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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.

Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.

Messenger RNA and small interfering RNA are attractive modalities for curing diseases by complementation or knock-down of proteins. For success of these RNAs, a drug delivery system (DDS) is required to control a pharmacokinetics, to enhance cellular uptake, to overcome biological membranes, and to release the cargo into the cytoplasm. Based on past research, developing nanoparticles that are neutrally charged have been the mainstream of their development. Also, the materials are further mounted with pH- and/or reducing environment-responsive units. In this review, we summarize progress made in the molecular design of these materials. We also focus on the importance of the hydrophobic scaffold for tissue/cell targeting, intracellular trafficking, and immune responses. As a practical example, the design concept of the SS-cleavable and pH-activated lipid-like material (ssPalm) and subsequent molecular modification tailored to the RNA-based medical application is discussed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The tumor microenvironment plays a key role in cancer progression, drug resistance, metastasis, etc. To establish a new therapeutic strategy based on control of the tumor microenvironment, I have developed a lipid nanoparticle (LNP)-based in vivo small interfering RNA (siRNA) delivery system equipped with a targeting ligand. First, I established an LNP that induces membrane fusion in response to acidification after internalization by cells using the original pH-sensitive cationic lipid YSK05. A modification of polyethylene glycol to YSK05-containing LNPs allowed significant gene silencing in the human renal cell carcinoma model. Then, I attempted to establish a tumor vasculature-targeting LNP because the vasculature is responsible for the tumor microenvironment. Cyclic RGD peptide is known to be a ligand against integrin αVβ3, which is highly expressed on tumor endothelial cells (TECs). Optimized cyclic RGD peptide-modified LNP (RGD-LNP) suppressed gene expression in TECs to 50%. The inhibition of vascular endothelial cell growth factor receptor 2 (VEGFR2), which is a dominant factor in angiogenesis, by the injection of RGD-LNP significantly delayed tumor growth. Finally, I examined the effect of RGD-LNP on the tumor microenvironment. The suppression of VEGFR2 increased pericyte coverage and endothelial junctions, which indicate maturation of the vasculature. In RGD-LNP-treated mice, systemically administered nanoparticles encapsulating doxorubicin were distributed in a larger area than in untreated mice. Moreover, the therapeutic effect of doxorubicin-loaded liposomes was significantly enhanced by RGD-LNP. In conclusion, I succeeded in developing a new therapy based on regulation of the tumor microenvironment.

Although anti-angiogenic therapy is predicted to be an effective therapy for treating cancer, selectively targeting tumor endothelial cells (TECs), and not normal endothelial cells, remains a major obstacle. Modifying a drug carrier with a targeting ligand is a popular strategy for developing an active-targeting type drug delivery system (DDS). We previously reported that a cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD)-equipped liposome that contains encapsulated siRNA (RGD-MEND) achieved an efficient therapeutic outcome in a murine cancer model. To develop a more efficient TEC-targeting DDS, we examined the effect of the length of the polyethylene glycol (PEG) that is used as a peptide-linker on the cholesterol-scaffold, and liposomal composition on the efficiency of delivery of siRNA to cRGD receptor α V β 3 integrin positive cells. An RGD-MEND modified with shorter linker/no-linker, PEG350 or no-PEG, showed a higher cellular uptake in vitro. However, a shorter or no-linker RGD-cholesterol-modified MEND showed no silencing effect despite its high, in vitro silencing efficiency. To examine the possibility that the cholesterol-scaffold ligand was removed from the surface of the RGD-MEND by interactions with serum proteins, the RGD-MEND was incubated in the presence of a 50% serum solution. The cellular uptake of the cholesterol-scaffold ligand was drastically reduced by the incubation in serum. Increasing the cholesterol ratio in the lipid envelope and adding a helper lipid improved the in vivo knockdown efficiency, probably due to an enhanced ligand retention, even in in vivo conditions. The findings reported herein suggest that the lipid composition and the ligand scaffold of the MEND are major factors in successfully developing an efficient active-targeting DDS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Plasmid DNA (pDNA) is expected to be a new class of medicine for treating currently incurable diseases. To deliver these nucleic acids, we developed a liposomal delivery system we have called a multifunctional envelope-type nano device (MEND). In this report, we demonstrate that a MEND containing a pH-sensitive cationic lipid, YSK05 (YSK-MEND), efficiently delivered pDNA via systemic injection, and that its expression was highly dependent on the encapsulation state of the pDNA. In the preparation, the pH, ionic strength, and sodium chloride (NaCl) concentration of the lipid/pDNA mixture strongly affected the encapsulation efficiency of pDNA. Additionally, the transgene expression of luciferase in the liver by the injected YSK-MEND was dependent on the encapsulation state of pDNA rather than the nature of the YSK-MEND. Confocal laser scanning microscopy findings revealed that injection of the YSK-MEND led to homogenous gene expression in the liver compared to injection via the hydrodynamic tail vein (HTV). Concerning the safety of the YSK-MEND, a transient increase in the activity of liver enzymes was observed. However, no significant adverse events were observed. Taken together, the YSK-MEND represents a potentially attractive therapy for the treatment of various hepatic diseases.

Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing., (Copyright © 2012 Elsevier B.V. All rights reserved.)

RNA vaccines are applicable to the treatment of various infectious diseases via the inducement of robust immune responses against target antigens by expressing antigen proteins in the human body. The delivery of messenger RNA by lipid nanoparticles (LNPs) has become a versatile drug delivery system used in the administration of RNA vaccines. LNPs are widely considered to possess adjuvant activity that induces a strong immune response. However, the properties of LNPs that contribute to their adjuvant activity continue to require clarification. To characterize the relationships between the lipid composition, particle morphology, and adjuvant activity of LNPs, the nanostructures of LNPs and their antibody production were evaluated. To simply compare the adjuvant activity of LNPs, empty LNPs were subcutaneously injected with recombinant proteins. Consistent with previous research, the presence of ionizable lipids was one of the determinant factors. Adjuvant activity was induced when a tiny cholesterol assembly (cholesterol-induced phase, ChiP) was formed according to the amount of cholesterol present. Moreover, adjuvant activity was diminished when the content of cholesterol was excessive. Thus, it is plausible that an intermediate structure of cholesterol (not in a crystalline-like state) in an intra-particle space could be closely related to the immunogenicity of LNPs. [ABSTRACT FROM AUTHOR]

Multiple sclerosis is a disease caused by autoantigen-responsive immune cells that disrupt the myelin in the central nervous system (CNS). Although immunosuppressive drugs are used to suppress symptoms, no definitive therapy exists. As in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis, a partial sequence of the myelin oligodendrocyte glycoprotein (MOG35–55) was identified as a causative autoantigen. This suggests that the induction of immune tolerance that is specific to MOG35–55 would be a fundamental treatment for EAE. We previously reported that lipid nanoparticles (LNPs) containing an anionic phospholipid, phosphatidylserine (PS), in their lipid composition, can be used to deliver mRNA and that this leads to proteins of interest to be expressed in the spleen. In addition to the targeting capability of PS, PS molecules avoid activating the immune system. Physiologically, the recognition of PS on apoptotic cells suppresses immune activation against these cells by releasing cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β that negatively regulate immunity. In this study, we tested whether mRNA delivery of autoantigens to the spleen by PS-LNPs causes the expression of MOG35–55 antigens with minimal immune stimulation and whether this could be used to treat an EAE model by inducing immune tolerance. [ABSTRACT FROM AUTHOR]

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs. [Display omitted] [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) are responsible for hepatic fibrosis and liver cirrhosis via their ability to produce extracellular matrices such as collagens and elastin. However, a strategy for delivering cargoes to HSCs has not been established yet. We herein report on attempts to deliver small interfering RNA (siRNA) to HSCs using several types of SS-cleavable proton-activated lipid-like materials (ssPalms) that contained myristic acid (ssPalmM) or hydrophobic vitamin A (ssPalmA) and E (ssPalmE) as hydrophobic scaffolds. We initially verified that hepatic fibrosis could induce the treatment with tetrachloromethane in terms of collagen fibrils and the expression of marker genes, type I collagen α-1, transforming growth factor β, heat shock protein 47, and α-smooth muscle actin. The siRNA silencing efficiency of the 3 LNPs was then compared using fibrosis-induced mice. Of the materials tested, LNP ssPalmA showed the highest efficiency, with an effective (ED)50 of approximately 0.25 mg siRNA/kg. The LNP ssPalmA showed a significant inhibitory effect on collagen production at a dose of 3.0 mg siRNA/kg with no evidence of any severe adverse effects. In conclusion, LNP ssPalmA holds considerable potential for use in the treatment of HSCs-mediated diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: siRNA delivery to tumor endothelial cells was achieved using arginyl-glycyl-aspartic acid (RGD)-modified lipid nanoparticles containing a novel pH-sensitive and biodegradable lipid. The anti-tumor efficacy of an immune checkpoint inhibitor was improved by the silencing of VEGFR2 using the delivery system, because the combination therapy induced vascular normalization and increased CD8+ T cell infiltration into tumors. The efficient delivery of nucleic acids is a promising strategy to improve therapeutic outcomes in immune checkpoint inhibitor-resistant cancers. Despite the promising anticancer effects of immune checkpoint inhibitors, their low objective response rate remains to be resolved; thus, combination therapies have been investigated. We investigated the combination of an anti-programmed cell death 1 (aPD-1) monoclonal antibody with the knockdown of vascular endothelial factor receptor 2 (VEGFR2) on tumor endothelial cells to overcome resistance to immune checkpoint inhibitors and improve the objective response rate. The successful delivery of small interfering RNA to tumor endothelial cells was achieved by RGD peptide-modified lipid nanoparticles composed of a novel, pH-sensitive, and biodegradable ssPalmO-Phe. RGD-modified lipid nanoparticles efficiently induced the knockdown of VEGFR2 in tumor endothelial cells (TECs), which induced vascular normalization. The combination of a PD-1 monoclonal antibody with Vegfr2 knockdown enhanced CD8+ T cell infiltration into tumors and successfully suppressed tumor growth and improved response rate compared with monotherapy. Our combination approach provides a promising strategy to improve therapeutic outcomes in immune checkpoint inhibitor-resistant cancers. [ABSTRACT FROM AUTHOR]