitution. Bubble stability was assessed by evaluating the decay of the US backscattering signal in vitro. Bubbles containing DSPG were more stable than bubbles with only DSPC. The composition DSPC:DSPG:DSPE-PEG2k 30:60:10 (molar ratio) was the most stable with an effective half-life of 9.12 min, compared to bubbles without DSPG, which had half-life of 2.05 min. Bubble attenuation of US depended highly on the compositions. Bubbles without DSPG had the highest attenuation indicating higher oscillation the most but were also destroyed by higher energy US. No bubbles with DSPG showed any indication of destruction but all had increased attenuations to varying degrees, DSPC:DSPG:DSPE-PEG2k 45:45:10 showed the least attenuation.

Ultrasound image-guided gene delivery using three-dimensional diagnostic ultrasound and lipid-based microbubbles.

Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered plasmid DNA (pDNA) to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.

Effect of lipid shell composition in DSPG-based microbubbles on blood flow imaging with ultrasonography.

Diagnostic ultrasound is non-invasive and provides real-time imaging. Microbubbles (MBs) are ultrasound contrast agents used to observe small blood flow, such as tumor tissue. However, MBs have short blood flow imaging time. This study developed lipid-based microbubbles (LMBs) with longer blood flow imaging time by focusing on their shell composition. Liposome research reported that addition 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) to the lipid composition enhances liposome membrane stability. Therefore, we introduced DSPG at different ratios into the LMBs lipid shell. Results showed that the lipid shell composition of MBs affects stability in vivo. 60% DSPG-containing LMBs (DSPG60-LMBs) have sustained blood flow imaging time compared with LMBs, which have other DSPG ratios, Sonazoid® and SonoVue®. DSPG60-LMBs also showed less uptake into the liver compared with Sonazoid®. Therefore, DSPG60-LMBs can have long blood flow imaging time and can be effective diagnostic agents in ultrasound imaging.

Characterization of Brain-Targeted Drug Delivery Enhanced by a Combination of Lipid-Based Microbubbles and Non-Focused Ultrasound.

The combination of focused ultrasound (FUS) and microbubbles, an ultrasound (US) contrast agent, has attracted much attention for its ability to open the blood brain barrier (BBB) and deliver drugs to the brain parenchyma. FUS can concentrate US energy in a restricted space, whereas non-focused US can affect a wide area of tissue. Non-focused US is also promising for drug delivery to the brain and other tissues. We have previously developed lipid-based microbubbles (LBs), and demonstrated that non-focused US and LBs have potential for drug delivery to tumor tissues. In this study, to achieve efficient and safe brain-targeted drug delivery, we evaluated the characteristics of BBB opening using non-focused US and LBs. Our results indicated that LBs could induce BBB opening with non-focused US. US frequency and intensity affected the efficiency of BBB opening and brain damage, and showed that the dose of LBs was also related to the efficiency of BBB opening. Furthermore, the combination of non-focused US and LBs could deliver macromolecules at 2000 kDa to the brain, and the induction of BBB opening was found to be reversible. These results suggest that the combination of non-focused US and LBs has potential as a brain-targeted drug delivery system.

Effects of encapsulated gas on stability of lipid-based microbubbles and ultrasound-triggered drug delivery.

The combination of Ultrasound (US) and US contrast agent (microbubbles, MBs), which is gas stabilized by a shell such as phospholipids or proteins, has potential as a useful innovative diagnostic and therapeutic tool. Previous studies have evaluated how particle size or shell components of MBs affect their physical characteristics, imaging ability, and drug delivery efficacy. We reported that MBs composed of neutral, anionic phospholipids, and polyethylene glycol-conjugated phospholipids at appropriate ratios were highly stable for US imaging. However, the effects of encapsulated gas on stability and drug delivery efficacy have not been characterized. Therefore, we developed several gas-loaded MBs with identical shell compositions and assessed their stability by US imaging (LOGIQ E9 with ML6-15 probe, MI 0.20). In addition, we assessed the effects of gas encapsulated in MBs on brain-targeted drug delivery, because the brain requires an efficient drug delivery system. Perfluoropropane and perfluorobutane-loaded MBs (MB-C3F8 and MB-C4F10) showed sustained US imaging in vitro and in vivo compared with sulfur hexafluoride-loaded MBs (MB-SF6). In addition, treatment of MB-C3F8 and MB-C4F10 with non-focused US efficiently delivered Evans blue, which was used as a model drug, to the brain to a greater extent than MB-SF6. In these treatments, notable damage to brain was not observed, which was assessed by HE staining and denatured neuron staining. Our results suggested that perfluoropropane and perfluorobutane could be useful for the production of MBs with high stability to allow for US imaging and drug delivery.