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.

A human cell orthogonal enzyme ß-D-galacturonidase for sensitive detection of antigen proteins.

Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the same (i.e., endogenous) activity in human cells and difficulty in simultaneous use of multiple enzymes because of differences in their required reaction conditions. In this report, we identify an enzyme that can overcome these problems: ß-D-galacturonidase (GalUAase) from Eisenbergiella tayi. GalUAase activity was confirmed to be absent from human cells. The substrate of GalUAase, galacturonic acid, is highly hydrophilic because of its anionic carboxylate group; high substrate hydrophilicity is an ideal characteristic for the substrate of an enzyme used for detection because it decreases nonspecific adsorption to biological samples. We show that E. tayi GalUAase could be used in the detection of antigen proteins on live human cells with lower background signal than the conventionally used enzyme ß-D-galactosidase. The combinatorial use of GalUAase with ß-D-galactosidase enabled simultaneous detection of two antigens on live cells.

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.

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.

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.

Increased Enzyme Loading in PICsomes via Controlling Membrane Permeability Improves Enzyme Prodrug Cancer Therapy Outcome.

Mesoscopic-sized polyion complex vesicles (PICsomes) with semi-permeable membranes are promising nanoreactors for enzyme prodrug therapy (EPT), mainly due to their ability to accommodate enzymes in their inner cavity. Increased loading efficacy and retained activity of enzymes in PICsomes are crucial for their practical application. Herein, a novel preparation method for enzyme-loaded PICsomes, the stepwise crosslinking (SWCL) method, was developed to achieve both high feed-to-loading enzyme efficiency and high enzymatic activity under in vivo conditions. Cytosine deaminase (CD), which catalyzes the conversion of the 5-fluorocytosine (5-FC) prodrug to cytotoxic 5-fluorouracil (5-FU), was loaded into PICsomes. The SWCL strategy enabled a substantial increase in CD encapsulation efficiency, up to ~44% of the feeding amount. CD-loaded PICsomes (CD@PICsomes) showed prolonged blood circulation to achieve appreciable tumor accumulation via enhanced permeability and retention effect. The combination of CD@PICsomes and 5-FC produced superior antitumor activity in a subcutaneous model of C26 murine colon adenocarcinoma, even at a lower dose than systemic 5-FU treatment, and showed significantly reduced adverse effects. These results reveal the feasibility of PICsome-based EPT as a novel, highly efficient, and safe cancer treatment modality.

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.

Dietary Problems Are Associated with Frailty Status in Older People with Fewer Teeth in Japan.

This study aimed to investigate the association between dietary problems and frailty according to tooth loss in older Japanese people. This cross-sectional study included 160 older people (mean age 82.6 years) from Japan. Frailty status was assessed using the Study of Osteoporotic Fractures (SOF) criteria, which consists of (i) weight loss > 5% in the past year, (ii) inability to perform five chair stands, and (iii) self-perceived reduced energy level. Frailty was defined as the presence of ≥2 items of SOF criteria. Multivariate logistic regression analyses were performed with frailty as the dependent variable and dietary problems as the independent variable, stratified according to having <20 teeth. Low appetite and no enjoyment of eating were associated with frailty after adjusting for covariates in participants with <20 teeth. Dietary problems, including low appetite, eating alone, and negative attitudes toward enjoyment of eating were associated with a self-perceived reduced energy level in participants with <20 teeth. However, this association was not observed in participants with ≥20 teeth. In older people with fewer teeth, dietary problems have been suggested to be associated with frailty. Therefore, it may be necessary to pay attention to dietary problems, especially in older people with tooth loss.

Nanostructure Control of an Antibiotic-Based Polyion Complex Using a Series of Polycations with Different Side-Chain Modification Rates.

Developing nanovehicles for delivering antibiotics is a promising approach to overcome the issue of antibiotic resistance. This study aims to utilize a polyion complex (PICs) system for developing novel nanovehicles for polymyxin-type antibiotics, which are known as last resort drugs. The formation of antibiotic-based PIC nanostructures is investigated using colistimethate sodium (CMS), an anionic cyclic short peptide, and a series of block catiomers bearing different amounts of guanidinium moieties on their side chains. In addition, only the modified catiomer, and not the unmodified catiomer, self-assembles with CMS, implying the importance of the guanidine moieties for enhancing the interaction between the catiomer and CMS via the formation of multivalent hydrogen bonding. Moreover, micellar and vesicular PIC nanostructures are selectively formed depending on the ratio of the guanidine residues. Size-exclusion chromatography reveals that the encapsulation efficiency of CMS is dependent on the guanidinium modification ratio. The antimicrobial activity of the PIC nanostructures is also confirmed, indicating that the complexation of CMS in the PICs and further release from the PICs successfully occurs.

Specific adsorption of a ß-lactam antibiotic in vivo by an anion-exchange resin for protection of the intestinal microbiota.

The fraction of antibiotics that are excreted from the intestine during administration leads to disruption of commensal bacteria as well as resulting in dysbiosis and various diseases. To protect the gut microbiota during treatment with antibiotics, use of activated carbon (AC) has recently been reported as a method to adsorb antibiotics. However, the antibiotic adsorption by AC is nonspecific and may also result in the adsorption of essential biological molecules. In this work, we reported that an anion exchange resin (AER) has better specificity than AC for adsorbing the ß-lactam antibiotic cefoperazone (CEF). Because CEF has a negatively charged carboxylate group and a conjugated system, the AER was used to adsorb CEF through electrostatic and π-π interactions. The AER was specific for CEF over biological molecules such as bile acids and vitamins in the intestine. The AER protected Escherichia coli from CEF in vitro. Furthermore, oral administration of the AER reduced the fecal free CEF concentration, and protected the gut microbiota from CEF-induced dysbiosis.

Effect of Size and Loading of Retinoic Acid in Polyvinyl Butyrate Nanoparticles on Amelioration of Colitis.

Butyrate has been used in the treatment of inflammatory bowel diseases (IBD). However, the controlled release of butyrate has been indicated to be necessary in order to avoid the side effects verified at high concentrations. We previously developed nanoparticles (NPs) of polyvinyl butyrate (PVBu) as an oral butyrate donor for the controlled release of butyrate for the treatment of colitis. To examine the effect of the size of NPs on the therapeutic effect of colitis, here we prepared PVBu NPs with different sizes (100 nm and 200 nm). Both sizes of PVBu NPs significantly suppressed the inflammatory response in macrophages in vitro. PVBu NPs with 200 nm showed better effects on the amelioration of colitis compared with the 100 nm-NPs. We found unexpectedly that 200 nm-NP incorporated with all-trans retinoic acid (ATRA) showed a much better therapeutic effect than those with unloaded 200 nm-NPs, although ATRA alone was reported to worsen the inflammation. The synergistic effect of ATRA with butyrate shows evidence of being a promising approach for IBD treatment.

[Challenges to Creation of Artificial Organelle for Effective Editing of Cell Function].

In "cell-function editing", the combination of biological methods with artificial methods is a promising way to effectively implement functions that live cells do not originally possess. In the present symposium review, two approaches with methodology of building "artificial organelle" were implemented for editing cellular functions. One approach is the "membrane-bound artificial organelle", which is mainly created from polymeric nanocapsules that function in cells, and the other approach mimics the "membraneless organelle", which has recently gained immense interest in the field of cell biology. Furthermore, some examples of artificial cells are also described, which were constructed by utilizing artificial organelles. In this context, some recent progress has been observed in the author's research on the development of polyion complex (PIC) materials, in particular, PICsome-based nanoreactors, designer coacervates for protein sequestration, and yolk-shell PIC structures that are reminiscent of artificial cells. These technologies may contribute to effective "cell editing" or "cell renovation", which enables the edited cells to show higher performance at the target site in the human body, compared to the native 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.

Polyvinyl Butyrate Nanoparticles as Butyrate Donors for Colitis Treatment.

Butyrate has been attracting attention for the suppression of inflammatory bowel disease (IBD). However, clinical trials of butyrate for IBD treatment have resulted in controversial outcomes, likely owing to the adverse effect of butyrate on the intestinal epithelium that was observed at high butyrate concentrations. Herein, we propose polyvinyl butyrate (PVBu) nanoparticles (NPs) as butyrate donors for delivery to the lower part of the intestine for the treatment of colitis. The PVBu NPs suppressed the inflammatory activation of macrophages in vitro, although sodium butyrate inversely further activated macrophages. Oral administration of NPs did not change the luminal concentration of free butyrate; however, NPs showed a therapeutic effect on a colitis mouse model. In addition, incorporation of vitamin D3 into the NPs enhanced the therapeutic effect on colitis. Hence, PVBu NPs are a promising therapeutic for IBD treatment, not only as a butyrate donor but also as a carrier for hydrophobic drugs like vitamin D3.