In vitro evaluation of novel SN-38 prodrug activated by α-rhamnosidase of exogenous enzyme.

This study introduces the α-rhamnose (Rham)-conjugated prodrug of SN-38 (Rham-SN-38) as a promising alternative to irinotecan. α-rhamnosidase, responsible for SN-38 release from Rham-SN-38, does not express in human cells, minimizing individual variability and side effects. The injection of the α-rhamnosidase into the tumor tissues makes it possible, for the first time, to activate the Rham-SN-38. Furthermore, α-rhamnosidase demonstrates significantly higher activity than carboxylesterase, the specific enzyme activating irinotecan. SN-38 release mediated by α-rhamnosidase completes within 2 h, with a kcat/Km value approximately 5.0 × 104-fold higher than that of irinotecan. The 50% inhibition concentration (IC50) of Rham-SN-38 against three types of cancer cells and one normal cell exceeds 4.5 × 103 nM. The addition of α-rhamnosidase significantly increases cytotoxicity, with IC50 comparable to free SN-38. The QIC50, an index reflecting the difference in cytotoxicity with and without α-rhamnosidase, exceeds approximately 1.0 × 102-fold. Rham-SN-38, synthesized in this study, demonstrates significant potential as a prodrug for cancer therapy.

Effective design of PEGylated polyion complex (PIC) nanoparticles for enhancing PIC internalisation in cells utilising block copolymer combinations with mismatched ionic chain lengths.

In nanomedicine, PEGylation of nanomaterials poses a dilemma since it inhibits their interaction with target cells and enables their retention in target tissues despite its biocompatibility and nonspecific internalisation suppression. PEGylated polypeptide-based polyion complexes (PICs) are fabricated via the self-assembly of PEGylated aniomers and homocatiomers based on electrostatic interactions. We propose that various parameters like block copolymer design and PIC domain characteristics can enhance the cell-PEGylated PIC interactions. Remarkably, the properties of the PIC domain were tuned by the matched/mismatched ionomer chain lengths, PIC domain crosslinking degree, chemical modification of cationic species after crosslinking, PIC morphologies (vesicles/micelles) and polyethylene glycol (PEG) chain lengths. Cellular internalisation of the prepared PICs was evaluated using HeLa cells. Consequently, mismatched ionomer chain lengths and vesicle morphology enhanced cell-PIC interactions, and the states of ion pairing, particularly cationic residues, affected the internalisation behaviours of PICs via acetylation or guanidinylation of amino groups on catiomers. This treatment attenuated the cell-PIC interactions, possibly because of reduced interaction of PICs with negatively charged species on the cell-surface, glycosaminoglycans. Moreover, morphology and PEG length were correlated with PIC internalisation, in which PICs with longer and denser PEG were internalised less effectively. Cell line dependency was tested using RAW 264.7 macrophage cells; PIC recognition could be maintained after capping amino groups on catiomers, indicating that the remaining anionic groups were still effectively recognised by the scavenger receptors of macrophages. Our strategy for tuning the physicochemical properties of the PEGylated PIC nanocarriers is promising for overcoming the PEG issue.

Development of novel tracers for sentinel node identification in cervical cancer.

Indocyanine green (ICG) with near-infrared (NIR) fluorescence imaging is used for lymphatic mapping. However, binding of ICG to blood proteins like serum albumin can shorten its retention time in sentinel lymph nodes (SLNs). Here, we investigated the efficacy and safety of a new fluorescence tracer comprising phytate and liposome (LP)-encapsulated ICG. Coadministration of phytate with LP containing phosphatidic acid promotes chelation mediated by Ca2+ in bodily fluids to enhance SLN retention. Uniformly sized LPs (100 nm) encapsulating ICG under conditions that minimized fluorescence self-quenching during storage were produced. We analyzed the behavior of the new tracer (ICG-phytate-LP) and control tracers (ICG and ICG-LP) in the lymphatic flow of mice in terms of lymph node retention time. We also tested lymphatic flow and safety in pigs that have a more human-like lymphatic system. LPs encapsulating stabilized ICG were successfully prepared. Mixing LP with phytate in the presence of Ca2+ increased both the particle size and negative surface charge. In mice, ICG-phytate-LP had the best lymph node retention, with a fluorescence intensity ratio that increased over 6 h and then decreased slowly over the next 24 h. In pigs, administration of ICG and ICG-phytate-LP resulted in no death or weight loss. There were no obvious differences between blood test results for the ICG and ICG-phytate-LP groups, and the overall safety was good. ICG-phytate-LP may be a useful new tracer for gynecological cancers that require time for lymph node identification due to a retroperitoneal approach.

Dynamic frustrated charge hotspots created by charge density modulation sequester globular proteins into complex coacervates.

This study presents a simple strategy for the sequestration of globular proteins as clients into synthetic polypeptide-based complex coacervates as a scaffold, thereby recapitulating the scaffold-client interaction found in biological condensates. Considering the low net charges of scaffold proteins participating in biological condensates, the linear charge density (σ) on the polyanion, polyethylene glycol-b-poly(aspartic acids), was reduced by introducing hydroxypropyl or butyl moieties as a charge-neutral pendant group. Complex coacervate prepared from the series of reduced-σ polyanions and the polycation, homo-poly-l-lysine, could act as a scaffold that sequestered various globular proteins with high encapsulation efficiency (>80%), which sometimes involved further agglomerations in the coacervates. The sequestration of proteins was basically driven by electrostatic interaction, and therefore depended on the ionic strength and charges of the proteins. However, based on the results of polymer partitioning in the coacervate in the presence or absence of proteins, charge ratios between cationic and anionic polymers were maintained at the charge ratio of unity. Therefore, the origin of the electrostatic interaction with proteins is considered to be dynamic frustrated charges in the complex coacervates created by non-neutralized charges on polymer chains. Furthermore, fluorescence recovery after photobleaching (FRAP) measurements showed that the interaction of side-chains and proteins changed the dynamic property of coacervates. It also suggested that the physical properties of the condensate are tunable before and after the sequestration of globular proteins. The present rational design approach of the scaffold-client interaction is helpful for basic life-science research and the applied frontier of artificial organelles.

Engineered macrophages acting as a trigger to induce inflammation only in tumor tissues.

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

Engineered macrophages acting as a trigger to induce inflammation only in tumor tissues based on arginase 1-responsive TNF-α accelerated release.

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

Characterization of polypropyleneimine as an alternative transfection reagent.

Polypropyleneimine (PPI) was examined as a transfection reagent comparing with most widely used polymer, polyethyleneimine (PEI). PPI had better responsiveness to the endosomal pH and showed more condensation ability of plasmid DNA than PEI. Although the cytotoxicity of PPI was somewhat higher than PEI, the transfection efficacy of PPI was comparable with PEI or higher than PEI in some cell line. Thus, PPI would be an alternative transfection reagent.

Facile preparation of hexagonal nanosheets via polyion complex formation from α-helical polypeptides and polyphosphate-based molecules.

The polyion complex-based supramolecular self-assembly of hexagonal nanosheets was achieved via the complexation of a PEGylated block catiomer with ATP and other polyphosphate-containing small molecules. The formation of hexagonal nanosheets required the presence of a polyethylene glycol block and α-helix formation in the catiomer block, which was induced by complexation with the polyphosphate moiety.

Quiescent B Cells Acquire Sensitivity to Cell Cycle Arresting Agents by B Cell Receptor Stimulation.

For the treatment of autoimmune diseases, depletion of B cells specific for auto-antigens is important because they will be a source of plasmablasts/plasma cells to produce autoantibodies. However, because some types of B cells like naïve B cells and memory B cells are at quiescent phase, they are insensitive to anticancer drugs which exert cytotoxicity by arresting the cell cycle. Here we show that B cell receptor (BCR) stimulation increases the sensitivity of anticancer drugs by promoting the proliferation of quiescent B cells. The BCR stimulation to primary naïve B cells enhanced sensitivity to several anticancer drugs which arrest the cell cycle through different mechanisms. The present results indicated that combination of the BCR stimulation and anticancer drugs is a promising strategy for the antigen-specific depletion of pathogenic quiescent B cells.

Synthesis and biological evaluation of a monocyclic Fc-binding antibody-recruiting molecule for cancer immunotherapy.

Antibody-recruiting molecules (ARMs) are bispecific molecules composed of an antibody-binding motif and a target-binding motif that redirect endogenous antibodies to target cells to elicit immune responses. To enhance the translational potential of ARMs, it is crucial to design antibody/target-binding motifs that have strong affinity and are easy to synthesize. Here, we synthesized a novel Fc-binding ARM (Fc-ARM) that targets folate receptor (FR)-positive cancer cells, Reo-3, using a recently developed monocyclic peptide 15-Lys8Leu, which binds strongly to the Fc region of an antibody. Reo-3 bound to the Fc region of the antibody with a K d of 5.8 nM, and recruited a clinically used antibody mixture to attack FR-positive IGROV-1 cells as efficiently as Fc-ARM2, in which a bicyclic Fc-binding peptide was used. These results indicate that 15-Lys8Leu, which can be synthesized readily, is suitable for various applications including the development of Fc-ARMs.

Protein Kinase C α-Responsive Gene Carrier for Cancer-Specific Transgene Expression and Cancer Therapy.

The presence of intracellular signal transduction and its abnormal activities in many cancers has potential for medical and pharmaceutical applications. We recently developed a protein kinase C α (PKCα)-responsive gene carrier for cancer-specific gene delivery. Here, we demonstrate an in-depth analysis of cellular signal-responsive gene carrier and the impact of its selective transgene expression in response to malfunctioning intracellular signaling in cancer cells. We prepared a novel gene carrier consisting of a linear polyethylenimine (LPEI) main chain grafted to a cationic PKCα-specific substrate (FKKQGSFAKKK-NH2). The LPEI-peptide conjugate formed a nanosized polyplex with pDNA and mediated efficient cellular uptake and endosomal escape. This polyplex also led to successful transgene expression which responded to the target PKCα in various cancer cells and exhibited a 10-100-fold higher efficiency compared to the control group. In xenograft tumor models, the LPEI-peptide conjugate promoted transgene expression showing a clear-cut response to PKCα. Furthermore, when a plasmid containing a therapeutic gene, human caspase-8 (pcDNA-hcasp8), was used, the LPEI-peptide conjugate had significant cancer-suppressive effects and extended animal survival. Collectively, these results reveal that our method has great potential for cancer-specific gene delivery and therapy.

A FRET-based Protein Kinase Assay Using Phos-tag-modified Quantum Dots and Fluorophore-labeled Peptides.

We have developed a novel FRET-based assay to monitor protein kinase activity using quantum dots (QDs) and fluorophore-labeled substrate peptides. To develop a FRET-based protein kinase assay, it is important to consider the phosphate group recognition strategy and to ensure that the FRET pairs are close enough because the FRET efficiency is highly dependent on the distance between the FRET pairs. Here, we incorporated a phos-tag, which captures phosphate groups strongly and selectively, into a protein kinase assay to recognize phosphorylation. Our detection system was composed of phos-tag-modified QDs and Cy5-labeled substrate peptides. Because the phos-tags capture phosphate groups by forming dinuclear complexes, the Cy5-labeled substrate peptides are captured by the phos-tags on the QD surface upon protein kinase-mediated phosphorylation, which induces FRET from the QDs to Cy5 because of the approximation of Cy5 to the QDs. On the basis of the difference of this FRET efficiency, we successfully measured protein kinase A activity, which demonstrated the feasibility of our assay.

Effect of a Chloroacetyl Modification on the Suppression of Dissociation of a Fluorescent Molecule from Cells for Antigen-Specific Cell Staining.

We previously developed a hydrolase-based fluorescence amplification method for antigen-specific cell labelling, in which fluorescent substrates stained cells by a non-covalent hydrophobic interaction. To improve the substrates retention in cells, we examined the effect of a chloroacetyl group modification on the substrate retention. We found that the chloroacetyl group suppressed the dissociation of the substrate after forming a covalent bond with intracellular proteins. However, the slow reaction speed of the chloroacetyl group allowed dissociation for cells in the early stage of the staining reaction.

Preparation of a PEGylated liposome that co-encapsulates l-arginine and doxorubicin to achieve a synergistic anticancer effect.

Strategies that combine chemotherapies with unconventional agents such as nitric oxide (NO) have been shown to enhance cancer therapies. Compared with small molecule chemotherapy drugs, nanosized particles have improved therapeutic efficacies and reduced systemic side effects because of the enhanced permeability and retention effect. In this report, we prepared PEGylated liposomes (LP) that incorporated l-arginine (Arg) and the anticancer drug doxorubicin (Dox) to yield a co-delivery system (Dox-Arg-LP). On the basis of our previous research, we hypothesized that Dox-Arg-LP should achieve a synergistic anticancer effect because Arg conversion to NO by activated M1 macrophages augments the chemotherapeutic activity of Dox. Dox-Arg-LP showed comparable physical properties to those of conventional Dox-only liposomes (Dox-LP). In vitro assessment revealed that the cytotoxicity of Dox-Arg-LP toward cancer cells was significantly higher than that of Dox-LP. In vivo application of Dox-Arg-LP in mice enhanced the chemotherapeutic effect with a 2 mg kg-1 dose of Dox-Arg-LP achieving the same therapeutic efficacy as a two-fold higher dose of Dox-LP (i.e., 4 mg kg-1). Therefore, co-encapsulation of dual agents into a liposome formulation is an efficient strategy to enhance chemotherapy while reducing systemic toxicity.

Fc-Binding Antibody-Recruiting Molecules Targeting Prostate-Specific Membrane Antigen: Defucosylation of Antibody for Efficacy Improvement*.

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc-binding antibody-recruiting molecules (Fc-ARMs). Fc-ARMs can theoretically recruit most endogenous antibodies, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc-ARMs that target prostate cancer (Fc-ARM-Ps). Fc-ARM-Ps recruited antibodies to cancer cells expressing prostate-specific membrane antigen but did so with lower efficiency compared with Fc-ARMs targeting the folate receptor. Upon recruitment by Fc-ARM-P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N-glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc-ARM strategy.

Acute palliative care unit-initiated interventions for advanced cancer patients at the end of life: prediction of impending death based on Glasgow Prognostic Score.

PURPOSE: Unexpected death occurred in an unexpectedly high proportion of advanced cancer patients in the acute palliative care unit (APCU) setting and associated with fewer signs of impending death. Recognition of patients at high risk of approaching death, especially immediately after admitting APCU among clinicians, can improve the end-of-life trajectory. Our objective was accurate prognostication within a few days of admission. METHODS: Patients admitted to an APCU of the NTT Medical Center Tokyo, Tokyo, Japan, between April 2009 and December 2016 were retrospectively examined. The Glasgow Prognostic Score (GPS) was optimized with concomitant neutrophilia, lymphocytopenia, thrombocytopenia, anemia, and monocytosis. Kaplan-Meier survival curves were estimated, and independent predictors for 3-day mortality were identified using univariate and multivariate analyses. The sensitivity, specificity, and likelihood ratios (LRs) associated with imminent death were also assessed. RESULTS: Nine hundred ninety-one patients were included; 52.9% was male. The median age was 72 years. The median survival was 13 days (IQ range 6 to 26), and 11.7% died within 3 days of admission. Significant difference in survival with a GPS of 2 was observed in GPS optimized with concomitant thrombocytopenia, and it was the only significant predictor associated with 3-day mortality (p = 0.004), which had high specificity (> 95%) and high positive LR (> 5). CONCLUSION: The prognostic value of the GPS was enhanced by adding thrombocytopenia. The concurrent use of the GPS and platelet count improved the prognostication of limited time of survival and could assist in the personal and clinical decisions for advanced cancer patients.

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.

Development of a novel molecular probe for the detection of liver mitochondrial redox metabolism.

Redox status influences the course of the inflammatory, metabolic, and proliferative liver diseases. Oxidative stress is thought to play a crucial and sustained role in the pathological progression of early steatosis to severe hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Oxidative stress induced by reactive oxygen species which are generated in the mitochondria can lead to chronic organelle damage in hepatocytes. Currently, the diagnosis of liver disease requires liver biopsy, which is invasive and associated with complications. The present report describes the development of a novel molecular probe, EDA-PROXYL, with higher reactivity and mitochondrial selectivity than standard carboxyl-PROXYL and carbamoyl-PROXYL probes. The membrane permeability of our probe improved in aqueous environments which led to increased accumulation in the liver and interaction of EDA-PROXYL with the carnitine transporter via the amine (NH3+) group further increased accumulation. This increased mitochondrial sensitivity and enhanced accumulation highlight the potential of EDA-PROXYL as a molecular probe for determining metabolic reactions of the mitochondria. Thus, this novel probe could be a tool for the evaluation of redox status of the mitochondria to assess the degree of liver injury and, ultimately, the response to pharmacological therapy.

Effect of an Endothelin B Receptor Agonist on the Tumor Accumulation of Nanocarriers.

Enhancing blood flow to tumors is a prominent strategy for improving the tumor accumulation of macromolecular drugs through the enhanced permeability and retention (EPR) effect. IRL-1620 is an agonist of the endothelin B receptor, and is a promising molecule to enhance tumor blood flow by activating endothelial nitric oxide synthase. However, contradictory effects on tumor blood flow modulation have been reported because the effects of IRL-1620 may differ in different animal models. Here, we examined for the first time the effect of IRL-1620 on the EPR effect for PEGylated liposomes in a CT-26 murine colon cancer model. Co-injection of IRL-1620 at an optimum dose (3 nmol/kg) nearly doubled the tumor accumulation of liposomes compared with controls, indicating that IRL-1620 enhanced the EPR effect in the present colon cancer model. Co-injection of IRL-1620 is a promising strategy to improve the therapeutic effects of macromolecular drugs while reducing their side effects.

Synthesis of peptide conjugates with vitamins for induction of antigen-specific immunotolerance.

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all-trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen-specific immunotolerance. We established a synthetic scheme for the preparation of the peptide-vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen-specific immunotolerance.