Small extracellular vesicles carrying reovirus, tumor antigens, interferon-ß, and damage-associated molecular patterns for efficient tumor treatment.

Small extracellular vesicles (SEV) have attracted much attention both as mediators of intercellular communication and as drug delivery systems. In addition, recent studies have shown that SEV containing virus components and virus particles are released from virus-infected cells. Oncolytic viruses, which efficiently kill tumor cells by tumor cell-specific replication, have been actively studied as novel anticancer agents in clinical and preclinical studies. However, it remains to be fully elucidated whether SEV released from oncolytic virus-infected cells are involved in the antitumor effects of oncolytic viruses. In this study, we examined the tumor cell killing efficiencies and innate immune responses following treatment with SEV released from oncolytic reovirus-infected tumor cells in vitro and in vivo. Reovirus-infected B16 cells secreted SEV associated with or containing reovirus particles (Reo-SEV) with a diameter of approximately 130 nm and a zeta potential of -17 mV, although death of reovirus-infected B16 cells was not observed. The secreted Reo-SEV also contained interferon (IFN)-ß, tumor antigens, and damage-associated molecular patterns (DAMPs), including heat shock proteins (HSPs). Reo-SEV were secreted from the tumor tissues of reovirus-injected mice. Inhibition of the SEV secretion pathway using GW4869, which is a neutral sphingomyelinase inhibitor, resulted in significant reduction in the infectious titers of reovirus in the culture supernatants, suggesting that the cells released progeny virus via the SEV secretion pathway. Reo-SEV more efficiently killed mouse tumor cells and induced innate immune responses in mouse bone marrow-derived dendritic cells than reovirus. Reovirus and Reo-SEV mediated efficient and comparable levels of growth suppression of B16 subcutaneous tumors and induction of tumor infiltration of CD8+ T cells following intravenous administration. These results indicate that Reo-SEV are a promising oncolytic agent and that SEV are an effective delivery vehicle for oncolytic virus.

One-Step Formation Method of Plasmid DNA-Loaded, Extracellular Vesicles-Mimicking Lipid Nanoparticles Based on Nucleic Acids Dilution-Induced Assembly.

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.

4-Iodobenzonitrile as a fluorogenic derivatization reagent for chromatographic analysis of L-p-boronophenylalanine in whole blood samples using Suzuki coupling reaction.

BACKGROUND: L-p-Boronophehylalanine (BPA) is used in boron neutron capture therapy (BNCT), which is a novel selective cancer radiotherapy technique. It is important to measure BPA levels in human blood for effective radiotherapy; a prompt gamma-ray spectrometer, ICP-AES, and ICP-MS have been used for this purpose. However, these methods require sophisticated and expensive apparatuses as well as experienced analysts. Herein, we propose an HPLC-FL method for the determination of BPA after precolumn derivatization. A new fluorogenic reagent for aryl boronic acid derivatives, namely, 4-iodobenzonitrile, was employed for the fluorogenic derivatization of BPA based on the Suzuki coupling reaction. RESULTS: After the fluorogenic derivatization, a fluorescent cyanobiphenyl derivative is formed with maximum fluorescence at 335 nm after excitation at 290 nm. The developed method showed good linearity (r2=0.997) over the concentration range of 0.5-1000 nmol/L, and the detection limit (S/N = 3) was 0.26 nmol/L. The proposed method is more sensitive than previously reported methods for the determination of BPA, including the ICP-MS. Finally, the proposed method was successively applied to the measurement of BPA in human whole blood samples with a good recovery rate (≥95.7 %) using only 10 µL of blood sample. The proposed method offers a simple and efficient solution for monitoring BPA levels in BNCT-treated patients. SIGNIFICANCE: 4-Iodobenzonitrile was investigated as a new fluorogenic reagent for BPA based on Suzuki coupling. A new HPLC-FL method for BPA in whole blood samples with ultrasensitivity was developed. The developed method is superior in sensitivity to all previously reported methods for BPA. The method requires only a very small sample volume, making it suitable for micro-blood analysis of BPA via fingerstick sampling.

Profile of Nagasaki Islands Study (NaIS): A Population-Based Prospective Cohort Study on Multi-disease.

In an aging society, it is important to visualize the conditions of people living with diseases or disabilities, such as frailty and sarcopenia, and determine the environmental and genetic factors underlying such conditions. Atherosclerosis and arterial stiffness are key conditions between these factors and noncommunicable diseases. In 2014, we launched a population-based prospective open-cohort study, the Nagasaki Islands Study (NaIS), which was conducted in Goto City, located in the remote islands of Nagasaki Prefecture, Japan, mostly involving middle-aged and older residents. We conducted our own health checkups along with the annual standardized checkups organized by the municipality; recruited study participants; and started to follow-up with them for vital status (death), migration, and occurrence of diseases such as myocardial infarction, stroke, fracture, and human T-cell leukemia virus type 1 (HTLV-1) -associated uveitis. Our checkups were conducted as baseline surveys in different areas of Goto City during the fiscal years 2014-2016, secondary surveys during 2017-2019, and tertiary surveys since 2021, consisting of medical interviews, physical examinations, blood and urine tests, body composition measurements, osteoporosis screening, arterial stiffness measurements, carotid ultrasonography, and dental examination. A total of 4,957 residents participated in either the baseline or secondary surveys and were followed-up; and 3,594 and 3,364 residents (aged 27-96 and 28-98 years) participated in the baseline and secondary surveys, respectively. In conclusion, the NaIS has been undertaken to reveal the influence of aging and risk factors of noncommunicable diseases and disabilities, with an aim to contribute towards better healthcare in the future.

Development of Triple-Negative Breast Cancer-Targeted Liposomes with MUC16 Binding Peptide Ligand in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a highly malignant tumor that does not express the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2). As molecular approaches to these targets have limited clinical utility in TNBC, novel strategies for the treatment of TNBC are urgently needed. MUC16 (Mucin-16) is a glycoprotein involved in cell proliferation and apoptosis and is overexpressed in breast cancer. To develop a clinically available strategy for TNBC treatment, we synthesized a MUC16 targeted peptide (EVQ)-grafted lipid derivative, EVQ-(SG)5-lipid, and prepared EVQ-(SG)5/PEGylated liposomes of 100 nm by size and a slightly negative ζ-potential value. Thus, we aimed at investigating the association between EVQ-(SG)5/PEGylated and TNBC cell lines by interacting with MUC16 using an in vitro model. In addition, we aimed at exploring the intracellular distribution and cellular uptake pathway of EVQ-(SG)5/PEGylated liposomes as novel drug delivery carriers for TNBC.

Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

Development of an apolipoprotein E mimetic peptide-lipid conjugate for efficient brain delivery of liposomes.

Liposomes are versatile carriers that can encapsulate various drugs; however, for delivery to the brain, they must be modified with a targeting ligand or other modifications to provide blood-brain barrier (BBB) permeability, while avoiding rapid clearance by reticuloendothelial systems through polyethylene glycol (PEG) modification. BBB-penetrating peptides act as brain-targeting ligands. In this study, to achieve efficient brain delivery of liposomes, we screened the functionality of eight BBB-penetrating peptides reported previously, based on high-throughput quantitative evaluation methods with in vitro BBB permeability evaluation system using Transwell, in situ brain perfusion system, and others. For apolipoprotein E mimetic tandem dimer peptide (ApoEdp), which showed the best brain-targeting and BBB permeability in the comparative evaluation of eight peptides, its lipid conjugate with serine-glycine (SG)5 spacer (ApoEdp-SG-lipid) was newly synthesized and ApoEdp-modified PEGylated liposomes were prepared. ApoEdp-modified PEGylated liposomes were effectively associated with human brain capillary endothelial cells via the ApoEdp sequence and permeated the membrane in an in vitro BBB model. Moreover, ApoEdp-modified PEGylated liposomes accumulated in the brain 3.9-fold higher than PEGylated liposomes in mice. In addition, the ability of ApoEdp-modified PEGylated liposomes to localize beyond the BBB into the brain parenchyma in mice was demonstrated via three-dimensional imaging with tissue clearing. These results suggest that ApoEdp-SG-lipid modification is an effective approach for endowing PEGylated liposomes with the brain-targeting ability and BBB permeability.

Selective fluorescence labeling of myristicin using Mizoroki-Heck coupling reaction. Application to nutmeg powder, oil, and human plasma samples.

Myristicin [5-allyl-1­methoxy-2,3-(methylenedioxy)benzene] is the major constituent of the seasoning nutmeg oil and powder. Sometimes myristicin is abused via its ingestion at high doses to cause hallucination. In these high doses, myristicin could cause severe adverse health effects, including convulsion, delirium, and palpitation. Hence there is a strong need for a sensitive method for its analysis, such as fluorescence determination. Myristicin has a very weak fluorescence and also lacks derivatizable groups like the carboxylic, hydroxyl, or amino group in its structure, which makes its fluorescence derivatization challenging. In this research, we developed a fluorescence labeling method for myristicin based on the Mizoroki-Heck coupling reaction of its terminal alkene with a fluorescent aryl iodide derivative, 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIB-I). Then, we developed an HPLC fluorescence detection method for the determination of myristicin utilizing this labeling reaction. The developed method showed a good linear response for myristicin (r = 0.995) in the range of 0.01-10 µmol/L with excellent sensitivity down to the detection limit of 2.9 nmol/L (9.6 fmol/injection). Finally, the developed method could be successfully applied to determine myristicin content in nutmeg powder, oil samples, and human plasma with simple extraction methods and good recoveries ranging from 89.3 to 106%.

Synthesis and evaluation of a novel adapter lipid derivative for preparation of cyclic peptide-modified PEGylated liposomes: Application of cyclic RGD peptide.

Peptide ligand modified nanoparticles can simply prepared by post-insertion method to mix pre-formed nanoparticles with peptide-lipid conjugates in an aqueous solution at an optimal temperature. Therefore, water dispersibility of peptide-lipid conjugates is a very important factor for implementing the post-insertion method. We proposed that highly water dispersible peptide-lipid conjugates can be easily synthesized by separately designing novel adapter lipids with different water dispersibility and reacting them with ligands in a highly efficient manner. Adapter lipids have three critical roles; as spacers of ligand-conjugated lipids for efficient ligand presentation, as structures that form discrete molecular weight distributions, and as providing water dispersibility. In this study, we developed a novel adapter-lipid derivative that enables a variety of cyclic peptide modifications using the click reaction. The integrin αvß3-targeted cyclic RGDfK (cRGD) peptide was selected as the cyclic peptide ligand. We designed a novel alkyne-tagged lipid with a discrete peptide spacer and bound the cRGD peptide using a click reaction to synthesize a cRGD-conjugated lipid with good water dispersibility for the preparation of cRGD-modified PEGylated liposomes using the post-insertion method. We also revealed that cRGD-modified PEGylated liposomes are efficiently associated with integrin αvß3-expressing murine colon carcinoma (Colon-26) cells in a modification amount- and peptide sequence-dependent manner, showing high cytotoxicity upon loading with doxorubicin. This novel adapter lipid derivative can be used to synthesize various cyclic peptides by click reactions and will provide useful insights for the future development of cyclic peptide-modified PEGylated liposomes.

Synthesis and Evaluation of High Functionality and Quality Cell-penetrating Peptide Conjugated Lipid for Octaarginine Modified PEGylated Liposomes In U251 and U87 Glioma Cells.

The use of peptide ligand modified PEGylated liposomes has been widely investigated for tumor targeting. Peptides are mainly inserted in the liposomal lipid bilayer using PEG2K-lipid spacer (Peptide-PEG2K-DSPE). However, a lower cellular uptake from longer nonlinear PEG2K spacer was reported, we here synthesized a high functionality and quality (HFQ) lipid with a short, linear serine-glycine repeated peptide [(SG)5] spacer. The objective of the current study is to develop novel octaarginine (R8) peptide-HFQ lipid grafted PEGylated liposomes for glioma cells targeting. In vitro liposomes characterization showed that the mean particle size of all liposomal formulations was in the nano-scale range < 120 nm, with a small PDI value (i.e. ∼0.2) and had a spherical shape under Transmission Electron Microscope, indicating a homogenous particle size distribution. The flow cytometry in vitro cellular association data with U251 MG and U87 cells revealed that 1.5% R8-(SG)5-lipid-PEGylated liposomes exhibited significantly higher cellular association of ∼15.87 and 7.59-fold than the conventional R8-PEG2K-lipid-PEGylated liposomes (10.4 and 6.19-fold), respectively, relative to the unmodified PEGylated liposomes. Moreover, intracellular distribution studies using confocal laser scanning microscopy (CLSM) corroborated the results of the in vitro cell association. The use of ligand-HFQ-lipid liposomes could be a potential alternative to ligand-PEG2K-lipid-modified liposomes as a drug delivery system for tumor targeting.

Stem Cell Therapy for Acute/Subacute Ischemic Stroke with a Focus on Intraarterial Stem Cell Transplantation: From Basic Research to Clinical Trials.

Stem cell therapy for ischemic stroke holds great promise for the treatment of neurological impairment and has moved from the laboratory into early clinical trials. The mechanism of action of stem cell therapy includes the bystander effect and cell replacement. The bystander effect plays an important role in the acute to subacute phase, and cell replacement plays an important role in the subacute to chronic phase. Intraarterial (IA) transplantation is less invasive than intraparenchymal transplantation and can provide more cells in the affected brain region than intravenous transplantation. However, transplanted cell migration was reported to be insufficient, and few transplanted cells were retained in the brain for an extended period. Therefore, the bystander effect was considered the main mechanism of action of IA stem cell transplantation. In most clinical trials, IA transplantation was performed during the acute and subacute phases. Although clinical trials of IA transplantation demonstrated safety, they did not demonstrate satisfactory efficacy in improving patient outcomes. To increase efficacy, increased migration of transplanted cells and production of long surviving and effective stem cells would be crucial. Given the lack of knowledge on this subject, we review and summarize the mechanisms of action of transplanted stem cells and recent advancements in preclinical and clinical studies to provide information and guidance for further advancement of acute/subacute phase IA stem cell transplantation therapy for ischemic stroke.

Microfluidic Post-Insertion Method for the Efficient Preparation of PEGylated Liposomes Using High Functionality and Quality Lipids.

Introduction: Targeted liposomes using ligand peptides have been applied to deliver therapeutic agents to the target sites. The post-insertion method is commonly used because targeted liposomes can be prepared by simple mixing of ligand peptide-lipid and liposomes. A large-scale preparation method is required for the clinical application of ligand-peptide-modified liposomes. Large-scale preparation involves an increase in volume and a change in the preparation conditions. Therefore, the physicochemical properties of liposomes may change owing to large alterations in the preparation conditions. To address this issue, we focused on a microfluidic device and developed a novel ligand peptide modification method, the microfluidic post-insertion method. Methods: We used integrin αvß3-targeted GRGDS (RGD) and cyclic RGDfK (cRGD)-modified high functionality and quality (HFQ) lipids, which we had previously developed. First, the preparation conditions of the total flow rate in the microfluidic device for modifying HFQ lipids to polyethylene glycol (PEG)-modified (PEGylated) liposomes were optimized by evaluating the physicochemical properties of the liposomes. The targeting ability of integrin αvß3-expressing colon 26 murine colorectal carcinoma cells was evaluated by comparing the cellular association properties of the liposomes prepared by the conventional post-insertion method. Results: When the RGD-HFQ lipid was modified into PEGylated liposomes by varying the total flow rate (1, 6, and 12 mL/min) of the microfluidic device, as the total flow rate increased, the polydispersity index also increased, whereas the particle size did not change. Furthermore, the RGD- and cRGD-modified PEGylated liposomes prepared at a total flow rate of 1 mL/min showed high cellular association properties equivalent to those prepared by the conventional post-insertion method. Conclusion: Microfluidic post-insertion method of HFQ lipids might be useful for clinical application and large-scale preparation of targeted liposomes.

Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice.

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney.

Evaluation of transgene expression characteristics and DNA vaccination against melanoma metastasis of an intravenously injected ternary complex with biodegradable dendrigraft poly-L-lysine in mice.

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and γ-polyglutamic acid (γ-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

Ligand Design for Specific MHC Class I Molecules on the Cell Surface.

We have validated that ligand peptides designed from antigen peptides could be used for targeting specific major histocompatibility complex class I (MHC-I) molecules on the cell surface. To design the ligand peptides, we used reported antigen peptides for each MHC-I molecule with high binding affinity. From the crystal structure of the peptide/MHC-I complexes, we determined a modifiable residue in the antigen peptides and replaced this residue with a lysine with an ε-amine group modified with functional molecules. The designed ligand peptides successfully bound to cells expressing the corresponding MHC-I molecules via exchange of peptides bound to MHC-I. We demonstrated that the peptide ligands could be used to transport a protein or a liposome to cells expressing the corresponding MHC-I. This strategy may be useful for targeted delivery to cells overexpressing MHC-I, which have been observed in autoimmune diseases.

Recent Strategies for Targeted Brain Drug Delivery.

Because the brain is the most important human organ, many brain disorders can cause severe symptoms. For example, glioma, one type of brain tumor, is progressive and lethal, while neurodegenerative diseases cause severe disability. Nevertheless, medical treatment for brain diseases remains unsatisfactory, and therefore innovative therapies are desired. However, the development of therapies to treat some cerebral diseases is difficult because the blood-brain barrier (BBB) or blood-brain tumor barrier prevents drugs from entering the brain. Hence, drug delivery system (DDS) strategies are required to deliver therapeutic agents to the brain. Recently, brain-targeted DDS have been developed, which increases the quality of therapy for cerebral disorders. This review gives an overview of recent brain-targeting DDS strategies. First, it describes strategies to cross the BBB. This includes BBB-crossing ligand modification or temporal BBB permeabilization. Strategies to avoid the BBB using local administration are also summarized. Intrabrain drug distribution is a crucial factor that directly determines the therapeutic effect, and thus it is important to evaluate drug distribution using optimal methods. We introduce some methods for evaluating drug distribution in the brain. Finally, applications of brain-targeted DDS for the treatment of brain tumors, Alzheimer's disease, Parkinson's disease, and stroke are explained.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

[Development of Nano-DDS Carriers for Control of Spatial Distribution Using Multi-color Deep Imaging].

Because active-targeted liposomes are very complex formulations, quality characteristics of functional lipids have not been defined yet, and this is a major obstacle in clinical application of active targeted liposomes. We have developed high functionality and quality (HFQ) lipids, which define quality characteristics of functional lipids for clinical drug delivery system (DDS) applications. Because HFQ lipids are designed to enable facile and rapid functionalization of DDS carrier by simple and one-step mixing, we are expanding applications for not only liposomes but also exosomes and cells. Recently, we developed multi-color deep imaging by tissue clearing for analysis of spatial distribution of DDS in various tissues. Nanocarriers are usually non-uniformly distributed in solid tumors because of their heterogeneity. Especially, in refractory cancer such as pancreatic cancer, the presence of collagen and blood vessels greatly affects intra-tumor distribution of DDS carrier. Therefore information on spatial relations between the tissue structure and DDS carrier is important to regulate precisely intra-tumor distribution of DDS carrier. Recently, our group has established multi-color deep imaging to analyze spatial distribution of stromal collagen, liposomes, and blood vessels in pancreatic tumor tissue. In this review, we present recent research in developing HFQ lipids. Moreover, current status of research on DDS for pancreatic cancer treatment is reviewed.

Nanobubble Mediated Gene Delivery in Conjunction With a Hand-Held Ultrasound Scanner.

Recent research has revealed that nanobubbles (NBs) can be an effective tool for gene transfection in conjunction with therapeutic ultrasound (US). However, an approach to apply commercially available hand-held diagnostic US scanners for this purpose has not been evaluated as of now. In the present study, we first compared in vitro, the efficiency of gene transfer (pCMV-Luciferase) with lipid-based and albumin-based NBs irradiated by therapeutic US (1MHz, 5.0 W/cm2) in oral squamous carcinoma cell line HSC-2. Secondly, we similarly examined if gene transfer in mice is possible using a clinical hand-held US scanner (2.3MHz, MI 1.0). Results showed that lipid-based NBs induced more gene transfection compared to albumin-based NBs, in vitro. Furthermore, significant gene transfer was also obtained in mice liver with lipid-based NBs. Sub-micro sized bubbles proved to be a powerful gene transfer reagent in combination with conventional hand-held ultrasonic diagnostic device.

Synthesis of a Novel Pyrazine⁻Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells.

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine-pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure-fluorescence relationship analysis of the substituent effects with/without Zn2+, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn2+ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn2+ toward understanding its functions in living systems.