Brain Delivery of Cisplatin Using Microbubbles in Combination with Ultrasound as an Effective Therapy for Glioblastoma.

Glioblastoma is a highly invasive and fatal disease. Temozolomide, a blood-brain barrier (BBB)-penetrant therapeutic agent currently used for glioblastoma, does not exhibit sufficient therapeutic effect. Cisplatin (CDDP), a versatile anticancer drug, is not considered a therapeutic option for glioblastoma due to its low BBB permeability. We previously investigated the utility of microbubbles (MBs) in combination with ultrasound (US) in promoting BBB permeability and reported the efficacy of drug delivery to the brain using a minimally invasive approach. This study aimed to evaluate the feasibility of CDDP delivery to the brain using the combination of MBs and US for the treatment of glioblastoma. We used mice that were implanted with glioma-261 GFP-Luc cells expressing luciferase as the glioblastoma model. In this model, after tumor inoculation, the BBB opening was induced using MBs and US, and CDDP was simultaneously administered. We found that the CDDP concentrations were higher at the glioblastoma site where the US was applied, although CDDP normally cannot pass through the BBB. Furthermore, the survival was longer in mice treated with CDDP delivered via MBs and US than in those treated with CDDP alone or those that were left untreated. These results suggest that the combination of MBs and US is an effective antitumor drug delivery system based on BBB opening in glioblastoma therapy.

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.

Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for ultrasound imaging and therapy.

Microbubble formulations have a long history for enhancement of ultrasound (US) imaging and recently also for therapeutic applications. Previously, a series of freeze-dried bubble formulations based on the lipids DSPC and DSPG were developed. Here, we have attempted to scale-up the production process for future more extensive studies. Bubbles were prepared by homogenization of a lipid dispersion in a perfluoropropane atmosphere in a medium size (300-500 mL) homogenizer and then freeze-dried for better storage stability. In total, 300 freeze-dried vials were prepared. The properties of the bubbles were similar to those previously prepared on a lab scale with the difference that they were slightly larger and also had a better stability. The re-entrapped gas concentration after re-constituted freeze-dried bubbles was 9.4 µL/µmol lipid. The re-entrapped rate was 72.3% of fresh bubble before freeze-drying (13.0 µL/µmol lipid). The half-life of US imaging signal of the re-constituted freeze-dried bubbles in water in vitro was shorter than that of the fresh bubbles (2.7 min vs. 3.3 min). A leak of Evans Blue, that binds to albumin, from mouse ear blood vessel was observed after combination of bubble and US irradiation of 1 MHz for 1 min. As a result of bubble vibration by US irradiation, vascular endothelial cell bond opened and Evans Blue leaked. Toxicity of bubble was tested in rats. No toxicity was found after a single injection in the dose range tested. No serious toxicity was seen after repeated injections (one daily injection during 15 days).

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.