Impact of in vivo fate of STING agonist-loaded lipid nanoparticles on antitumor immunity.

Therapeutically manipulating the stimulator of interferon genes (STING) pathway has promising potential for enhancing antitumor immunity. Agonists of this pathway (STING agonists) are being evaluated in clinical trials. Loading the STING agonists into lipid nanoparticles (LNPs) increases their safety and efficacy. We previously developed STING agonists loaded LNPs consisting of the ionizable lipid YSK12-C4 (YSK12-LNPs), which showed significant antitumor effects. However, it is largely unclear how the in vivo fate of STING agonists loaded LNPs affects the antitumor immune responses. In this study, we compared the YSK12-LNPs with LNPs composed of DLin-MC3-DMA (MC3-LNPs) showing different in vivo fates. Biodistribution and flow cytometry analyses of mouse tissues revealed that the MC3-LNPs delivered higher amounts of STING agonists to the liver than the YSK12-LNPs. Additionally, significantly more liver leukocytes internalized the MC3-LNPs than the YSK12-LNPs. In contrast, the YSK12-LNPs delivered higher amounts of STING agonists to the liver leukocytes than the MC3-LNPs, leading to the effective induction of innate immunity and inflammation in the tumors. However, the antitumor effects in the B16-F10 lung metastasis and CT26 tumor models were comparable. Interestingly, flow cytometry analyses suggested that the YSK12-LNPs were more likely to activate natural killer cells and M1 macrophages, while the MC3-LNPs were more likely to activate CD8+ T cells. Our data suggest that different antitumor immune response mechanisms may operate depending on the characteristics and distribution of the LNPs.

The impact of, and expectations for, lipid nanoparticle technology: From cellular targeting to organelle targeting.

The success of mRNA vaccines against COVID-19 has enhanced the potential of lipid nanoparticles (LNPs) as a system for the delivery of mRNA. In this review, we describe our progress using a lipid library to engineer ionizable lipids and promote LNP technology from the viewpoints of safety, controlled biodistribution, and mRNA vaccines. These advancements in LNP technology are applied to cancer immunology, and a potential nano-DDS is constructed to evaluate immune status that is associated with a cancer-immunity cycle that includes the sub-cycles in tumor microenvironments. We also discuss the importance of the delivery of antigens and adjuvants in enhancing the cancer-immunity cycle. Recent progress in NK cell targeting in cancer immunotherapy is also introduced. Finally, the impact of next-generation DDS technology is explained using the MITO-Porter membrane fusion-based delivery system for the organelle targeting of the mitochondria. We introduce a successful example of the MITO-Porter used in a cell therapeutic strategy to treat cardiomyopathy.

Understanding Gene Involvement in Hepatocellular Carcinoma: Implications for Gene Therapy and Personalized Medicine.

Hepatocellular carcinoma (HCC) is the dominant type of liver cancers and is one of the deadliest health threats globally. The conventional therapeutic options for HCC are hampered by low efficiency and intolerable side effects. Gene therapy, however, now offers hope for the treatment of many disorders previously considered incurable, and gene therapy is beginning to address many of the shortcomings of conventional therapies. Herein, we summarize the involvement of genes in the pathogenesis and prognosis of HCC, with a special focus on dysregulated signaling pathways, genes involved in immune evasion, and non-coding RNAs as novel two-edged players, which collectively offer potential targets for the gene therapy of HCC. Herein, the opportunities and challenges of HCC gene therapy are discussed. These include innovative therapies such as genome editing and cell therapies. Moreover, advanced gene delivery technologies that recruit nanomedicines for use in gene therapy for HCC are highlighted. Finally, suggestions are offered for improved clinical translation and future directions in this area of endeavor.

Investigation of the Nanoparticulation Method and Cell-Killing Effect following the Mitochondrial Delivery of Hydrophobic Porphyrin-Based Photosensitizers.

Photodynamic therapy is expected to be a less invasive treatment, and strategies for targeting mitochondria, the main sources of singlet oxygen, are attracting attention to increase the efficacy of photodynamic therapy and reduce its side effects. To date, we have succeeded in encapsulating the photosensitizer rTPA into MITO-Porter (MP), a mitochondria-targeted Drug Delivery System (DDS), aimed at mitochondrial delivery of the photosensitizer while maintaining its activity. In this study, we report the results of our studies to alleviate rTPA aggregation in an effort to improve drug efficacy and assess the usefulness of modifying the rTPA side chain to improve the mitochondrial retention of MITO-Porter, which exhibits high therapeutic efficacy. Conventional rTPA with anionic side chains and two rTPA analogs with side chains that were converted to neutral or cationic side chains were encapsulated into MITO-Porter. Low-MP (MITO-Porter with Low Drug/Lipid) exhibited high drug efficacy for all three types of rTPA, and in Low-MP, charged rTPA-encapsulated MP exhibited high drug efficacy. The cellular uptake and mitochondrial translocation capacities were similar for all particles, suggesting that differences in aggregation rates during the incorporation of rTPA into MITO-Porter resulted in differences in drug efficacy.

Vaccination with a combination of STING agonist-loaded lipid nanoparticles and CpG-ODNs protects against lung metastasis via the induction of CD11bhighCD27low memory-like NK cells.

BACKGROUND: Natural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction, identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles (STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of the inducted memory-like NK cells to prevent melanoma lung metastasis. METHODS: The antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluated using a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed through their preventative effect. RESULTS: The combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12) and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, the combination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells could be a phenotype of memory-like NK cells. CONCLUSION: As far as could be ascertained, this is the first report of the in vivo induction, identification, and confirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low memory-like NK cells thus paving the way for the development of efficient immunotherapies.

Human cardiosphere-derived cells with activated mitochondria for better myocardial regenerative therapy.

Cell transplantation is a promising therapeutic strategy for myocardial regeneration therapy. To improve therapeutic effects, we developed a culture medium additive that enhances the mitochondrial function of cardiomyocytes for transplantation. A mitochondrial targeting drug delivery system (MITO-Porter system) was used to deliver mitochondrial activation molecules to mouse-derived cardiac progenitor cells. In this study, we investigated whether the mitochondrial function of human-derived myocardial precursor cells could be enhanced using MITO-Porter. Human cardiosphere-derived cells (CDCs) were isolated from myocardium which was excised during surgery for congenital heart disease. MITO-Porter was added to the cell culture medium to generate mitochondrial activated CDCs (human MITO cells). The human MITO cells were transplanted into myocardial ischemia-reperfusion model rat, and the effect was investigated. The transplanted human MITO cells improved the cardiac function and suppressed myocardial fibrosis compared to conventional cell transplantation methods. These effects were observed not only with myocardial administration but also by intravenous administration of human MITO cells. This study is the first study that assessed whether the mitochondrial delivery of functional compounds improved the outcome of human-derived myocardial cell transplantation therapy.

Innovative System for Delivering Nucleic Acids/Genes Based on Controlled Intracellular Trafficking as Well as Controlled Biodistribution for Nanomedicines.

This review paper summarizes progress that has been made in the new field of "Controlled Intracellular Trafficking." This involves the development of new systems for delivering plasmid DNA (pDNA), small interfering RNA (siRNA), mRNA, proteins, their escape from endosomes, the mechanism for how they enter the nucleus, how they enter mithochondria and how materials subsequently function within a cell. In addition, strategies for delivering these materials to a selective tissue after intravenous administration was also intensively investigated not only to the liver but also to tumors, lungs, adipose tissue and the spleen. In 2020, a new mRNA vaccine was developed against coronavirus disease 2019 (COVID-19), where ionizable cationic lipids were used as a delivery system. Our strategy to identify an efficient ionizable cationic lipids (iCL) based on a lipid library as well as their applications concerning the delivery of siRNA/mRNA/pDNA is also described.

Microenvironmental Changes in Mediastinal Fat-associated Lymphoid Clusters and Lungs in Early and Late Stages of Metastatic Lung Cancer Induction.

The prognosis of metastatic lung melanoma (MLM) has been reported to be poor. An increasing number of studies have reported the function of several immune cells in cancer regression. Although the function of mediastinal fat-associated lymphoid clusters (MFALCs) in the progression of inflammatory lung lesions has been previously reported, the association between MLM progression and MFALCs development has remained unexplored. Herein, we compared the microenvironmental changes in the lungs and MFALCs among phosphate-buffered saline (PBS) and cancer groups at early (1 week) and late (2 weeks) stages following the intravenous injection of B16-F10 melanoma cells into C57BL/6 mice. Except for lung CD4+ helper T-cells and Iba1+ macrophage populations of early stage, we observed a significant increase in the proliferating and immune cell (CD20+ B-lymphocytes, CD3+ T-lymphocytes, CD8+ cytotoxic T-cells, CD16+ natural killer (NK) cells populations, area of high endothelial venules, and lung lymphatic vessels in cancer groups at both the stages as compared with the PBS groups. Furthermore, a significant positive correlation was observed between immune cell populations in MFALCs and the lungs (B- and T-lymphocytes, and NK cells in both stages). Collectively, our findings suggest a promising cancer therapeutic strategy via targeting immune cells in MFALCs.

Delivering mRNA to a human NK cell line, NK-92 cells, by lipid nanoparticles.

In cancer immunotherapy, therapeutic methods targeting NK are highly expected. NK cell-based therapy using NK-92, a human NK cell line, has been clinically evaluated. Delivering mRNA into NK-92 cells is a potent strategy for enhancing its functions. However, the use of lipid nanoparticles (LNP) for this purpose has not yet been evaluated. We previously developed a LNP that was composed of CL1H6 (CL1H6-LNP) for the efficient delivery of siRNA to NK-92 cells, and the use of this material for delivering mRNA to NK-92 cells is reported in this study. Compared with a DLin-MC3-DMA based LNP, used as a benchmark, the CL1H6-LNP caused a high mRNA expression intensity and a cell transfection efficiency of 100%. The efficient mRNA delivery by this CL1H6-LNP is attributed to the high affinity for NK-92 cells and the intense, rapid fusion with the endosomal membrane. It therefore appears that the CL1H6-LNP could be a useful non-viral vector for modifying the NK-92 functions by mRNA. Our findings also provide some insights into the design and development of LNPs for delivering mRNA to NK-92 and NK cells.

Development of a Mitochondrial Targeting Lipid Nanoparticle Encapsulating Berberine.

Delivering drugs to mitochondria, the main source of energy in neurons, can be a useful therapeutic strategy for the treatment of neurodegenerative diseases. Berberine (BBR), an isoquinoline alkaloid, acts on mitochondria and is involved in mechanisms associated with the normalization and regulation of intracellular metabolism. Therefore, BBR has attracted considerable interest as a possible therapeutic drug for neurodegenerative diseases. While BBR has been reported to act on mitochondria, there are few reports on the efficient delivery of BBR into mitochondria. This paper reports on the mitochondrial delivery of BBR using a lipid nanoparticle (LNP), a "MITO-Porter" that targets mitochondria, and its pharmacological action in Neuro2a cells, a model neuroblastoma. A MITO-Porter containing encapsulated BBR (MITO-Porter (BBR)) was prepared. Treatment with MITO-Porter (BBR) increased the amount of BBR that accumulated in mitochondria compared with a treatment with naked BBR. Treatment with MITO-Porter (BBR) resulted in increased ATP production in Neuro2a cells, which are important for maintaining life phenomena, compared with treatment with naked BBR. Treatment with MITO-Porter (BBR) also increased the level of expression of mitochondrial ubiquitin ligase (MITOL), which is involved in mitochondrial quality control. Our findings indicate that increasing the accumulation of BBR into mitochondria is important for inducing enhanced pharmacological actions. The use of this system has the potential for being important in terms of the regulation of the metabolic mechanism of mitochondria in nerve cells.

Fine-tuning the encapsulation of a photosensitizer in nanoparticles reveals the relationship between internal structure and phototherapeutic effects.

Photodynamic therapy (PDT) is a cancer therapy that uses a photosensitizer (PS) in the presence of oxygen molecules. Since singlet oxygen is highly reactive, it is important to deliver it to the target site. Thus, an efficient drug delivery system (DDS) is essential for enhancing the efficacy of such a treatment and protecting against the side effects of PDT. Here, we report on attempts to increase the therapeutic effect of PDT by using a DDS, a lipid nanoparticle (LNP). We prepared a porphyrin analog, rTPA (PS) that was encapsulated in LNPs using a microfluidic device. The findings indicated that the internal structure of the prepared particles changed depending on the amount of rTPA in LNPs. The photoactivity and cell-killing effect of PS in LNPs also changed when the amount of the cargo increased. These results suggest that the internal structure of LNPs is important factors that affect drug efficacy.

Cancer Immunotherapy with Lipid Nanoparticles Loaded with a Stimulator of Interferon Genes Agonist against Renal Tumor Lung Metastasis.

Metastatic renal cell carcinoma (RCC) has a poor prognosis, and the major organ of metastasis is the lung. Immunotherapy with immune checkpoint inhibitors (ICIs) is the first-line therapy, but the response rates are low. Thus, the development of a more effective immunotherapy against metastatic RCC would be highly desirable. We previously demonstrated how a stimulator of an interferon gene (STING) agonist-loaded lipid nanoparticles (STING-LNPs) significantly activates natural killer (NK) cells and induces an antitumor effect against cases of melanoma lung metastasis that have shown ICI resistance. In this study, we evaluated the potential of using STING-LNPs in the treatment of lung metastatic RCC (Renca). An intravenous injection of STING-LNPs drastically decreased the amount of Renca tumor colonies. In contrast, monotherapies using ICIs showed no antitumor effect, and even a combination of ICI and STING-LNP therapies failed to enhance the antitumor effects. The main effector cells would be NK cells, and the activation of NK cells by the STING-LNPs may avoid the increased expression of immune checkpoint molecules. These findings provide useful insights into the development of an effective immunotherapy against metastatic RCC.

Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery.

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

mRNA-Loaded Lipid Nanoparticles Targeting Immune Cells in the Spleen for Use as Cancer Vaccines.

mRNA delivery has recently gained substantial interest for possible use in vaccines. Recently approved mRNA vaccines are administered intramuscularly where they transfect antigen-presenting cells (APCs) near the site of administration, resulting in an immune response. The spleen contains high numbers of APCs, which are located near B and T lymphocytes. Therefore, transfecting APCs in the spleen would be expected to produce a more efficient immune response, but this is a challenging task due to the different biological barriers. Success requires the development of an efficient system that can transfect different immune cells in the spleen. In this study, we report on the development of mRNA-loaded lipid nanoparticles (LNPs) targeting immune cells in the spleen with the goal of eliciting an efficient immune response against the antigen encoded in the mRNA. The developed system is composed of mRNA loaded in LNPs whose lipid composition was optimized for maximum transfection into spleen cells. Dendritic cells, macrophages and B cells in the spleen were efficiently transfected. The optimized LNPs produced efficient dose-dependent cytotoxic T lymphocyte activities that were significantly higher than that produced after local administration. The optimized LNPs encapsulating tumor-antigen encoding mRNA showed both prophylactic and therapeutic antitumor effects in mice.

mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy.

Dendritic cells (DCs) are attractive antigen-presenting cells to be targeted for vaccinations. However, the systemic delivery of mRNA to DCs is hampered by technical challenges. We recently reported that it is possible to regulate the size of RNA-loaded lipid nanoparticles (LNPs) to over 200 nm with the addition of salt during their formation when a microfluidic device is used and that larger LNPs delivered RNA more efficiently and in greater numbers to splenic DCs compared to the smaller counterparts. In this study, we report on the in vivo optimization of mRNA-loaded LNPs for use in vaccines. The screening included a wide range of methods for controlling particle size in addition to the selection of an appropriate lipid type and its composition. The results showed a clear correlation between particle size, uptake and gene expression activity in splenic DCs and indicated that a size range from 200 to 500 nm is appropriate for use in targeting splenic DCs. It was also found that it was difficult to predict the transgene expression activity and the potency of mRNA vaccines in splenic DCs using the whole spleen. A-11-LNP, which was found to be the optimal formulation, induced better transgene expression activity and maturation in DCs and induced clear therapeutic antitumor effects in an E.G7-OVA tumor model compared to two clinically relevant LNP formulations.

Extrahepatic targeting of lipid nanoparticles in vivo with intracellular targeting for future nanomedicines.

A new era of nanomedicines that involve nucleic acids/gene therapy has been opened after two decades in 21st century and new types of more efficient drug delivery systems (DDS) are highly expected and will include extrahepatic delivery. In this review, we summarize the possibility and expectations for the extrahepatic delivery of small interfering RNA/messenger RNA/plasmid DNA/genome editing to the spleen, lung, tumor, lymph nodes as well as the liver based on our studies as well as reported information. Passive targeting and active targeting are discussed in in vivo delivery and the importance of controlled intracellular trafficking for successful therapeutic results are also discussed. In addition, mitochondrial delivery as a novel strategy for nucleic acids/gene therapy is introduced to expand the therapeutic dimension of nucleic acids/gene therapy in the liver as well as the heart, kidney and brain.

Interval- and cycle-dependent combined effect of STING agonist loaded lipid nanoparticles and a PD-1 antibody.

Programmed cell death 1 (PD-1) blockade combination to other drugs have attracted the interest of scientists for treating tumors resistant to PD-1 blockade. In this study, the impact of the interval, order of administration, and number of cycles of immunotherapeutic combination of stimulator of interferon genes (STING) pathway agonist loaded lipid nanoparticle (STING-LNP) and PD-1 antibody for inducing the optimal combined antitumor activity against a melanoma lung metastasis is reported. One cycle had no effect, but two and three cycles resulted in a combinedantitumor effect. The interval between the administration was found to influence the induction of the combined effect. The second and third doses increased the gene expression of the NK cell activation marker, interferon γ (IFN-γ), PD-1 and a ligand of PD-1 (PD-L1), whereas the first dose failed. NK cells in the lung showed an increase in the expression of the activation markers and PD-1 after the second dose. The combined antitumor effect of this combination therapy against melanoma lung metastasis model could be dependent on the interval as well as the number of doses of STING-LNP.These findings suggest the importance of the protocol setting when combining a nano system loaded with an immune adjuvant and PD-1 antibody.

On the size-regulation of RNA-loaded lipid nanoparticles synthesized by microfluidic device.

The use of lipid nanoparticles (LNPs) for nucleic acid delivery is now becoming a promising strategy with a number of clinical trials as vaccines or as novel therapies against a variety of genetic and infectious diseases. The use of microfluidics for the synthesis of the LNPs has attracted interest because of its considerable advantages over other conventional synthetic methods including scalability, reproducibility, and speed. However, despite the potential usefulness of large particles for nucleic acid delivery to dendritic cells (DCs) as a vaccine, the particle size of the LNPs prepared using microfluidics is typically limited to approximately from 30 to 100 nm. In this study, focusing on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the effect of some synthetic parameters, including total flow rate, flow rate ratio, buffer pH, lipid concentration, molar ratio of PEG-lipid as well as salt concentration, on particle size was systematically examined by means of the design of experiment approaches. The findings indicated that the simple addition of salt (e.g. NaCl) to a buffer containing nucleic acids contributed greatly to the synthesis of large LNPs over 200 nm and this effect was concentration-dependent with respect to the salt. The effect of salt on particle size was consistent with a Hofmeister series. The systemic injection of larger mRNA-loaded LNPs resulted in a higher transgene expression in mouse splenic DCs, a higher activation of various splenic immune cells, and had a superior effect as a therapeutic cancer vaccine in a syngeneic mouse model compared to the smaller-sized counterpart with constant lipid composition prepared with lower NaCl concentration. Collectively, size-regulation by the simple addition of salt is a promising strategy for developing potent LNPs.

Innovative cancer nanomedicine based on immunology, gene editing, intracellular trafficking control.

The recent rapid progress in the area of drug delivery systems (DDS) has opened a new era in medicine with a strong linkage to understanding the molecular mechanisms associated with cancer survival. In this review, we summarize new cancer strategies that have recently been developed based on our DDS technology. Cancer immunotherapy will be improved based on the concept of the cancer immunity cycle, which focuses on dynamic interactions between various types of cancer and immune cells in our body. The new technology of genome editing will also be discussed with reference to how these new DDS technologies can be used to introduce therapeutic cargoes into our body. Lastly, a new organelle, mitochondria will be the focus of creating a new cancer treatment strategy by a MITO-Porter which can deliver macromolecules directly to mitochondria of cancer cells via a membrane fusion approach and the impact of controlled intracellular trafficking will be discussed.

Combined nano cancer immunotherapy based on immune status in a tumor microenvironment.

Since the effect of cancer immunotherapy is largely dependent on the status of the immune system in the tumor microenvironment (TME), choice of therapy and the development of new therapies based on the immune status in the TME would be predicted to be effective. Unfortunately, the development of delivery systems for such therapy has been slow. Here, we defined a parameter of immune status in TME showing antitumor effects and demonstrated the cancer immunotherapy with an adjuvant loaded lipid nanoparticle (LNP), which was taken advantage the parameter. An analysis was carried out to determine the relationship between antitumor effects and gene expression (22 target genes) in tumors (MC38 and E.G7-OVA) that respond to the programmed cell death 1 (PD-1) antibody and non-responding tumors (B16-F10 and 4T1). The immune status showing an effective antitumor effect, which consisted of 10 genes, was then extracted. Treatment with the adjuvant loaded LNP caused a significant antitumor effect against an E.G7-OVA tumor, and the gene expression in the E.G7-OVA tumor was completely within the range of gene expression for showing an effective antitumor effect, as defined by the identified immune status panel (IS-panel-10). Although the treatment with the adjuvant loaded LNP failed to induce a sufficient antitumor effect against the 4T1 tumor, we succeeded in enhancing the antitumor effect by using a combination therapy that was adopted based on the analysis by the IS-panel-10 in the TME. The 10 genes were found to affect the prognosis in a variety of human cancers. Collectively, the findings reported herein demonstrate the potential of immune status analysis in the TME for developing cancer immunotherapies using a delivery system.