Generation of a Rat Monoclonal Antibody for Human PAICS, a de novo Purine Biosynthetic Enzyme.

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.

NF9 peptide specific cytotoxic T lymphocyte clone cross react to Y453F mutation of SARS-CoV-2 virus spike protein.

The recognition by cytotoxic T cells (CTLs) is essential for the clearance of SARS-CoV-2 virus-infected cells. Several viral proteins have been described to be recognized by CTLs. Among them, the spike (S) protein is one of the immunogenic proteins. The S protein acts as a ligand for its receptors, and several mutants with different affinities for its cognate receptors have been reported, and certain mutations in the S protein, such as L452R and Y453F, have been found to inhibit the HLA-A24-restricted CTL response. In this study, we conducted a screening of candidate peptides derived from the S protein, specifically targeting those carrying the HLA-A24 binding motif. Among these peptides, we discovered that NF9 (NYNYLYRLF) represents an immunogenic epitope. CTL clones specific to the NF9 peptide were successfully established. These CTL clones exhibited the ability to recognize endogenously expressed NF9 peptide. Interestingly, the CTL clone demonstrated cross-reactivity with the Y453F peptide (NYNYLFRLF) but not with the L452R peptide (NYNYRYRLF). The CTL clone was able to identify the endogenously expressed Y453F mutant peptide. These findings imply that the NF9-specific CTL clone possesses the capability to recognize and respond to the Y453F mutant peptide.

Widely distributable and retainable in-situ gelling material for treating myocardial infarction.

Intramyocardial hydrogel injection is a promising therapy to prevent negative remodeling following myocardial infarction (MI). In this study, we report a mechanism for in-situ gel formation without external stimulation, resulting in an injectable and tissue-retainable hydrogel for MI treatment, and investigate its therapeutic outcomes. A liquid-like polymeric solution comprising poly(3-acrylamidophenylboronic acid-co-acrylamide) (BAAm), polyvinyl alcohol (PVA), and sorbitol (S) increases the viscous modulus by reducing the pre-added sorbitol concentration is developed. This solution achieves a sol-gel transition in-vitro in heart tissue by spontaneously diffusing the sorbitol. After intramyocardial injection, the BAAm/PVA/S with lower initial viscous modulus widely spreads in the myocardium and gelate compared to a viscoelastic alginate (ALG) hydrogel and is retained longer than the BAAm/S solution. Serial echocardiogram analyses prove that injecting the BAAm/PVA/S into the hearts of subacute MI rats significantly increases the fraction shortening and ejection shortening and attenuates the expansion of systolic LV diameter for up to 21 d after injection compared to the saline injection as a control, but the ALG injection does not. In addition, histological evaluation shows that only the BAAm/PVA/S decreases the infarct size and increases the wall thickness 21 d after injection. The BAAm/PVA/S intramyocardial injection is better at restraining systolic ventricular dilatation and cardiac failure in the rat MI model than in the control groups. Our findings highlight an effective injectable hydrogel therapy for MI by optimizing injectability-dependent distribution and retention of injected material. STATEMENT OF SIGNIFICANCE: In-situ gelling material is a promising strategy for intramyocardial hydrogel injection therapy for myocardial infarction (MI). Since the sol-gel transition of reported materials is driven by external stimulation such as temperature, pH, or ultraviolet, their application in vivo remains challenging. In this study, we first reported a synthetic in-situ gelling material (BAAm/PVA/S) whose gelation is stimulated by spontaneously reducing pre-added sorbitol after contacting the heart tissue. The BAAm/PVA/S solution spreads evenly, and is retained for at least 21 d in the heart tissue. Our study demonstrated that intramyocardial injection of the BAAm/PVA/S with more extensive distribution and longer retention had better effects on preventing LV dilation and improving cardiac function after MI than that of viscoelastic ALG and saline solution. We expect that these findings provide fundamental information for the optimum design of injectable biomaterials for treating MI.

HER-2-Targeted Boron Neutron Capture Therapy with Carborane-integrated Immunoliposomes Prepared via an Exchanging Reaction.

Boron neutron capture therapy (BNCT) is a promising modality for cancer treatment because of its minimal invasiveness. To maximize the therapeutic benefits of BNCT, the development of efficient platforms for the delivery of boron agents is indispensable. Here, carborane-integrated immunoliposomes were prepared via an exchanging reaction to achieve HER-2-targeted BNCT. The conjugation of an anti-HER-2 antibody to carborane-integrated liposomes successfully endowed these liposomes with targeting properties toward HER-2-overexpressing human ovarian cancer cells (SK-OV3); the resulting BNCT activity toward SK-OV3 cells obtained using the current immunoliposomal system was 14-fold that of the l-BPA/fructose complex, which is a clinically available boron agent. Moreover, the growth of spheroids treated with this system followed by thermal neutron irradiation was significantly suppressed compared with treatment with the l-BPA/fructose complex.

SARS-CoV-2 spike protein-derived immunogenic peptides that are promiscuously presented by several HLA-class II molecules and their potential for inducing acquired immunity.

The current coronavirus disease 2019 (COVID-19) pandemic that is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a significant threat to public health. Although vaccines based on the mRNA of the SARS-CoV-2 spike protein have been developed to induce both cellular and humoral immunity against SARS-CoV-2, there have been some concerns raised about their high cost, particularly in developing countries. In the present study, we aim to identify an immunogenic peptide in the SARS-CoV-2 spike protein to activate cellular immunity, particularly CD4+ helper T lymphocytes (Th cells), which are a commander of immune system. SARS-CoV-2 spike protein-derived peptides Spike448-477 and Spike489-513(N501Y)-specific CD4+ Th cell lines were generated by repetitive stimulation of healthy donor-derived CD4+T-cells with each peptide. Their HLA-restrictions were addressed by using blocking antibodies against HLA and HLA-transfected L-cells. The epitopes of Spike448-477-specific CD4+ Th cell lines were defined using a series of 7-14-mer overlapping truncated peptides and alanine-substituted epitope peptides. To address responsiveness of these CD4+ Th cell lines to several SARS-CoV-2 variants, we stimulated the CD4+ Th cell lines with mutated peptides. We addressed whether these identified peptides were useful for monitoring T-cell-based immune responses in vaccinated donors using the IFN-γ ELISpot assay. The Spike448-477 peptide was found to be a promiscuous peptide presented by HLA- DRB1*08:02, DR53, and DPB1*02:02. Although HLA-DPB1*02:02-restricted CD4+ Th cells did not response to some peptides with the L452R and L452Q mutations, the other CD4+ Th cells were not affected by any mutant peptides. We developed two tetramers to detect HLA-DRB1*08:02/Spike449-463- and Spike449-463(L452R/Y453F)-recognizing CD4+ Th cells. Spike489-513(N501Y) peptide was also a promiscuously presented to HLA-DRB1*09:01 and DRB1*15:02. The T-cell responses specific to both peptides Spike448-477 and Spike489-513 were detected in PBMCs after vaccinations. In addition, we observed that the Spike448-477 peptide activated both CD8+ T-cells and CD4+ Th cells in individuals receiving mRNA vaccines. SARS-CoV-2 spike protein-derived peptides, Spike448-477 and Spike489-513, include several epitopes that are presented by multiple HLA-class II alleles to activate CD4+ Th cells, which are considered useful for monitoring the establishment of acquired immunity after vaccination.

HER-2-targeted boron neutron capture therapy using an antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex.

The development of boron agents with integrated functionality, including biocompatibility, high boron content, and cancer cell targeting, is desired to exploit the therapeutic efficacy of boron neutron capture therapy (BNCT). Here, we report the therapeutic efficacy of BNCT using a HER-2-targeted antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex. The anticancer effect of BNCT using our system was 30-fold that of the clinically available boron agent l-BPA/fructose complex.

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy.

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.

Carborane bearing pullulan nanogel-boron oxide nanoparticle hybrid for boron neutron capture therapy.

Boron neutron capture therapy shows is a promising approach to cancer therapy, but the delivery of effective boron agents is challenging. To address the requirements for efficient boron delivery, we used a hybrid nanoparticle comprising a carborane = bearing pullulan nanogel and hydrophobized boron oxide nanoparticle (HBNGs) enabling the preparation of highly concentrated boron agents for efficient delivery. The HBNGs showed better anti-cancer effects on Colon26 cells than a clinically boron agent, L-BPA/fructose complex, by enhancing the accumulation and retention amount of the boron agent within cells in vitro. The accumulation of HBNGs in tumors, due to the enhanced permeation and retention effect, enabled the delivery of boron agents with high tumor selectivity, meeting clinical demands. Intravenous injection of boron neutron capture therapy (BNCT) using HBNGs decreased tumor volume without significant body weight loss, and no regrowth of tumor was observed three months after complete regression. The therapeutic efficacy of HBNGs was better than that of L-BPA/fructose complex. BNCT with HBNGs is a promising approach to cancer therapeutics.

Prevention of anastomotic stenosis for decellularized vascular grafts using rapamycin-loaded boronic acid-based hydrogels mimicking the perivascular tissue function.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induces graft anastomotic stenosis, resulting in graft failure. Herein, we developed a drug-loaded tissue-adhesive hydrogel as artificial perivascular tissue to suppress VSMCs proliferation. Rapamycin (RPM), an anti-stenosis drug, is selected as the drug model. The hydrogel was composed of poly (3-acrylamidophenylboronic acid-co-acrylamide) (BAAm) and polyvinyl alcohol. Since phenylboronic acid reportedly binds to sialic acid of glycoproteins which is distributed on the tissues, the hydrogel is expected to be adherent to the vascular adventitia. Two hydrogels containing 25 or 50 mg/mL of BAAm (BAVA25 and BAVA50, respectively) were prepared. A decellularized vascular graft with a diameter of <2.5 mm was selected as a graft model. Lap-shear test indicates that both hydrogels adhered to the graft adventitia. In vitro release test indicated that 83 and 73 % of RPM in BAVA25 and BAVA50 hydrogels was released after 24 h, respectively. When VSMCs were cultured with RPM-loaded BAVA hydrogels, their proliferation was suppressed at an earlier stage in RPM-loaded BAVA25 hydrogels compared to RPM-loaded BAVA50 hydrogels. An in vivo preliminary test reveals that the graft coated with RPM-loaded BAVA25 hydrogel shows better graft patency for at least 180 d than the graft coated with RPM-loaded BAVA50 hydrogel or without hydrogel. Our results suggest that RPM-loaded BAVA25 hydrogel with tissue adhesive characteristics has potential to improve decellularized vascular graft patency.

Tumor-targeting hyaluronic acid/fluorescent carborane complex for boron neutron capture therapy.

In cancer therapeutics, boron neutron capture therapy (BNCT) requires a platform for selective and efficient 10B delivery into tumor tissues for a successful treatment. However, the use of carborane, a promising candidate with high boron content and biostability, has significant limitations in the biomedical field due to its poor water-solubility and tumor-selectivity. To overcome these hurdles, we present in this study a fluorescent nano complex, combining fluorescent carborane and sodium hyaluronate for high boron concentration and tumor-selectivity. Tumor cells actively internalized the complex through binding hyaluronan to CD44, overexpressed on the tumor cell surface. Furthermore, the subcellular distribution of this complex could also be detected due to its fluorescent properties. Moreover, after thermal neutron irradiations, the complex produced excellent cytotoxicity, equal to or greater than that of the clinically-used BPA-fructose. Therefore, this novel complex could be potentially more suitable for BNCT than the boron agent.

Tumor Growth Suppression With Novel Intra-arterial Chemotherapy Using Epirubicin-entrapped Water-in-oil-in-water Emulsion In Vivo.

BACKGROUND/AIM: A mixture of anticancer agents and iodized poppy seed oil (IPSO) has been widely used for intra-arterial chemotherapy of hepatocellular carcinoma. However, the anticancer agents can easily separate from IPSO, so the therapeutic potential is limited. We developed epirubicin-entrapped water-in-oil-in-water emulsion (WOW-Epi) using a double-membrane emulsification technique. MATERIALS AND METHODS: We delivered WOW-Epi through a hepatic arterial injection to VX2 hepatic tumor rabbit model (1.2 mg/kg). RESULTS: VX2 tumor growth was selectively suppressed in the WOW-Epi-treated group compared with the control treated groups. The accumulation of WOW in nearby cancer cells was confirmed via electron-microscopy. Endocytosis seemed to be the mechanism underlying the uptake of WOW. CONCLUSION: WOW-Epi led to tumour growth suppression in vivo. WOW does not cause toxicity to arterial vessels. WOW-Epi will be hopefully used for repeated intra-arterial chemotherapy to HCC patients in the near future.

Mechanism toward Turn-on of Polysaccharide-Porphyrin Complexes for Fluorescence Probes and Photosensitizers in Photodynamic Therapy in Living Cells.

ß-(1,3-1,6)-D-Glucan, λ-carrageenan, tamarind gum, and pullulan can dissolve various porphyrin derivatives via the formation of complexes in water using a high-speed vibration milling method. The aqueous solutions of the resulting complexes exhibit long-term stability. Despite the adverse effects of the self-quenching process, notable fluorescence and improved photodynamic activity of the polysaccharide-complexed porphyrin derivatives were observed in the presence of liposomes, micelles, cyclodextrins, and HeLa cells. It was noted that the type of porphyrins was more important than the type of polysaccharides present in the complex. Porphyrin self-aggregates were monodispersed in the lipid membranes of the liposomes and lysosomes. The polysaccharide-complexed porphyrin derivatives showed increased photodynamic activity toward HeLa cells under photoirradiation between 610 and 740 nm.

Improved Stability and Photodynamic Activity of Water-Soluble 5,15-Diazaporphyrins Incorporated in ß-(1,3-1,6)-d-Glucan with On-Off Switch.

5,15-Diazaporphyrins, which have a large absorption at wavelengths over 600 nm, were dissolved in water by complex formation with ß-(1,3-1,6)-d-glucans. Aqueous solutions of these complexes were relatively stable compared with their trimethyl-ß-cyclodextrin-complexed analogues. ß-Glucan-complexed diazaporphyrins showed quenched fluorescence and had low singlet-oxygen-generation abilities owing to random self-aggregation. However, external stimuli, such as the presence of liposomes or intracellular uptake, restored the fluorescence and singlet-oxygen-generation abilities of ß-glucan-complexed diazaporphyrins. Consequently, ß-glucan-complexed diazaporphyrins showed very high photodynamic activities toward HeLa cells.

Turn-on fluorescence and photodynamic activity of ß-(1,3-1,6)-d-glucan-complexed porphyrin derivatives inside HeLa cells.

Fluorescence intensities of water-soluble ß-(1,3-1,6)-d-glucan (ß-1,3-glucan)-complexed porphyrin derivatives were very weak as a result of self-quenching. However, ß-1,3-glucan-complexed tetra(aminophenyl)porphyrin exhibited 'off-state' to 'on-state' fluorescence switching activity by intracellular uptake. Furthermore, the internalised complex showed a high level of photodynamic activity toward HeLa cells under photoirradiation at long wavelengths.

Correction: Formation of ß-(1,3-1,6)-d-glucan-complexed [70]fullerene and its photodynamic activity towards macrophages.

Correction for 'Formation of ß-(1,3-1,6)-d-glucan-complexed [70]fullerene and its photodynamic activity towards macrophages' by Atsushi Ikeda et al., Org. Biomol. Chem., 2017, 15, 1990-1997.

Water Solubilization of Fullerene Derivatives by ß-(1,3-1,6)-d-Glucan and Their Photodynamic Activities toward Macrophages.

Anionic and neutral fullerene derivatives were dissolved in water by using ß-(1,3-1,6)-d-glucan (ß-1,3-glucan) as a solubilizing agent. In the water-solubilized complexes, the concentrations of fullerene derivatives were ≈0.30 mm and the average particle sizes were ≈90 nm. The ß-1,3-glucan-complexed fullerene derivative with a carboxylic acid was found to have higher photodynamic activity toward macrophages under visible-light irradiation (λ>610 nm) than other ß-1,3-glucan-complexed fullerene derivatives. This result suggests that carboxylic acid moieties in the complex enhance the binding affinity with ß-1,3-glucan receptors on the surface of macrophages when the ß-1,3-glucan is recognized. In contrast, all ß-1,3-glucan-complexed fullerene derivatives showed no photodynamic activity toward HeLa cells under the same conditions.

Formation of ß-(1,3-1,6)-d-glucan-complexed [70]fullerene and its photodynamic activity towards macrophages.

[70]Fullerene was dissolved in water by complexation with ß-1,3-glucan using a mechanochemical high-speed vibration milling apparatus. The photodynamic activity of ß-1,3-glucan-complexed C70 was highly dependent on the expression level of dectin-1 on the cell surfaces of macrophages. The photodynamic activity increased as a result of a synergistic effect between ß-1,3-glucan-complexed 1'-acetoxychavicol acetate and the C70 complex.

Lipid-membrane-incorporated arylboronate esters as agents for boron neutron capture therapy.

Arylboronate esters bearing methyl groups in both of their ortho positions were stably incorporated into lipid membranes at high concentrations without undergoing hydrolysis to the corresponding boronic acids. This method could be used in combination with previous methods to increase the maximum ratio of boron atoms in liposomal boron carriers.

Anti-inflammatory effect of water-soluble complex of 1'-acetoxychavicol acetate with highly branched ß-1,3-glucan on contact dermatitis.

The anti-inflammatory effect on contact dermatitis of the water solubilized 1'-Acetoxychavicol Acetate (ACA) by complexation with ß-1,3-glucan isolated form Aureobasidium pullulans black yeast is reported. It is well-known that ACA possesses a function to inhibit the activation of NF-κB by which genes encoding proinflammatory cytokines, chemokines, and growth factors are regulated. However, because ACA is quite insoluble in water, its usefulness has been extremely limited. On the other hand, a triple-helical polysaccharide ß-1,3-glucan can include hydrophobic compounds into intrastrand hydrophobic cavity and solubilize poorly water-soluble compounds. In this study, solubilization of ACA by complexation with highly branched ß-1,3-glucan was achieved. The effect of anti-inflammatory response of water-soluble ACA complex with ß-1,3-glucan was confirmed in vitro and in vivo.

Tumor growth suppression by gadolinium-neutron capture therapy using gadolinium-entrapped liposome as gadolinium delivery agent.

Neutron capture therapy (NCT) is a promising non-invasive cancer therapy approach and some recent NCT research has focused on using compounds containing gadolinium as an alternative to currently used boron-10 considering several advantages that gadolinium offers compared to those of boron. In this study, we evaluated gadolinium-entrapped liposome compound as neutron capture therapy agent by in vivo experiment on colon-26 tumor-bearing mice. Gadolinium compound were injected intravenously via tail vein and allowed to accumulate into tumor site. Tumor samples were taken for quantitative analysis by ICP-MS at 2, 12, and 24 h after gadolinium compound injection. Highest gadolinium concentration was observed at about 2 h after gadolinium compound injection with an average of 40.3 µg/g of wet tumor tissue. We performed neutron irradiation at JRR-4 reactor facility of Japan Atomic Energy Research Institute in Tokaimura with average neutron fluence of 2×10¹² n/cm². The experimental results showed that the tumor growth suppression of gadolinium-injected irradiated group was revealed until about four times higher compared to the control group, and no significant weight loss were observed after treatment suggesting low systemic toxicity of this compound. The gadolinium-entrapped liposome will become one of the candidates for Gd delivery system on NCT.