key: cord-0715694-vrl0jf8u
authors: Yamada, Yuma; Sato, Yusuke; Nakamura, Takashi; Harashima, Hideyoshi
title: Evolution of drug delivery system from viewpoint of controlled intracellular trafficking and selective tissue targeting toward future nanomedicine
date: 2020-09-08
journal: J Control Release
DOI: 10.1016/j.jconrel.2020.09.007
sha: ab57641a9a60a7806e2dfd86641dcbd3d36ce248
doc_id: 715694
cord_uid: vrl0jf8u

Due to the rapid changes that have occurred in the field of drug discovery and the recent developments in the early 21st century, the role of drug delivery systems (DDS) has become increasingly more important. For the past 20 years, our laboratory has been developing gene delivery systems based on lipid-based delivery systems. One of our efforts has been directed toward developing a multifunctional envelope-type nano device (MEND) by modifying the particle surface with octaarginine, which resulted in a remarkably enhanced cellular uptake and improved intracellular trafficking of plasmid DNA (pDNA). When we moved to in vivo applications, however, we were faced with the PEG-dilemma and we shifted our strategy to the incorporation of ionizable cationic lipids into our system. This resulted in some dramatic improvements over our original design and this can be attributed to the development of a new lipid library. We have also developed a mitochondrial targeting system based on a membrane fusion mechanism using a MITO-Porter, which can deliver nucleic acids/pDNA into the matrix of mitochondria. After the appearance of antibody medicines, Opdivo, an immune checkpoint inhibitor, has established cancer immunology as the 4th strategy in cancer therapy. Our DDS technologies can also be applied to this new field of cancer therapy to cure cancer by controlling our immune mechanisms. The latest studies are summarized in this review article.

We initiated our gene delivery research project in 2000 at Hokkaido University by focusing on the delivery of plasmid DNA (pDNA). By introducing cell penetrating peptides, especially octaarginine (R8) into a system, cellular uptake as well as endosomal escape was increased efficiently [1] and a multifunctional envelope-type nano device (MEND) modified with R8 induced transfection activities as high as that for the adenovirus in tumor cell lines [2] . To apply the R8-MEND for in vivo gene delivery, we shielded the positive charge of R8 with polyethyleneglycol (PEG), which improved the in vivo pharmacokinetics of the R8-MEND, resulting in an enhanced tumor delivery as well as an extended circulation time, however, the transfection activities of the R8-MEND were greatly decreased in tumor tissue, causing us to face the PEG-dilemma [3] . To clarify this problem, we proposed a new strategy for selectively cleaving PEG from the MEND in tumor tissue, and this resulted in an enhanced transfection activity of PPD-MEND [4] .

Due to a Nobel Prize for RNA interference that was awarded in 2006, gene delivery research has shifted from pDNA to short interfering RNA (siRNA), since siRNA medicine can be classified not as gene therapy in which the regulatory process requires much less time compared to that for gene therapy. We also shifted our focus from pDNA delivery to siRNA delivery and we improved the MEND by introducing GALA to overcome the PEG-dilemma, which succeeded to enhancing gene silencing in tumor tissue [5] . In 2010, a breakthrough technology appeared when it was found that the use of a pH-sensitive (ionizable) cationic lipid, a cationic lipid as DLin-KC2-DMA, resulted in an enhanced in vivo gene silencing efficiency of a marker gene in the liver by two orders of magnitudes (50% effective dose (ED 50 ): 0.02 mg siRNA/kg from 2 mg siRNA/kg) from the original compound DLin-DMA [6] . Based on our analysis, the endosomal escape efficiency of the We also developed a MITO-Porter to deliver encapsulated compounds to mitochondria via a membrane fusion mechanism. R8 played an important role in enhancing cellular uptake as well as in mitochondrial targeting. We then examined the targeting of a small molecular drug (propidium iodide), a peptide (mastoparan), a protein (DNase), to mitochondria however it was difficult to prove that we successfully delivered the compounds into the matrix of mitochondria [9] . We then applied an antisense oligo nucleotide to knock down gene expression in mitochondria, because silencing can only be detected when an oligo nucleotide is delivered to the mitochondrial matrix [10] . At that time, there was no reporter gene available for mitochondria gene delivery, but we have many reporter genes for the nucleus such as luciferase, green fluorescent protein (GFP), etc.

We were waiting for the development of a reporter gene for mitochondria, but it did not appear. Therefore, we decided to develop it by ourselves and how we succeeded in developing mitochondrial reporter gene is described in this article. In addition, recent progress in mitochondrial delivery technology for translational studies are also explained.

In 2014, Opdivo was approved in Japan for use as an antibody drug for the treatment of melanomas and was subsequently extended to lung cancer, renal cell carcinoma, Hodgkin's lymphoma, etc. Professor Tasuku Honjo was awarded a Nobel Prize in 2018, along with James Patrick Allison for his work on the mechanism of immune checkpoint inhibition. The

Nobel Prize as well as the clinical success of cancer immunotherapy by immune checkpoint inhibitors established cancer immunotherapy as the 4 th strategy. We are also in the process of developing nano devices for cancer immunotherapy, since a DNA vaccine was the first goal of our gene delivery research because we thought it may be the easiest way to reach the goal for gene therapy. We initially focused on delivering pDNA to dendritic cells (DC). However, DC are completely different from tumor cell lines and we thought that the nuclear membrane might be a key step in developing DNA vaccines, since tumor cell lines frequently divide while DC do not. We developed a T-MEND [11] to overcome this barrier and succeeded in developing a KALA-MEND to transfect DC, antigen presentation,

cytotoxic T-cell induction and antitumor activity under ex vivo conditions [12] . We attempted to find a way to apply the KALA-MEND in vivo, however, the KALA-MEND

Disease phenotypes of various diseases including genetic diseases, infectious diseases, and cancer, may be derived from abnormal genetic status including upregulation, downregulation, mutation and the deletion of the responsible genes [13] [14] [15] [16] . These therapeutic targets are sometimes resistant to treatment by small molecule drugs [17] . [18] , and can suppress the expression of a specific gene through RNA interference (RNAi), which was initially discovered by Fire and Mello in C. elegans in 1998 [19] . Many efforts have been made to apply siRNAs for therapeutics to date [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . In 2018, only 20 years from the discovery of RNAi, Patisiran (ONPATTRO TM ), a treatment for familial amyloid polyneuropathy (FAP) caused by mutations in the transthyretin (TTR) gene, was approved by the U.S. Food and Drug Administration as a first-ever RNAi medicine [30] . Patisiran is also a first-ever approved lipid nanoparticle (LNP) containing therapeutic RNA. Patisiran rapidly accumulates in liver tissue and is taken up in hepatocytes, which mainly produce TTR proteins, by endocytosis upon

The liver is the primary target of RNA-loaded LNPs because it is estimated that genetic disorders in hepatocytes, parenchymal cells in liver tissue are the cause of thousands of human diseases [31] . [38] . They showed a 3.6-fold faster blood clearance and over a 20-fold higher accumulation in hepatocytes of neutral liposomes in wild-type mice compared to ApoE-deficient mice, which was not observed for negatively-charged liposomes. In a that ionizable amino lipids composed of linear saturated scaffolds or a constrained adamantane scaffold have the ability to avoid accumulation in hepatocytes, indicating that the ApoE-mediated pathway is lacking [40, 41] . Further investigations will be needed to develop a precise understanding of the relationships between chemical structure requirements and ApoE-mediated accumulation to hepatocytes. Akinc et al. also showed that decoration of N-acetyl-D-garactosamine (GalNAc) clusters on the surface of the siRNA-loaded iLNPs restored gene silencing in hepatocytes in ApoE-deficient mice or LDLR knockout mice. It is known that GalNAc and galactose are recognized by asialoglycoprotein receptors (ASGPRs), which are expressed in hepatocytes [42] . Due to following impressive features of ASGPRs, decorating GalNAc on nanoparticles is an alternative strategy for targeting hepatocytes [43] [44] [45] : 1) a specific and high (~500,000 molecules per individual cell) expression in hepatocytes, 2) a rapid (~15 min) recycling time. It has also been reported that decorating GalNAc on siRNA-loaded LNPs contributed to improving hepatocyte-specificity and reduced cationic lipid-associated hepatic toxicity which is caused by the off-target accumulation of the LNPs to LSECs and activation of the same cell type followed by neutrophil-mediated inflammation [46] .

GalNAc-ASGPR (exogenous) pathway are useful for efficiently delivering RNAs to hepatocytes so far. However, little was known regarding the kinetics of the delivery process including blood clearance, hepatic accumulation and cellular uptake in hepatocytes between the endogenous and exogenous targeting mechanisms. We recently investigated this using siRNA-loaded LNPs, the surfaces of which were modified differently so as to modify the mechanism for hepatocyte targeting ( Fig. 1 ) [47] . The blood half-life of unmodified (bare) LNPs that target hepatocytes through the endogenous mechanism was only 1.8 min, which was much (approx. 18-fold) faster than that of GalNAc/Shielded LNPs (modified with both GalNac and PEG) that target hepatocytes though the exogenous mechanism. The bare LNPs accumulated rapidly in liver tissue within 10 min upon intravenous administration, were located in or near LSECs for approximately 10 min, and were then endocytosed 30 min after the administration. Electron microscopic observations revealed that most of the LNPs were endocytosed by hepatocytes in wild-type mice. The ApoE is known to bind to heparan sulfate proteoglycans (HSPGs) as well as LDLR through the arginine-rich receptor-binding domain [48] . Treatment with heparin, which can compete with HSPGs, even 10 min after administration of the bare LNPs significantly suppressed both the accumulation and gene silencing of the LNPs in hepatocytes, suggesting that HSPGs in the liver are involved in the rapid accumulation of the bare LNPs. The relatively slow uptake of the bare or GalNAc/Shielded LNPs could be explained by assuming that receptors are mainly distributed on microvilli but not on coated pits and therefore a certain time would be required for the particles to migrate to coated pits for internalization [49, 50] .

The endosomal escape process is now recognized as the rate-limiting step for the cytosolic delivery of RNAs [51] . LNPs that contain ionizable amino lipids are known be processed through membrane fusion following electrostatic interaction with the negatively charged endosomal membranes in acidified endosomes. In this step, phase transition of endosomal membranes from a lamellar (L) to an inverted hexagonal (H II ) phase is required for successful membrane fusion [52] . Heyes et al. reported that the presence of cis-unsaturated bond(s) in hydrophobic scaffolds of ionizable cationic lipids are important for facilitating membrane fusion and for inducing the cytosolic delivery of siRNAs [53] .

The unsaturated bond can allow complexes between ionizable cationic lipids and anionic lipids to adopt a cone-shape, which can disrupt the bilayer structure and thus facilitate membrane fusion. The subsequent rational design of linker structures between hydrophilic head groups and hydrophobic scaffolds of ionizable amino lipid successfully maximized their fusogenic activity and the cytosolic delivery of siRNA [6, 54] . The attachment of two hydrophobic scaffolds from a single carbon atom is currently adopted as a linker of the ionizable amino lipids. Jayaraman et al. screened ~100 types of ionizable amino lipids and found DLin-MC3-DMA (MC3) to be the most potent lipid with an ED 50 of 0.005 mg/kg in a mouse factor 7 (F7) model, which was approximately a 100-fold higher efficiency

We recently developed an original library of ionizable amino lipids and identified the most potent lipid for the hepatic delivery of siRNAs, referred to as CL4H6 [41] . The CL4H6 lipid contains two very long hexyl oleate chains (C24+O1) ( Fig. 2A) . The CL4H6-LNPs exhibited an ED 50 of 0.0025 mg/kg in a mouse F7 model. We found that 4.2% of the siRNA was loaded in the RNA-induced silencing complex (RISC) at 24 hours after intravenous administration, suggesting that endosomal escape was better compared to MC3. The CL4H6 also exhibited a better tolerability compared to MC3-LNPs due to the fact that the CL4H6 is biodegradable. It is known that phosphocholine (PC)-containing neutral lipids, including phosphatidylcholines and sphingomyelins (SMs), are cylindrical-shaped and therefore stabilize bilayers and strongly inhibit membrane fusion [52, 57] . We revealed that the long hexyl oleate scaffolds can overcome the inhibitory effect of 

The functional breakdown of mitochondria can affect various functions and cause a variety of diseases. It has been reported that mutations / deletions in mitochondrial DNA (mtDNA) cause to the onset of some of these diseases [59, 60] . Therefore, research focusing on mitochondrial gene therapy is expected to lead to the development of innovative medicines. This section focuses on our research efforts on "mitochondrial drug delivery system (DDS) for mitochondrial RNA therapy". Research directed toward the development of nanomedicines based on MITO-Porter technology includes fields such as mitochondrial gene therapy [77] [78] [79] , cancer therapy [80] [81] [82] [83] [84] , ischemic diseases therapy [85, 86] and cell therapy [87] . Our research outcomes regarding validating a mitochondrial gene therapeutic strategy are summarized in the following section, with a particular focus on mitochondrial RNA therapy.

Mitochondrial genomic abnormalities such as the accumulation of mtDNA mutations would be predicted to cause the onset of various types of diseases. Mitochondrial genetic material including mtDNA, mitochondrial RNA (mRNA, tRNA, rRNA) is concentrated inside the mitochondrial matrix, that is tightly enclosed by double mitochondrial membranes. Delivery of therapeutic cargoes into the mitochondrial matrix is needed to successfully achieve gene therapy targeting mitochondria. Our previous findings showed that the MITO-Porter achieved mitochondrial matrix delivery [88] .

It is also noteworthy that therapeutic macromolecules such as nucleic acids can be efficiently packaged in the carrier. Controlling mitochondrial function is necessary for J o u r n a l P r e -p r o o f sufficient amounts of cargoes to be delivered to mitochondria. To date, we succeeded in efficiently packaging nanoparticles of therapeutic nucleic acids with polycations in the MITO-Porter. These cargoes include circular DNA [89, 90] , an antisense RNA oligonucleotide (ASO) [91, 92] , tRNA [93] and mRNA [94, 95] . In the following, we summarize our efforts in mitochondrial gene therapy based on the MITO-Porter technology.

With the goal of achieving gene therapy targeting mitochondria, we have mainly The membrane potential of mitochondria was also evaluated using the JC-1 dye.

Using JC-1, when the membrane potential was decreased, a green fluorescence is observed in the cytoplasm, red colored mitochondria are observed in the case where cells possess normal mitochondria with a membrane potential (Fig. 4C (a) ) [91] . In cells where the mitochondrial transfection of ASO was performed, a green fluorescence was observed in the cytoplasm (Fig. 4C (b) ). These findings suggest that the direct mitochondrial transfection of ASO could control mitochondrial functions via mitochondrial knock down. The next topic is the validation of the mitochondrial RNA therapeutic strategy targeting diseased cells. This study was initiated as an independent clinical research "Study for establishment of a drug treatment for a mitochondrial disease" and was a collaborative effort between three facilities, namely, the Faculty of Pharmaceutical Sciences in Hokkaido University, Hokkaido University Hospital and Sapporo City General Hospital. As the first validation, we used G625A cells obtained from a patient with a mitochondrial disease, who possesses a large amount of mtDNA with a G625A point mutation located in the region coding for tRNA Phe (heteroplasmic mutation) [101] . The mutation rate of the mtDNA is 80%, the mitochondrial complex III activity is reduced, and the mitochondrial membrane potential / ATP production ability is reduced.

As shown in Figure 5A , we carried out the mitochondrial transfection of wild-type mitochondrial tRNA Phe (WT-tRNA) into G625A cells that carried the mutant tRNA Phe (MT-tRNA) using the MITO-Porter system, in an attempt to reduce the ratio of the MT-tRNA content in mitochondria. The quantification of the content ratio indicated that a decrease in the content ratio of MT-tRNA in mitochondria was observed when the mitochondrial transfection of WT-tRNA was performed, whereas the transfection of the control sequence had no effect on the content ratio (Fig. 5B) . Furthermore, mitochondrial function (mitochondrial respiratory activity) after the mitochondria transfection of the therapeutic RNA was significantly improved compared to that of control RNA (Fig. 5C ) [93] . As the next challenge, we verified a gene therapy strategy that targets a disease 

In this section, we focus on our research dealing with RNA therapy targeting mitochondria. In the near future, we plan to initiate a research project for "Drug discovery targeting mitochondria". As part of this activity, we will participate in the drug discovery project "7 SEAS PROJECT", the aim of which is to conduct research and development for 

Immunotherapy is now well established in the field of cancer therapy based on the success of immune checkpoint inhibitors (ICIs). However, the clinical benefits of ICIs are limited in minor portion of cancer patient. To overcome this problem, combination therapies of ICIs with other types of therapies have been investigated. In this situation, the demand for nano DDS technology has been increasing.

Our immune system protects us from cancer invasion, which is represented by a series of spatiotemporal events, namely the Cancer-Immunity Cycle [102] . Cancer cell derived antigens (neoantigens) are recognized by DCs, followed by DC maturation and antigen presentation. The mature DCs then move to the lymph nodes (LNs) and prime T

The ICIs such as the programmed cell death 1 (PD-1) and the programmed cell death ligand

That is, PD-1/PD-L1 antibodies improve the latter steps after T cell activation. Therefore, therapies that can effectively induce T cell activation should be suitable candidates for such combination therapies.

The priming of T cells by mature DCs is essential for inducing effective T cell activation. Antigens and adjuvants are often employed to enhance this process. Antigens are supplied in various ways such as proteins, peptides, mRNA and DNA [103] . In any case, since they are easily degraded, when used naked, they would not be expected to be effective. Various types of adjuvants are currently available, but agonists of innate immune receptors such as toll-like receptors (TLRs) have been used most recently [104] . Most of the agonists are also unstable in the body or are insoluble, because they are nucleic acids or lipid components. Furthermore, the drawbacks associated with the use of antigens and adjuvants significantly reduce the efficiency of their delivery to target cells and tissues. Therefore, the use of a nano DDS technology should be a promising strategy for their efficient delivery [105] . We have been developing lipid-based nano DDS such as liposomes and LNPs for delivering antigens and adjuvants [76, 105, 106] Delivering antigens to the cytosol in APCs is essential for MHC-I antigen presentation, namely cross-presentation. The use of a combination of the R8 peptide, a cell-penetrating peptide, and fusogenic liposomes would allow efficient endosomal escape to occur, resulting in the cytosolic delivery of antigens [108] . An ovalbumin (OVA) loaded R8 peptide modified liposome (R8-Lip/OVA) was found to induce MHC-I specific antigen presentation in mouse DCs. Interestingly, the enhancement in the C-terminal trimming of the antigen peptide by the R8-Lip contributes to the efficient MHC-I antigen presentation [109] . Furthermore, the co-encapsulation of polyinosinic-polycytidylic acid (polyI:C), a TLR3 and melanoma differentiation-associated gene 5 (Mda5) agonist, into the R8-Lip/OVA drastically enhanced the OVA-specific CTL response and antitumor activity against E.G7-OVA tumor [110] . The effect of polyI:C appeared to be maximized by its CD1 molecules present lipid antigens that are different from MHC molecules, leading to T cell activation and the activation of natural killer T (NKT) cells [111] .

Glycolipids derived from tuberculosis are well-known lipid antigens that are presented on CD1 molecules, but the insolubility of these molecules in aqueous media has hampered research progress in this area and their application to tuberculosis vaccines. To overcome this problem, we incorporated glycolipids such as glucose monomycolate (GMM) and glycerol monomycolate (GroMM) into the lipid membrane of the R8-Lip, resulting in the water-dispersion and efficient uptake by APCs [112, 113] . We demonstrated that GMM and GroMM are potent target antigens for T cell responses against tuberculosis by using GMM and GroMM loaded R8-Lips, and the GMM and GroMM loaded R8-Lips were found to be useful as tuberculosis vaccines in rhesus macaques and guinea pigs [112] [113] [114] [115] . On the other hand, alpha-galactosylceramide (αGC) is a lipid antigen that is presented on CD1d molecules and is expected to act as an adjuvant for NKT cell activation [116] . We also J o u r n a l P r e -p r o o f incorporated αGC into the lipid membrane of the R8-Lip [117] . The αGC-loaded R8-Lip drastically enhanced αGC presentation on CD1d in APCs, the activation of NKT cells and exerted antitumor effect in a B16-F10 melanoma lung metastasis mouse model, compared with solution types of αGC. These findings suggest that the R8-Lip represents a potent lipid-based nano DDS for delivering lipid antigens.

Since mycobacterium bovis Bacille Calmette-Guerin (BCG) contains various antigens and adjuvants, BCG is a very successful immunotherapeutic drug. In addition to its use as a tuberculosis vaccine, BCG is intravesically infused in non-muscle invasive bladder cancer patients. The therapeutic rate is more than 70% and is significantly higher than that of ICIs [118, 119] . However, patients suffer from a high frequency of serious side effects due to the use of the live mycobacterium [120] . In this situation, the BCG cell wall skeleton (BCG-CWS) is potent candidate for use instead of BCG, because it is the main immune active component in the BCG drug [121] . Clinical applications of BCG-CWS have, however, been limited due to the huge molecular size and the insolubility of the cell wall material in both aqueous solutions and organic solvents. To overcome the unfavorable properties of BCG-CWS, we encapsulated BCG-CWS into an R8-modified LNP (CWS-LNP) by the liposome evaporated via emulsified lipid (LEEL) method [122] . The CWS-LNP is a nano-sized particle formulation and is a highly homogenous dispersion in water. Treatment with the CWS-LNP induces strong antitumor responses in MBT-2 (mouse bladder cancer) tumor mouse model, a naturally developed balder cancer rat model, and human immune cells. A study using an MBT-2 tumor mouse model indicates that the antitumor immune responses against bladder cancer by the CWS-LNP is initiated by the internalization of the CWS-LNP into bladder cancer cells, but not APCs [123] . This mechanism appears to be similar to that for BCG-mediated antitumor immunity. Thus, we applied the CWS-LNP to a systemic cancer adjuvant, because, unlike BCG, the CWS-LNP is non-infectious (Fig. 6A) . The intravenous administration of CWS-LNP enhanced the induction of OVA-specific CTL and the growth suppression of E.G7-OVA tumor [124] . On the other hand, a biochemical analysis in blood after the intravenous administration of CWS-LNP and an evaluation of weight change during the immunization of CWS-LNP suggest that the CWS-LNP caused no side effects. Furthermore, an investigation of the distribution of the CWS-LNP in the spleen after the intravenous administration revealed that the CWS-LNP is mainly taken up by B cells and a part of CWS-LNP is taken up by DCs. Interestingly, the internalization of CWS-LNP by DCs, but not B cells may contribute J o u r n a l P r e -p r o o f to the effective induction of CTL [125] . Collectively, the CWS-LNP can be expected to be applied not only as an immunotherapeutic drug against bladder cancer, but also as a systemically administered cancer adjuvant.

Nucleic acids are a satisfactory modality that functions as antigens, adjuvants and controllers at the gene level. DNA and cyclic dinucleotides (CDNs) are recognized by cyclic GMP-AMP synthase (cGAS), the stimulator of interferon genes (STING) pathway, and also function as adjuvants [126] . Stimulation of the cGAS-STING pathway induces the production of type I interferons (IFNs) and proinflammatory cytokines. We previously demonstrated the potential of DNA or CDN loaded LNPs in cancer immunotherapy [127] [128] [129] . Since the cGAS-STING pathway is operative in the cytosol, the agonists must be delivered to the cytosol. We encapsulated a cyclic di-GMP (c-di-GMP), a type of CDNs, into the YSK05-LNP for enhancing the cytosolic delivery of c-di-GMP [127] . The c-di-GMP-loaded YSK05-LNP (cdGMP/YSK05-LNP) induced higher type I IFN production than commercially available transfection reagents in a macrophage cell line. In addition, the subcutaneous administration of the cdGMP/YSK05-LNP with OVA induced an OVA-specific CTL response and a strong inhibition of E.G7-OVA tumor growth. On the other hand, a cdGMP/YSK05-LNP treatment strongly activated NK cells, resulting in the therapeutic effect in a B16-F10 melanoma lung metastasis [128] . B16-F10 cells are nearly lacking in MHC-I molecules. These findings suggest that the cdGMP/YSK05-LNP could show antitumor activity against not only immunogenic tumors, but also tumors that escape from CTL killing. In addition to cancer immunotherapy, the strong production of systemic type I IFNs by the cdGMP/YSK05-LNP may show therapeutic effects against virus pneumonia such as COVID-19 pneumonia, because inflammatory type 2 conventional DCs that optimally prime T cell responses in the lung are generated by type I IFNs [130] .

The regulation of gene expression in immune cells by siRNA appears to be a useful method for controlling complicated cancer immune responses. However, it is well known that it is difficult to introduce siRNA to immune cells using non-viral vectors. We previously developed siRNA-loaded LNPs that target various immune cells [131] [132] [133] [134] [135] [136] . An 136] . Moreover, mouse DCs that contain immune suppressive genes such as the suppressor of cytokine signaling 1 (SOCS1) and indoleamine 2,3-dioxygenase 1 (IDO1) that were silenced by the YSK12-LNP resulted in a significantly strengthened antitumor effect in DC-based therapy [133, 136] . In particular, IDO1 is intimately associated with the formation of immune suppressive tumor microenvironments, leading to a poor prognosis of cancer patients [137, 138] . Because the production of IDO1 results in the inhibition of effector T cells, the induction of regulatory T (Treg) cells and the activation of myeloid-derived suppressor cells (MDSCs) [139] . Thus, IDO1 is a potent target for a combination therapy with ICIs. In our study, the treatment of IDO1-silenced DCs by the YSK12-LNP appeared to be accompanied by a decrease in the Treg cell population in tumor tissue (Fig. 6B ) [136] . We conclude that the YSK12-LNP can be potent nano DDS for siRNA delivery to immune cells.

Given the Cancer-Immunity Cycle, LNs are central organs for initiating T cell responses against tumors [102] . Thus, LNs are attractive delivery targets for cancer immunotherapy. For direct delivery via the lymphatic system by using nano DDS, particles size is a critical factor and the suitable size appears to be in the range from 20 nm to 50 nm [140] [141] [142] . An important technology in the success of the Patisiran preparation is microfluidic mixing. Microfluidic devices are used for this purpose, since they can continuously mix a lipid solution and a drug solution, resulting in manufacturing on an industrial scale [143] [144] [145] . Moreover, the continuous rapid mixing with microfluidic devices can generate LNPs with sizes below 30 nm [143, 146, 147] . It therefore appears that LNP technology will be increasingly applied to the field of LN targeting.

We recently reported on the effect of LNP properties prepared by a microfluidic device on LN delivery (Fig. 6C ) [148] . To our knowledge, only a few such studies have been reported [149, 150] Lilac Pharma is another venture company originated from Hokkaido University and is now focusing on microfluidic technology for scaling up the production of lipid nanoparticles.

We are currently creating networking relationships with companies who are interested in our technologies and in developing Nanomedicines. We hope to see a new era of 

High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma

Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid

A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo

Rational design of cationic lipids for siRNA delivery

In vivo therapeutic Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection

Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases

Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process

KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine

MicroRNA therapeutics: towards a new era for the management of cancer and other diseases

Therapeutic Applications of CRISPR/Cas for Duchenne Muscular Dystrophy

In vivo gene editing in dystrophic mouse muscle and muscle stem cells

Rapid modelling of cooperating genetic events in cancer through somatic genome editing

RNAi therapies: drugging the undruggable

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery

RNAi-mediated gene silencing in non-human primates

Cationic lipids and polymers mediated vectors for delivery of siRNA

Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target

Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone

The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice

Delivery materials for siRNA therapeutics

Relationship Between the

Physicochemical Properties of Lipid Nanoparticles and the Quality of siRNA Delivery to Liver Cells

Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery

Genetic heterogeneity in human disease

The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer

The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice

Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA

Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting

Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E

Apolipoprotein E: cholesterol transport protein with expanding role in cell biology

The role of apolipoprotein E in the elimination of liposomes from blood by hepatocytes in the mouse

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands

Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo

Asialoglycoprotein receptor mediated hepatocyte targeting -strategies and applications

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection

pH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus

Different kinetics for the hepatic uptake of lipid nanoparticles between the apolipoprotein E/low density lipoprotein receptor heparan sulfate proteoglycan-binding activity of apolipoprotein E

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice

Distribution of an asialoglycoprotein receptor on rat hepatocyte cell surface

Breaking down the barriers: siRNA delivery and endosome escape

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids

Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo

Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape

Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown

A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing

Mitochondrial diseases, Nat Rev Dis Primers

Mitochondrial pharmacology

Mitochondrial diseases in man and mouse

A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies

Mitochondrial disease--its impact, etiology, and pathology

Mitochondrial DNA mutations in human disease

The genetics and pathology of oxidative phosphorylation

Mitochondrial dysfunction in

Parkinson's disease: molecular mechanisms and pathophysiological consequences

Mitochondrial diabetes: molecular mechanisms and clinical presentation

Mitochondrial mutations in cancer

Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes

A Dual-Ligand Liposomal System Composed of a Cell-Penetrating Peptide and a Mitochondrial RNA Aptamer Synergistically Facilitates Cellular Uptake and Mitochondrial Targeting

Packaging of the Coenzyme Q(10) into a Liposome for Mitochondrial Delivery and the Intracellular Observation in Patient Derived Mitochondrial Disease Cells

The Use of a Microfluidic Device to Encapsulate a Poorly Water-Soluble Drug

Lipid Nanoparticles and an Attempt to Regulate Intracellular Trafficking to Reach Mitochondria

Enhanced -cyclodextrin-threaded polyrotaxanes using a MITO-Porter

Innovative nanotechnologies for enhancing nucleic acids/gene therapy: Controlling intracellular trafficking to targeted biodistribution

Targeting the Mitochondrial Genome Through a Nanocarrier and the Regulation of Mitochondrial Gene Expression

Innovative Technologies in Nanomedicines: From Passive Targeting to Active

Targeting/From Controlled Pharmacokinetics to Controlled Intracellular Pharmacokinetics

Future of human mitochondrial DNA editing technologies, Mitochondrial DNA A DNA Mapp Seq Anal

Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells

Mitochondrial Delivery of Doxorubicin Using MITO-Porter Kills Drug-Resistant Renal Cancer Cells via Mitochondrial Toxicity

Optical control of mitochondrial reductive reactions in living cells using an electron donor-acceptor linked molecule

The optimization of cancer photodynamic therapy by utilization of a pi-extended porphyrin-type photosensitizer in combination with MITO-Porter

Mitochondrial delivery of an anticancer drug via systemic administration using a mitochondrial delivery system that inhibits the growth of drug-resistant cancer engrafted on mice

Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver

Therapeutic Strategies for Regulating Mitochondrial Oxidative Stress

Cardiac progenitor cells activated by damaged myocardium

Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

A mitochondrial delivery system using liposome-based nanocarriers that target myoblast cells

Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing

Development of a nanoparticle that releases nucleic acids in response to a mitochondrial environment

Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter

A nanocarrier for the mitochondrial delivery of nucleic acids to cardiomyocytes

Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene

Dual function MITO-Porter, a nano carrier integrating both efficient cytoplasmic delivery and mitochondrial macromolecule delivery

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier

Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an

Mitochondrial transgene expression via an artificial mitochondrial DNA

Successful cochlear implantation in a patient with mitochondrial hearing loss and m.625G>A transition

Oncology meets immunology: the cancer-immunity cycle

Comparison of DNA and mRNA vaccines against cancer, Drug discovery today

Innate immune pattern recognition: a cell biological perspective

Integration of nano drug-delivery system with cancer immunotherapy, Therapeutic delivery

Development of a multifunctional envelope-type nano device and its application to nanomedicine

Efficient MHC class I presentation by controlled intracellular trafficking of antigens in octaarginine-modified liposomes

Octaarginine-modified liposomes enhance cross-presentation by promoting the C-terminal trimming of antigen peptide

Incorporation of polyinosine-polycytidylic acid enhances cytotoxic T cell activity and antitumor effects by octaarginine-modified liposomes encapsulating antigen, but not by octaarginine-modified antigen complex

Mechanisms and Consequences of Antigen Presentation by CD1

A microbial glycolipid functions as a J o u r n a l P r e -p r o o f new class of target antigen for delayed-type hypersensitivity

Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs

Major T cell response to a mycolyl glycolipid is mediated by CD1c molecules in rhesus macaques

Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria-infected rhesus macaques

Harnessing invariant NKT cells in vaccination strategies

The nanoparticulation by octaarginine-modified liposome improves alpha-galactosylceramide-mediated antitumor therapy via systemic administration

Ablative and prophylactic effects of BCG Tokyo 172 strain for intravesical treatment in patients with superficial bladder cancer and carcinoma in situ of the bladder. Bladder cancer BCG Study Group

Incidence and Treatment of J o u r n a l P r e -p r o o f Complications of Bacillus Calmette-Guerin Intravesical Therapy in Superficial Bladder Cancer

Development of immunoadjuvants for immunotherapy of cancer

Nanoparticulation of BCG-CWS for application to bladder cancer therapy

Mechanism responsible for the antitumor effect of BCG-CWS using the LEEL method in a mouse bladder cancer model

Application of BCG-CWS as a

Systemic Adjuvant by Using Nanoparticulation Technology

Distribution of BCG-CWS-Loaded Nanoparticles in the Spleen After Intravenous Injection Affects Cytotoxic T Lymphocyte Activity

Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing

A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy

Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma

DNA-loaded nano-adjuvant formed with a vitamin E-scaffold intracellular environmentally-responsive lipid-like material for cancer immunotherapy

Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection

Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: programmed endosomal escape and dissociation

A20 silencing by lipid envelope-type nanoparticles enhances the efficiency of lipopolysaccharide-activated dendritic cells

A lipid nanoparticle for the efficient delivery of siRNA to dendritic cells

Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines

Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core

The silencing of indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells by siRNA-loaded lipid nanoparticles enhances cell-based cancer immunotherapy

IDO in the Tumor Microenvironment: Inflammation, Counter-Regulation, and Tolerance

3-dioxygenase expression in human cancers: clinical and immunologic perspectives

Discovery of IDO1 Inhibitors: From Bench to Bedside

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Nanoparticles target distinct dendritic cell populations according to their size

Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing

Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation

Advances in microfluidics for lipid nanoparticles and extracellular vesicles and applications in drug delivery systems

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

Understanding the formation mechanism of lipid nanoparticles in microfluidic devices with chaotic micromixers

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution

Formulation and manufacturing of lymphatic targeting liposomes using microfluidics

Scale-Independent Microfluidic Production of Cationic Liposomal Adjuvants and Development of Enhanced Lymphatic Targeting Strategies

The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes

The conduit system exports locally secreted IgM from lymph nodes

Lymph node conduits transport virions for rapid T cell activation

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes

The host STING pathway at the interface of cancer and immunity