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Lymphatic endothelial cells (LECs) that form lymphatic vessels play a pivotal role in immune regulation. It was recently reported that LECs suppress the antigen-dependent anti-tumor immunity in cancer tissues. Thus, regulating the function of LECs is a promising strategy for cancer therapy. The objective of this study was to develop a method for the selective delivery of small interfering RNA (siRNA) to LECs. For this purpose, the siRNA was formulated into nanoparticles (LNPs) to prevent them from being degraded in body fluids and to facilitate their penetration of the cell membrane. A breakthrough technology for achieving this is ONPATTRO®, a world's first siRNA drug. Since LNPs are taken up by hepatocytes relatively well via low-density lipoprotein receptors, most of the LNP systems that have been developed so far target hepatocytes. In this study, we report on the development of a new method for the rapid and convenient method for modifying LNPs with antibodies using the CLick reaction on the Interface of the nanoParticle (CLIP). The CLIP approach was faster and more versatile than the conventional method using amide coupling. As a demonstration, we report on the LEC-targeted siRNA delivery by using antibody-modified LNPs both in vitro and in vivo. The method used for the modification of LNPs is highly promising and has the potential for expanding the LNP-based delivery of nucleic acids in the future., Competing Interests: Declaration of Competing Interest This research was conducted as joint research between Chiba University, Tohoku University and the NOF Corporation. The other funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nucleic acid drugs can control gene expression and function in a manner different from that of conventional compounds. On the other hand, nucleic acids can be easily degraded in the in vivo circumstances. In addition, nucleic acids cannot penetrate cell membranes. Therefore, a drug delivery system (DDS) is essential to protect nucleic acid molecules until they reach the target cell and to release them efficiently inside the cell. In order to apply nucleic acid drugs to new cancer therapeutic strategies, the author has been developing a DDS that enables functional control of vascular endothelial cells that consist of the tumor microenvironment. The aim of my study is to develop lipid nanoparticles (LNPs) were modified with functional molecules that control their pharmacokinetics in vivo and intracellular fate to delivered small interfering RNA (siRNA) to tumor vasculature. By imparting pH-responsive membrane fusion properties to lipid nanoparticles, I have developed a system that responds to acidification in endosomes within cells and subsequently efficiently releases siRNA into the cytoplasm via membrane fusion, where siRNA molecules exhibit their function. In addition, by developing a method for presenting functional molecules, such as peptides, saccharides and so on, that recognize target cells on the surface of LNPs, I succeeded in establishing LNPs which internalize more efficiently into specific cells than off-target cells. Finally, by integrating these technologies, I developed an in vivo siRNA DDS that enables in vivo control of genes of interest in tumor vascular endothelial cells and succeeded in cancer therapy by regulating vascular function.

Mitochondrial delivery of an anticancer drug targeting cancer cells would eventually result in cell death. To achieve this, a drug delivery system targeting mitochondria is needed. We recently developed a MITO-Porter, a liposome that delivers its cargo to mitochondria. We reported that such a MITO-Porter could deliver doxorubicin (DOX), an anticancer drug, to mitochondria in OS-RC-2 cells, a drug resistant cancer cell, resulting in inhibiting the cell growth, based in in vitro experiments. Herein, we report on validating the benefit of such a therapeutic strategy for treating drug resistant cancers by the in vivo targeting of mitochondria. We prepared a DOX-MITO-Porter, in which DOX was encapsulated in the MITO-Porter and optimized its retention in blood circulation. When the DOX-MITO-Porter was administered to mice bearing OS-RC-2 cells via tail vein injection, tumor size was significantly decreased, compared to DOX itself and to the DOX-encapsulated polyethylene glycol-modified liposome (DOX-PEG-LP). Intracellular observation confirmed that the DOX-MITO-Porter had accumulated in tumor mitochondria. It was also found a relationship between anti-tumor effect and the mitochondrial function, as indicated by the depolarization of mitochondrial membrane potential. This study provides support for the utility of an in vivo mitochondrial delivery system in drug resistant cancer therapies., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

For achieving efficient cancer treatment, it is important to elucidate the mechanism responsible for the accumulation of nanoparticles in tumor tissue. Recent studies suggest that nanoparticles are not delivered merely through gaps between tumor endothelial cells. We previously reported that the maturation of the vascular structure by the vascular endothelial cell growth factor receptor 2 (VEGFR2) using a previously developed siRNA delivery technology (RGD-MEND) significantly enhanced the accumulation of nanoparticles in types of cancers that area vessel-rich (renal cell carcinoma). This result was completely inconsistent with the generally accepted theory of the enhanced permeability and retention (EPR) effect. We hypothesized that a caveolin-1 (Cav1)-mediated transcellular route would be involved with the penetration of nanoparticles into tumor vasculature. To reveal the exact mechanism responsible for this enhancement, we observed the delivery of long-circulating liposomes (LPs) after Cav1 was co-suppressed by RGD-MEND with VEGFR2. The enhanced delivery of LPs by siRNA against VEGFR2 (siVEGFR2) was accompanied by the elevated expression of the Cav1 protein. In addition, Cav1 knockdown by siRNA against Cav1 (siCav1) canceled the enhanced delivery of LPs by siVEGFR2. The injection of siCav1 had no effect on the formation of alpha smooth muscle actin or vascular endothelial cell adhesion molecules. These results suggest that a Cav1-induced transcellular route and not a paracellular route, at least partially, contributes to the accumulation of nanoparticles in tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Because angiogenesis is a major contributor to cancer progression and metastasis, it is an attractive target for cancer therapy. Although a diverse number of small compounds for anti-angiogenic therapy have been developed, severe adverse effects commonly occur, since small compounds can affect not only tumor endothelial cells (TECs), but also normal endothelial cells. This low selectivity for TECs has motivated researchers to develop alternate types of drug delivery systems (DDSs). In this review, we summarize the current state of knowledge concerning the delivery of nano DDSs to TECs. Their payloads range from small compounds to nucleic acids. Perspectives regarding new therapeutic targets are also mentioned., Competing Interests: The authors declare no conflict of interest.

Introduction : Hyaluronan (HA), a natural polysaccharide, is produced in large amounts by the human body. Since its receptor CD44 is highly expressed in many types of cancers, HA is a promising ligand for cancer-targeting nanoparticles (NPs). Since the enhanced permeability and retention (EPR) effect-based strategy faces difficulties in terms of efficiency in clinical studies, studies focusing on HA-modified NPs that can actively target cancer cells should be prominent for further progress in NP-based cancer treatment. Areas covered : Various materials combined with HA composed of lipid nanoparticles and polymers as well as iron, gold and other metals have been examined. In addition, their cargos have been quite diverse and include small molecule drugs, imaging agents, proteins and nucleic acids. We summarize recent studies on varieties of NPs and describe the properties of HA as a ligand of CD44. Expert opinion : In spite of a number of studies on HA-based NPs, there is few examples of HA-based NPs in clinical. In order to make a progress in clinical study, an evaluation methodology of HA-NPs should be unified and normalized.

Navigating intratumoral drug distribution has proven to be one of the most challenging aspects of drug delivery. The barriers are significant and varied; increased diffusional distances, elevated interstitial fluid pressure, regions of dense extracellular matrix and high cell density, and overall heterogeneity. Such a long list imposes significant requirements on nano-sized carriers. Unfortunately, other capabilities are eclipsed by the distribution requirements. A drug can do no good until it reaches its target. Numerous strategies to improve drug distribution have been developed, taking account of various unique characteristics of solid tumors, including some mechanisms that are still not fully understood. Most of these strategies were from small animal tumor models which are our primary tool for understanding cancer physiology. The small animal tumor model is the most versatile and effective means of understanding tumor transport, but its prevalence belies some of its weaknesses. Tumors grown under lab conditions are developed much more quickly than naturally developed cancers, potentially impacting tumor heterogeneity, blood vessel development, extracellular matrix organization, cell diversity, and many other features of structure and physiology that may impact transport. These problems come in addition to the difficulties of making precise measurements within a living tumor. Resolving these problems is best done by improving our analysis methods, and by finding complementary models that can clarify and expound the details. In this review, we will first discuss some of the strategies employed to improve transport and then highlight some of the new models that have recently been developed in the Bae lab and how they may aid in the study of tumor transport in the future., (Copyright © 2017. Published by Elsevier B.V.)

To date anti-angiogenic therapy has been used for cancer therapy widely, yielding promising results. However, it has been elucidated that current anti-angiogenic drug has several issues to be solved, such as side-effects and drug resistance. It has been reported that tumor endothelial cells (TECs) differ from normal counterparts. In addition, it was shown that the TECs are heterogeneous according to the malignancy status of tumor. The development of novel strategy for targeting tumor vasculature is required. Recently, we have developed an active targeting system, which targets TECs specifically. In this review, we will discuss how TECs in tumor vasculature are heterogeneous and offer new perspectives on a drug delivery system, which can target heterogeneous tumor blood vessels from a viewpoint of personalized medicine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The delivery of SiRNA is not only a challenging strategy for developing new remedies, but is also useful as an analytic tool for an in vivo phenotypic alteration by loss-of-function. Specifically, ligand-mediated SiRNA active targeting can be used to silence any gene in any organ of interest. In this chapter, we describe the preparation of an active targeting system to tumor endothelial cells (TECs) using liposomal SiRNA modified with cyclic RGD peptides. The procedure consists of essentially three steps: (1) the synthesis of a cyclic RGD peptide derivative, (2) SiRNA encapsulation into a liposomal delivery system, and (3) modification of liposomal SiRNA with a cyclic RGD derivative.

Sophisticated drug delivery systems (DDS) are required for delivering drugs, especially macromolecules such as nucleic acids or proteins, to their sites of action. Therefore it is a prerequisite that future DDS are designed to selectively target a tissue. In this review, we focus on systems that actively target the vasculature in tumors or adipose tissues. For targeting tumor vasculatur, a new strategy referred to as dual-targeting is proposed that uses a combination of a receptor specific ligand and a cell penetrating peptide, which can induce the synergistic enhancement of tissue selectivity under in vivo conditions. A novel pH-sensitive cationic lipid was designed to enhance the endosomal release of encapsulated compounds such as siRNA as well as to improve the stability in blood circulation after intravenous administration. A cyclic RGD peptide is used as an active targeting ligand. For targeting adipose vasculature, prohibitin, which is expressed on the surface of adipose endothelial cells, was targeted with KGGRAKD peptides on the surface of PEGylated nanoparticles. Prohibitin targeted nanoparticles (PTNP) encapsulating Cytochrome c (CytC) can selectively target adipose vasculature by optimizing the lengths of the PEG linkers and can deliver CytC to adipose endothelial cells. PTNP can successfully induce anti-obese effects as well as apoptosis by delivering CytC to the cytosol in endothelial cells. Unexpectedly, the EPR (enhanced permeability and retention) effect, which is usually observed in tumor tissue, was also observed in the adipose vasculature, especially in obese mice, where PEGylated nanoparticles can pass through the endothelial barriers in adipose tissue. We believe that these achievements in active targeting will allow a greatly expanded use of DDS for nanomedicines.

Introduction: Data reported during the last decade of the twentieth century indicate that passive targeting is an efficient strategy for delivering nanocarrier systems to tumor tissues. The focus of this review is on active targeting as a next-generation strategy for extending the capacity of a drug delivery system (DDS)., Areas Covered: Tumor vasculature targeting was achieved using arginine- glycine-aspartic acid, asparagine-glycine-arginine and other peptides, which are well-known peptides, as ligand against tumor vasculature. An efficient system for delivering small interfering RNA to the tumor vasculature involved the use of a multifunctional envelope-type nanodevice based on a pH-modified cationic lipid and targeting ligands. The active-targeting system was extended from tumor delivery to adipose tissue delivery, where endothelial cells are tightly linked and are impermeable to nanocarriers. In mice, prohibitin-targeted nanoparticles can be used to successfully deliver macromolecules to induce anti-obese effects. Finally, the successful delivery of nanocarriers to adipose tissue in obese mice via the enhanced permeability and retention-effect is reported, which can be achieved in tumor tissue., Expert Opinion: Unlike tumor tissues, only a few reports have appeared on how liposomal carriers accumulate in adipose tissues after systemic injection. This finding, as well as active targeting to the adipose vasculature, promises to extend the capacity of DDS to adipose tissue. Since the site of action of nucleic acids is the cytosol, the intracellular trafficking of carriers and their cargoes as well as cellular uptake must be taken into consideration.

Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to αVβ3 integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy., (© 2013.)

We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer-polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured Kd value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature.

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.