Nobuhito Hamano

Quantification in molecular ultrasound imaging: a comparative study in mice between healthy liver and a human hepatocellular carcinoma xenograft.

The purpose of this study was to quantitatively assess the contrast kinetics of vascular endothelial growth factor receptor 2 (VEGFR2)‐targeted microbubbles (BR55; Bracco Suisse, Geneva, Switzerland) compared to clinically used microbubbles (SonoVue; Bracco SpA, Milan, Italy) in both normal liver and human hepatocellular carcinoma xenograft tumors in mice.

Modification of the C16Y peptide on nanoparticles is an effective approach to target endothelial and cancer cells via the integrin receptor

Liposomes have been explored as potential drug and gene-delivery particles. In recent years, tumor-targeted liposomes have been developed to improve the efficacy of antitumor treatment. The C16Y peptide is a modified C16 peptide, which is derived from the laminin γ1 chain, and binds to integrins α(v)β3 and α5β1 on endothelial cells. In this study, we prepared integrin-targeted C16Y peptide-modified liposomes (C16Y-L) to enhance the intracellular uptake of drugs and genes specifically into tumor tissues. The selectivity of C16Y-L for endothelial cells and cancer cells, which strongly express integrins α(v)β3 and α5β1, was assessed by flow cytometry and confocal microscopy. The cellular uptake of C16Y-L by both cell types was higher than uptake of the un-labeled and scramble peptide-modified liposomes. Next, to ascertain the involvement of receptor-mediated endocytosis in the process, these cells were treated with C16Y-L for 1h at 37°C or at 4°C. We found that uptake was also dependent on the temperature. Moreover, to evaluate whether the uptake depended on an integrin-ligand interaction, we examined the inhibition of C16Y-L uptake using recombinant integrin αVβ3 and found that the cellular uptake of C16Y-L treated with αVβ3 integrin decreased. This result suggests that C16Y-L can selectively target cells that highly express integrin αVβ3. Thus, the modification of the C16Y peptide on a Drug Delivery System (DDS) carrier may be a feasible approach for drug or gene delivery into tumors.

Bubble liposomes and ultrasound exposure improve localized morpholino oligomer delivery into the skeletal muscles of dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies.

Effects of doxorubicin-encapsulating AG73 peptide-modified liposomes on tumor selectivity and cytotoxicity

Doxorubicin-encapsulating liposomal formulations, known as Doxil, have been used for the treatment of Kaposis sarcoma and ovarian cancer. However, there is still a need for a drug delivery system for doxorubicin that limits the treatments side effects, namely, mucositis and hand-and-foot syndrome. The AG73 peptide derived from the laminin α1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in some cancer cells. In this study, to develop a safer and more selective liposomal formulation, we prepared doxorubicin-encapsulating AG73 peptide-modified liposomes (AG73-Dox). First, we assessed the selectivity of AG73-Dox for cancer cells, including syndecan-2 over-expressing cells, using flow cytometry and confocal microscopy. AG73-Dox showed selective cellular uptake on cancer cells and enhancement of the intracellular uptake. Next, we examined the cytotoxicity of AG73-Dox using a WST assay. AG73-Dox exhibited a higher cytotoxicity against cancer cells than other control liposomes. In addition, we showed that the antitumor efficacy of AG73-Dox in vivo was better than that of free Dox. When we examined the biodistribution of liposomes, AG73 peptide-modified liposomes (AG73-L) tended to bind to intratumoral vessels and extravasated in the tumor tissue. Thus, further optimization of AG73-L toward tumor targeting may lead to a development of a useful tool for cancer therapy.

Development of a Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections

Liposomes have been used as targeting carriers for drug delivery systems (DDSs), and the carriers are able to be modified with targeting ligands, such as antibodies and peptides. To evaluate the targetability of DDS carriers modified with a targeting ligand, culture cells expressing the targeting molecules as well as small animals are used. Furthermore, in vitro and in vivo screening analyses must be repeatedly performed. Therefore, it is important to establish an easy and high-precision screening system for targeting carriers. With this aim, we focused that whether this ex vivo system could easily support assessment of interaction between targeting ligand and its receptor under physiological environment and further screen the DDS carrier-modified with targeting moiety. We examined targeting ability via in vitro, ex vivo, and in vivo analyses using integrin αvβ3-targeting C16Y-L. For the in vitro analysis, the cellular uptake of C16Y-L was higher than that of control liposomes in colon26 cells. For the ex vivo analysis, we performed an immunohistochemical analysis using colon26 tumor sections. C16Y-L was specifically attached to the tumor sections, as found in the in vitro analysis. Moreover, to evaluate the ex vivo-in vivo correlation, we examined the intratumoral localization of C16Y-L. This result showed that C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature after the intravenous injection of C16Y-L, suggesting that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. Thus, the ex vivo peptide-modified liposomal analysis may be an easy and rapid screening system with high-precision and for consideration in in vivo conditions.