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.

Viability variation of T-cells under ultrasound exposure according to adhesion condition with bubbles.

PURPOSE: Although cellular immunotherapy is expected as a new cancer treatment, its therapeutic efficiency is limited in solid tumors, because most cells return to the bloodstream rather than adhere to the target site. Therefore, we are motivated to develop a technique to concentrate the cells in the blood flow using active control of bubble-surrounded cells under ultrasound exposure considering both aspects of cell controllability and viability. METHODS: We prepared a lipid bubble conjugating ligand to adhere to the surface of the T-cells. First, we evaluated the cell controllability by retaining the cells on a wall of an artificial blood vessel through continuous ultrasound exposure. Next, we investigated the cell viability under ultrasound exposure in a suspension with various bubble concentrations. RESULTS: We estimated the concentration of bubbles when the adhesion to the cell surface was saturated. Then, we evaluated the cell viability with various conditions of ultrasound exposure and bubble concentrations. However, it was confirmed that cell damage occurred under conditions that achieved proper control of the cells. Therefore, we exposed the cells to burst waves to reduce the applied ultrasound intensity. Consequently, the significant increase in cell viability was confirmed to be inversely proportional to the duty ratio. CONCLUSION: To retain cells on a vessel wall, determining the appropriate ultrasound condition including sound pressure and waveform is important to maintain cell viability.

Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives.

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized ß-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme ß-glucuronidase. To trigger the cell release of ß-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of ß-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact ß-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer.

Ultrasound and microbubble-mediated drug delivery and immunotherapy.

Ultrasound induces the oscillation and collapse of microbubbles such as those of an ultrasound contrast agent, where these behaviors generate mechanical and thermal effects on cells and tissues. These, in turn, induce biological responses in cells and tissues, such as cellular signaling, endocytosis, or cell death. These physiological effects have been used for therapeutic purposes. Most pharmaceutical agents need to pass through the blood vessel walls and reach the parenchyma cells to produce therapeutic effects in drug delivery. Therefore, the blood vessel walls act as an obstacle to drug delivery. The combination of ultrasound and microbubbles is a promising strategy to enhance vascular permeability, improving drug transport from blood to tissues. This combination has also been applied to gene and protein delivery, such as cytokines and antigens for immunotherapy. Immunotherapy, in particular, is an attractive technique for cancer treatment as it induces a cancer cell-specific response. However, sufficient anti-tumor effects have not been achieved with the conventional cancer immunotherapy. Recently, new therapies based on immunomodulation with immune checkpoint inhibitors have been reported. Immunomodulation can be regarded as a new strategy for cancer immunotherapy. It was also reported that mechanical and thermal effects induced by the combination of ultrasound and microbubbles could suppress tumor growth by promoting the cancer-immunity cycle via immunomodulation in the tumor microenvironment. In this review, we provide an overview of the application of ultrasound and microbubble combination for drug delivery and activation of the immune system in the microenvironment of tumor tissue.

Feasibility study of novel nanoparticles derived from Glycyrrhizae radix as vaccine adjuvant for cancer immunotherapy.

Aims: The feasibility of using nanoparticles derived from Glycyrrhizae radix extract (Glycyrrhiza NPs) as a vaccine adjuvant for cancer immunotherapy was evaluated. Methods: C57BL/6J mice were immunized with ovalbumin (OVA) and Glycyrrhiza NPs. After immunization, splenocytes were incubated with the H-2Kb epitope peptide of OVA (SL8) and the production of IFN-γ was evaluated. Moreover, an OVA-expressing lymphoma cell line (E.G7-OVA cells) was inoculated into mice after immunization to evaluate the antitumor effect. Results: The immunization of OVA with Glycyrrhiza NPs induced IFN-γ production and completely rejected E.G7-OVA cells. Conclusion: Glycyrrhiza NPs could prime antigen-specific CD8+ T-cells resulting in antitumor effects. Therefore, Glycyrrhiza NPs can be an effective vaccine adjuvant for cancer immunotherapy.

Glycyrrhizae radix is a medical plant that contains anti-inflammatory compounds such as glycyrrhizin. Nanoparticles (NPs) derived from Glycyrrhizae radix extract induced the production of proinflammatory cytokines. Therefore, these NPs could be used as a vaccine adjuvant. Here, a feasibility study on the use of Glycyrrhiza NPs as a vaccine adjuvant in cancer immunotherapy is reported. T-cell responses and antitumor effects were evaluated after the immunization of ovalbumin (OVA) with Glycyrrhiza NPs. The immunization of OVA with Glycyrrhiza NPs effectively induced OVA-specific T-cells and completely rejected OVA-expressing tumor cells. Therefore, Glycyrrhiza NPs could induce antitumor immunity and be an effective vaccine adjuvant in cancer immunotherapy.

Enhanced Vascular Permeability by Microbubbles and Ultrasound in Drug Delivery.

Ultrasound and microbubbles, an ultrasound contrast agent, have recently increased attention to developing novel drug delivery systems. Ultrasound exposure can induce mechanical effects derived from microbubbles behaviors such as an expansion, contraction, and collapse depending on ultrasound conditions. These mechanical effects induce several biological effects, including enhancement of vascular permeability. For drug delivery, one promising approach is enhancing vascular permeability using ultrasound and microbubbles, resulting in improved drug transport to targeted tissues. This approach is applied to several tissues and drugs to cure diseases. This review describes the enhancement of vascular permeability by ultrasound and microbubbles and its therapeutic application, including our recent study. We also discuss the current situation of the field and its potential future perspectives.

Enhanced antitumor activity of combined lipid bubble ultrasound and anticancer drugs in gynecological cervical cancers.

Chemotherapy plays an important role in the treatment of patients with gynecological cancers. Delivering anticancer drugs effectively to tumor cells with just few side effects is key in cancer treatment. Lipid bubbles (LB) are compounds that increase the vascular permeability of the tumor under diagnostic ultrasound (US) exposure and enable the effective transport of drugs to tumor cells. The aim of our study was to establish a novel drug delivery technique for chemotherapy and to identify the most effective anticancer drugs for the bubble US-mediated drug delivery system (BUS-DDS) in gynecological cancer treatments. We constructed xenograft models using cervical cancer (HeLa) and uterine endometrial cancer (HEC1B) cell lines. Lipid bubbles were injected i.v., combined with either cisplatin (CDDP), pegylated liposomal doxorubicin (PLD), or bevacizumab, and US was applied to the tumor. We compared the enhanced chemotherapeutic effects of these drugs and determined the optimal drugs for BUS-DDS. Tumor volume reduction of HeLa and HEC1B xenografts following cisplatin treatment was significantly enhanced by BUS-DDS. Both CDDP and PLD significantly enhanced the antitumor effects of BUS-DDS in HeLa tumors; however, volume reduction by BUS-DDS was insignificant when combined with bevacizumab, a humanized anti-vascular endothelial growth factor mAb. The BUS-DDS did not cause any severe adverse events and significantly enhanced the antitumor effects of cytotoxic drugs. The effects of bevacizumab, which were not as dose-dependent as those of the two drugs used prior, were minimal. Our data suggest that BUS-DDS technology might help achieve "reinforced targeting" in the treatment of gynecological cancers.

Lipid bubbles combined with low-intensity ultrasound enhance the intratumoral accumulation and antitumor effect of pegylated liposomal doxorubicin in vivo.

Pegylated liposomal doxorubicin (PLD) is a representative nanomedicine that has improved tumor selectivity and safety profile. However, the therapeutic superiority of PLD over conventional doxorubicin has been reported to be insignificant in clinical medicine. Combination treatment with microbubbles and ultrasound (US) is a promising strategy for enhancing the antitumor effects of chemotherapeutics by improving drug delivery. Recently, several preclinical studies have shown the drug delivery potential of lipid bubbles (LBs), newly developed monolayer microbubbles, in combination with low-intensity US (LIUS). This study aimed to elucidate whether the combined use of LBs and LIUS enhanced the intratumoral accumulation and antitumor effect of PLD in syngeneic mouse tumor models. Contrast-enhanced US imaging using LBs showed a significant decrease in contrast enhancement after LIUS, indicating that LIUS exposure induced the destruction of LBs in the tumor tissue. A quantitative evaluation revealed that the combined use of LBs and LIUS improved the intratumoral accumulation of PLD. Furthermore, tumor growth was inhibited by combined treatment with PLD, LBs, and LIUS. Therefore, the combined use of LBs and LIUS enhanced the antitumor effect of PLD by increasing its accumulation in the tumor tissue. In conclusion, the present study provides important evidence that the combination of LBs and LIUS is an effective method for enhancing the intratumoral delivery and antitumor effect of PLD in vivo.

Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression.

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3'-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

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.

A Pilot Study on Efficacy of Lipid Bubbles for Theranostics in Dogs with Tumors.

The combined administration of microbubbles and ultrasound (US) is a promising strategy for theranostics, i.e., a combination of therapeutics and diagnostics. Lipid bubbles (LBs), which are experimental theranostic microbubbles, have demonstrated efficacy in vitro and in vivo for both contrast imaging and drug delivery in combination with US irradiation. To evaluate the clinical efficacy of LBs in combination with US in large animals, we performed a series of experiments, including clinical studies in dogs. First, contrast-enhanced ultrasonography using LBs (LB-CEUS) was performed on the livers of six healthy Beagles. The hepatic portal vein and liver tissue were enhanced; no adverse reactions were observed. Second, LB-CEUS was applied clinically to 21 dogs with focal liver lesions. The sensitivity and specificity were 100.0% and 83.3%, respectively. These results suggested that LB-CEUS could be used safely for diagnosis, with high accuracy. Finally, LBs were administered in combination with therapeutic US to three dogs with an anatomically unresectable solid tumor in the perianal and cervical region to determine the enhancement of the chemotherapeutic effect of liposomal doxorubicin; a notable reduction in tumor volume was observed. These findings indicate that LBs have potential for both therapeutic and diagnostic applications in dogs in combination with US irradiation.

Lipid-based microbubbles and ultrasound for therapeutic application.

Microbubbles with diagnostic ultrasound have had a long history of use in the medical field. In recent years, the therapeutic application of the combination of microbubbles and ultrasound, called sonoporation, has received increased attention as microbubble oscillation or collapse close to various barriers in the body was recognized to potentially open those barriers, increasing drug transport across them. In this review, we aimed to describe the development of lipid-stabilized microbubbles equipped with functions, such as long circulation and drug loading, and the therapeutic application of sonoporation for tumor-targeted therapy, brain-targeted therapy, and immunotherapy. We also attempted to discuss the current status of the field and potential future developments.

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.

Lipid nanoparticles of Type-A CpG D35 suppress tumor growth by changing tumor immune-microenvironment and activate CD8 T cells in mice.

Type-A CpG oligodeoxynucleotides (ODNs), which have a natural phosphodiester backbone, is one of the highest IFN-α inducer from plasmacytoid dendritic cells (pDC) via Toll-like receptor 9 (TLR9)-dependent signaling. However, the in vivo application of Type-A CpG has been limited because the rapid degradation in vivo results in relatively weak biological effect compared to other Type-B, -C, and -P CpG ODNs, which have nuclease-resistant phosphorothioate backbones. To overcome this limitation, we developed lipid nanoparticles formulation containing a Type-A CpG ODN, D35 (D35LNP). When tested in a mouse tumor model, intratumoral and intravenous D35LNP administration significantly suppressed tumor growth in a CD8 T cell-dependent manner, whereas original D35 showed no efficacy. Tumor suppression was associated with Th1-related gene induction and activation of CD8 T cells in the tumor. The combination of D35LNP and an anti-PD-1 antibody increased the therapeutic efficacy. Importantly, the therapeutic schedule and dose of intravenous D35LNP did not induce apparent liver toxicity. These results suggested that D35LNP is a safe and effective immunostimulatory drug formulation for cancer immunotherapy.

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.

Tumor growth suppression by the combination of nanobubbles and ultrasound.

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti-tumor mechanism of this cancer therapy. Colon-26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0-4 W/cm2 , 2 min). The anti-tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti-tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3-4 W/cm2 of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2-4 W/cm2 . In vivo depletion of CD8+ T cells (not NK or CD4+ T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8+ T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti-tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.

Development of fluorous lipid-based nanobubbles for efficiently containing perfluoropropane.

Nano-/microbubbles are expected not only to function as ultrasound contrast agents but also as ultrasound-triggered enhancers in gene and drug delivery. Notably, nanobubbles have the ability to pass through tumor vasculature and achieve passive tumor targeting. Thus, nanobubbles would be an attractive tool for use as ultrasound-mediated cancer theranostics. However, the amounts of gas carried by nanobubbles are generally lower than those carried by microbubbles because nanobubbles have inherently smaller volumes. In order to reduce the injection volume and to increase echogenicity, it is important to develop nanobubbles with higher gas content. In this study, we prepared 5 kinds of fluoro-lipids and used these reagents as surfactants to generate "Bubble liposomes", that is, liposomes that encapsulate nanobubbles such that the lipids serve as stabilizers between the fluorous gas and water phases. Bubble liposome containing 1-stearoyl-2-(18,18-difluoro)stearoyl-sn-glycero-3-phosphocholine carried 2-fold higher amounts of C3F8 compared to unmodified Bubble liposome. The modified Bubble liposome also exhibited increased echogenicity by ultrasonography. These results demonstrated that the inclusion of fluoro-lipid is a promising tool for generating nanobubbles with increased efficiency of fluorous gas carrier.

[Novel dianostics and therapeutics with ultrasound technologies and nanotechnologies].

Ultrasound is a good tool for theranostics due to have multi-potency both of diagnostics with sonography and therapeutics with high intensity focused ultrasound (HIFU). In addition, microbubbles and nanobubbles are utilized as not only contrast imaging agent but also enhancer of drug and gene delivery by combination of ultrasound. Recently, we developed novel liposomal nanobubbles (Bubble liposomes) which were containing perfluoropropane. Bubble liposomes induced jet stream by low intensity ultrasound exposure and resulted in enhancing permeability of cell membrane. This phenomenon has been utilized as driving force for drug and gene delivery. On the other hand, the combination of Bubble liposomes and high intensity ultrasound induces strong jet stream and increase temperature. This condition can directly damage to tumor cells, we are applying this for cancer therapy. Therefore, their combination has potency for various cancer therapies such as gene therapy, immunotherapy and hyperthermia. In this review, we discuss about cancer therapy by the combination of Bubble liposomes and ultrasound.

Ultrasound-mediated gene delivery systems by AG73-modified Bubble liposomes.

Targeted gene delivery to neovascular vessels in tumors is considered a promising strategy for cancer therapy. We previously reported that "Bubble liposomes" (BLs), which are ultrasound (US) imaging gas-encapsulating liposomes, were suitable for US imaging and gene delivery. When BLs are exposed to US, the bubble is destroyed, creating a jet stream by cavitation, and resulting in the instantaneous ejection of extracellular plasmid DNA (pDNA) or other nucleic acids into the cytosol. We developed AG73 peptide-modified Bubble liposomes (AG73-BL) as a targeted US contrast agent, which was designed to attach to neovascular tumor vessels and to allow specific US detection of angiogenesis (Negishi et al., Biomaterials 2013, 34, 501-507). In this study, to evaluate the effectiveness of AG73-BL as a gene delivery tool for neovascular vessels, we examined the gene transfection efficiency of AG73-BL with US exposure in primary human endothelial cells (HUVEC). The transfection efficiency was significantly enhanced if the AG73-BL attached to the HUVEC was exposed to US compared to the BL-modified with no peptide or scrambled peptide. In addition, the cell viability was greater than 80% after transfection with AG73-BL. These results suggested that after the destruction of the AG73-BL with US exposure, a cavitation could be effectively induced by the US exposure against AG73-BL binding to the cell surface of the HUVEC, and the subsequent gene delivery into cells could be enhanced. Thus, AG73-BL may be useful for gene delivery as well as for US imaging of neovascular vessels.

AG73-modified Bubble liposomes for targeted ultrasound imaging of tumor neovasculature.

Ultrasound imaging is a widely used imaging technique. The use of contrast agents has become an indispensible part of clinical ultrasound imaging, and molecular imaging via ultrasound has recently attracted significant attention. We recently reported that "Bubble liposomes" (BLs) encapsulating US imaging gas liposomes were suitable for ultrasound imaging and gene delivery. The 12 amino acid AG73 peptide derived from the laminin α1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in blood vessels. In this study, we prepared AG73 peptide-modified BLs (AG73-BLs) and assessed their specific attachment and ultrasound imaging ability for blood vessels in vitro and in vivo. First, we assessed the specific attachment of AG73-BLs in vitro, using flow cytometry and microscopy. AG73-BLs showed specific attachment compared with non-labeled or control peptide-modified BLs. Next, we examined ultrasound imaging in tumor-bearing mice. When BLs were administered, contrast imaging of AG73-BLs was sustainable for up to 4 min, while contrast imaging of non-labeled BLs was not observed. Thus, it is suggested that AG73-BLs may become useful ultrasound contrast agents in the clinic for diagnosis based on ultrasound imaging.