Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells.

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Therapeutic effect and mechanism of Daikenchuto in a model of methotrexate-induced acute small intestinal mucositis.

BACKGROUND: Daikenchuto (DKT) has positive therapeutic effects on improving various gastrointestinal disorders. The present study investigated whether or not DKT has a potential therapeutic effect on chemotherapy-induced acute small intestinal mucositis (CIM) in a rat model. METHODS: Intraperitoneal injection of 10 mg/kg methotrexate (MTX) every 3 days for a total of 3 doses was used for induction of CIM in a rat model. The MTX and DKT-MTX groups were injected with MTX as above from the first day, and the DKT-MTX and DKT groups were administered 2.7% DKT via the diet at the same time. The rats were euthanized on day 15. RESULTS: The DKT-MTX group showed an improvement in the body weight and conditions of gastrointestinal disorders as well as increased levels of diamine oxidase in plasma and in the small intestinal villi. The pathology results showed that small intestinal mucosal injury in the DKT-MTX group was less severe than that in the MTX group. Immunohistochemistry for myeloperoxidase and malondialdehyde and quantitative real-time polymerase chain reaction (RT-qPCR) for TGF-ß1 and HIF-1α showed that DKT attenuated peroxidative damage. The crypts in the DKT-MTX group contained more Ki-67-positive cells than MTX group. The zonula occluden-1 and claudin-3 results showed that DKT promoted repair of the mucosal barrier. RT-qPCR for the amino acid transporters EAAT3 and BO+AT also confirmed that DKT promoted mucosal repair and thus promoted nutrient absorption. CONCLUSION: DKT protected against MTX-induced CIM in a rat model by reducing inflammation, stimulating cell proliferation, and stabilizing the mucosal barrier.

Possible Use of Non-purine Selective Xanthine Oxidoreductase Inhibitors for Prevention of Exercise-induced Acute Kidney Injury Associated with Renal Hypouricemia.

Exercise-induced acute kidney injury (EIAKI) is frequently complicated with renal hypouricemia (RHUC). In patients with RHUC, limiting anaerobic exercise can prevent EIAKI. However, it is challenging to reduce exercise intensity in athletes. We herein report a 16-year-old Japanese football player with familial RHUC with compound heterozygous mutations in urate transporter 1 (URAT1) who presented with recurrent EIAKI. As prophylaxis (hydration during exercise) could not prevent EIAKI, febuxostat was initiated. EIAKI was not observed for 16 months despite exercising intensively. Hence, non-purine-selective xanthine oxidoreductase inhibitors may decrease the incidence of EIAKI in athletes with RHUC.

Stress exacerbates pancreatic cancer both directly and indirectly by creating an immunosuppressive environment.

BACKGROUND/PURPOSE: Sympathetic nerve stimulation by stress exacerbates various solid tumors, including pancreatic cancer (PCa). The relationship between cancer and immunity has been suggested; however, there is limited information about the effects of nerve stimulation on immunity and cancer. We aimed to investigate the involvement of sympathetic nerve stimulation in immune cells and its effects on PCa using a restraint stress mouse model. METHODS: In the in vitro experiment, the mouse-derived PCa cell line (LTPA) was cultured in a noradrenalin-supplemented medium. In the in vivo experiment, mice were divided into non-stress and stress groups. RESULTS: LTPA proliferated significantly more when cultured in a noradrenalin-supplemented medium than in a normal medium. Flow cytometry analysis of blood immune cells revealed a significant decrease in B cells, T cells, and macrophages and a significant increase in myeloid-derived suppressor cells (MDSCs) in the stress group. Furthermore, a significant increase in blood noradrenaline levels was observed in the stress group (p < .01). In the PCa mice model, immune cells in the blood showed a similar trend, and the stress group had a poor prognosis. Furthermore, immunostaining at the tumor site showed that there was a lower number of B and T cells in the stress group. In addition, MDSCs were present at the tumor margins. CONCLUSION: These results suggest that sympathetic nerve stimulation is not only directly involved in PCa growth but also exacerbates PCa by creating an immunosuppressive environment in the blood and tumor tissue.

Airway management in the pediatric emergency department in Japan: A multicenter prospective observational study.

Aim: Tracheal intubation is a vital resuscitation procedure in the pediatric emergency department (ED). Despite its importance, little is known about the current status of emergency airway management in Japan. In this context, we aimed to investigate the airway management characteristics-particularly the location, patient, and provider factors-in the pediatric ED. Methods: We conducted a multicenter, prospective study of five pediatric EDs in Japan from October 2018 to June 2020. The study included all children (aged ≤18 years) who underwent intubation at the pre-ED or ED setting by physicians and those who were transferred from the ED to the operation room (OR) or pediatric intensive care unit (PICU) for intubation. We described the airway management characteristics according to the location, patient, and provider factors. Results: Of 231 children, 9 (4%) were transferred to the OR or PICU for airway management. Among the remaining 222 children, 45 were intubated at the pre-ED setting and 177 were intubated in the ED. The overall first-attempt success rate was 72%, with the rate varying by location, patient, and provider factors-for example, 68% at the pre-ED setting, 67% for children <2 years, 56% for children with airway-related anatomical anomalies, and 61% with intubation by a resident physician. Intubation-related adverse events were observed in 17%, most of which were hypoxemia (14%). Conclusions: Based on data from a multicenter prospective study, the overall first-attempt intubation success rate in pediatric EDs in Japan was 72%, with large variations by location, patient, and provider factors.

Analysis of Purine Metabolism to Elucidate the Pathogenesis of Acute Kidney Injury in Renal Hypouricemia.

Renal hypouricemia is a disease caused by the dysfunction of renal urate transporters. This disease is known to cause exercise-induced acute kidney injury, but its mechanism has not yet been established. To analyze the mechanism by which hypouricemia causes renal failure, we conducted a semi-ischemic forearm exercise stress test to mimic exercise conditions in five healthy subjects, six patients with renal hypouricemia, and one patient with xanthinuria and analyzed the changes in purine metabolites. The results showed that the subjects with renal hypouricemia had significantly lower blood hypoxanthine levels and increased urinary hypoxanthine excretion after exercise than healthy subjects. Oxidative stress markers did not differ between healthy subjects and hypouricemic subjects before and after exercise, and no effect of uric acid as a radical scavenger was observed. As hypoxanthine is a precursor for adenosine triphosphate (ATP) production via the salvage pathway, loss of hypoxanthine after exercise in patients with renal hypouricemia may cause ATP loss in the renal tubules and consequent tissue damage.

Successful induction of human chemically induced liver progenitors with small molecules from damaged liver.

BACKGROUND: Human chemically induced liver progenitors (hCLiP) induced by small molecules produced by mature hepatocytes can potentially overcome issues related to hepatocyte transplantation, such as graft rejection or donor shortage. However, to our knowledge, no studies have explored the induction of hCLiP from mature hepatocytes (MHs) in damaged liver, indicated for liver transplantation. METHODS: Liver tissues were collected from surgically resected livers, including damaged livers, of 86 patients at our department, and hepatocytes were isolated using the collagenase perfusion method. Hepatocytes isolated from 33 of these 86 donors were cultured in YAC medium containing Y-27632 (ROCK inhibitor), A-83-01 (TGF-ß type I receptor inhibitor), and CHIR99021 (GSK-3 inhibitor) to induce hCLiP, and their functions were assessed. RESULTS: Hepatocytes were isolated regardless of the liver fibrosis classifications (viability: F0,1: 87.2 ± 13.2%; F2,3: 87.8 ± 13.1%; and F4: 86.3 ± 4.2%). Most hepatocytes cultured in the YAC medium acquired the liver progenitor cell (LPC) gene. The expression of MH markers (ALB, HNF4α, G6PC, and CYP1A2) was lower in hCLiP than in MHs before reprogramming. Reverse transcription-polymerase chain reaction revealed that hCLiP markers (e.g., EpCAM, SOX9, CK19, and CD133) exhibited higher expression in LPCs than in MHs. Furthermore, hCLiPs had the ability to differentiate into hepatocytes, and were engrafted on the liver surface as mature hepatocytes. CONCLUSION: Hepatocytes could be isolated from damaged liver. Furthermore, hCLiP may be obtained from hepatocytes isolated from damaged liver and may differentiate into MHs in vitro. Autologous hCLiP can potentially be transplanted without tumorigenesis and remodel damaged liver.

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A.

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

Effect of Pore Size of Honeycomb-Patterned Polymer Film on Spontaneous Formation of 2D Micronetworks by Coculture of Human Umbilical Vein Endothelial Cells and Mesenchymal Stem Cells.

The geometrical control of micronetwork structures ( µ NSs) formed by endothelial cells is an important topic in tissue engineering, cell-based assays, and fundamental biological studies. In this study, µ NSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb-patterned poly-l-lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in µ NS formation when the pore size is more than 20  µ m. Furthermore, µ NS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that µ NSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

Bioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluorescein assay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile.

Regenerative medicine for the hepatobiliary system: A review.

Liver transplantation, the only proven treatment for end-stage liver disease and acute liver failure, is hampered by the scarcity of donors. Regenerative medicine provides an alternative therapeutic approach. Tremendous efforts dedicated to liver regenerative medicine include the delivery of transplantable cells, microtissues, and bioengineered whole livers via tissue engineering and the maintenance of partial liver function via extracorporeal support. This brief review summarizes the current status of regenerative medicine for the hepatobiliary system. For liver regenerative medicine, the focus is on strategies for expansion of transplantable hepatocytes, generation of hepatocyte-like cells, and therapeutic potential of engineered tissues in liver disease models. For biliary regenerative medicine, the discussion concentrates on the methods for generation of cholangiocyte-like cells and strategies in the treatment of biliary disease. Significant advances have been made in large-scale and long-term expansion of liver cells. The development of tissue engineering and stem cell induction technology holds great promise for the future treatment of hepatobiliary diseases. The application of regenerative medicine in liver still lacks extensive animal experiments. Therefore, a large number of preclinical studies are necessary to provide sufficient evidence for their therapeutic effectiveness. Much remains to be done for the treatment of hepatobiliary diseases with regenerative medicine.

The Efficacy of Autologous Myoblast Sheet Transplantation to Prevent Perforation After Duodenal Endoscopic Submucosal Dissection in Porcine Model.

The recent advent of endoscopy has enabled the endoscopic submucosal dissection (ESD) of superficial nonampullary duodenal epithelial tumors. However, the substantially thin wall and presence of bile and pancreatic juice make it technically difficult to perform duodenal ESD without perforation, which leads to lethal complications. The present study evaluated the efficacy of autologous myoblast sheet transplantation for the prevention of late perforation after duodenal ESD in a porcine model. Two weeks before ESD, skeletal muscle was surgically excised from the femur of pigs, and myoblasts were isolated and seeded in temperature-responsive culture dishes to prepare sheets. Immediately after ESD, the autologous myoblast sheets were attached to the serosal surface at the ESD site with omentopexy. The pigs were divided into two groups: the autologous myoblast sheet group (n = 5), where the myoblast cell sheet was attached to the ESD ulcer part from the duodenal serous side, and the Omentum group (n = 5), where only the omentum was used. The pigs were sacrificed and analyzed macroscopically and histologically on postoperative day 3. The macroscopic examination of the abdominal cavity revealed perforation in the ESD ulcer area and leakage of bile in the Omentum group but no perforation in the Sheet group. A histopathological examination revealed that continuity of the duodenal wall at the ESD site was maintained with dense connective tissue in the Sheet group. In conclusion, autologous myoblast sheets were useful for preventing perforation after duodenal ESD.

Differentiation of chemically induced liver progenitor cells to cholangiocytes: Investigation of the optimal conditions.

Chemically induced liver progenitor (CLiP) cells, converted in vitro from mature hepatocytes, possess the bipotentiality to differentiate into both hepatocytes and cholangiocytes. Here, we aimed to investigate the optimal conditions for bile duct (BD) induction from rat CLiPs. A two-step induction protocol was used for the differentiation of cholangiocytes. We investigated the effects of passage number, preincubation times, Matrigel, and mouse embryonic fibroblast (MEF) feeder cells on the induction of cholangiocytes. Earlier passages of CLiPs were better for BD induction compared with stable CLiPs. Extending the preincubation time of CLiPs before induction delayed the formation of the BD. Matrigel provided cells with space to form three-dimensional (3D) structures, but the long-term use of Matrigel from the induction step did not benefit the differentiation of CLiPs to cholangiocytes. MEF feeder cells, through the Jag/Notch pathway, affected BD formation and function, as well as gene and protein expression. CLiPs were a good cell source for cholangiocyte differentiation under appropriate conditions and may offer a key vehicle for the study of cholangiopathies in vitro.

Changes in HepG2 spheroid behavior induced by differences in the gap distance between spheroids in a micropatterned culture system.

Micropatterning is a promising technique for modulating culture environments. In this study, we investigated the effect of spheroid separation distance on their properties in a micropatterned chip of HepG2 spheroids. The basic chip design consisted of 37 collagen spots (300 µm in diameter) in a hexagonal arrangement on a glass substrate; the region without collagen-spots was modified by polyethylene glycol to create the non-adhesive surface. Three similar chips were fabricated with gap distances between collagen-spots of 500, 1000, and 1500 µm. HepG2 cells adhered on the collagen spots and then formed spheroids via cell proliferation. Although the albumin secretion activities of HepG2 spheroids were almost the same in all chips, inhibition of spheroid growth and anaerobic metabolism were intensified when the gap distance was less than 1000 µm. Additionally, such phenomena which are induced by interference effects between spheroids, were more pronounced at the inside region of the chip than at the outside region. However, the interference effect between spheroids was nearly avoided when the gap distance was at least 1500 µm. Furthermore, the concentration of dissolved oxygen between neighboring spheroids decreased as the gap distance decreased, indicating that the spheroids competed for oxygen and became hypoxic in a way that depended on the spheroid separation distance. These results indicate that the spheroid separation distance is an important factor that can modulate the spheroid properties.

Identification of repellent odorants to the body louse, Pediculus humanus corporis, in clove essential oil.

The control of body lice is an important issue for human health and welfare because lice act as vectors of disease such as typhus, relapsing fever, and trench fever. Body lice exhibit avoidance behavior to some essential oils, including clove essential oil. Therefore, odorants containing clove essential oil components may potentially be useful in the development of repellents to body lice. However, such odorants that induce avoidance behavior in body lice have not yet been identified from clove essential oil. Here, we established an analysis method to evaluate the avoidance behavior of body lice to specific odorants. The behavioral analysis of the body lice in response to clove essential oil and its constituents revealed that eugenol, a major component of clove essential oil, has strong repellent effect on body lice, whereas the other components failed to induce obvious avoidance behavior. A comparison of the repellent effects of eugenol with those of other structurally related odorants revealed possible moieties that are important for the avoidance effects to body lice. The repellent effect of eugenol to body lice was enhanced by combining it with the other major component of clove essential oil, ß-caryophyllene. We conclude that a synthetic blend of eugenol and ß-caryophyllene is the most effective repellent to body lice. This finding will be valuable as the potential use of eugenol as body lice repellent.

Rapid evaluation of fibrinogen levels using the CG02N whole blood coagulation analyzer.

Rapid evaluation of fibrinogen (Fbg) levels is essential for maintaining homeostasis in patients with massive bleeding during severe trauma and major surgery. This study evaluated the accuracy of fibrinogen levels measured by the CG02N whole blood coagulation analyzer (A&T Corporation, Kanagawa, Japan) using heparinized blood drawn for blood gas analysis (whole blood-Fbg). A total of 100 matched pairs of heparinized blood samples and citrated blood samples were simultaneously collected from patients in the intensive care unit. Whole blood-Fbg results were compared with those of citrated plasma (standard-Fbg). The whole blood coagulation analyzer measured fibrinogen levels within 2 minutes. Strong correlations between standard-Fbg and whole blood-Fbg were observed (ρ = 0.91, p < 0.001). Error grid analysis showed that 88% of the values were clinically acceptable, and 12% were in a range with possible effects on clinical decision-making; none were in a clinically dangerous range without appropriate treatment. Using a fibrinogen cutoff value of 1.5 g/L for standard-Fbg, the area under the receiver operating characteristic curve of whole blood-Fbg was 0.980 (95% confidence interval 0.951-1.000, p < 0.001). The whole blood coagulation analyzer can rapidly measure fibrinogen levels in heparinized blood and could be useful in critical care settings where excessive bleeding is a concern.

GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

Behavioural effects of antidepressants are dependent and independent on the integrity of the dentate gyrus.

The dentate gyrus (DG), a part of the hippocampal formation, is a candidate target of antidepressants and may play a role in the development of depressive syndrome; however, there is no direct neurobiological evidence supporting this theory. Here, we examined whether DG integrity is necessary for the behavioural effects of acute or chronic antidepressant treatment. Microinjection of colchicine into DG severely damaged the granule cells, as confirmed by morphological, electrophysiological, and behavioural analyses. Acute treatment with desipramine and fluoxetine decreased the immobility of saline-treated rats in the forced swimming test, whereas this decrease was inhibited in colchicine-treated rats. Chronic treatment with desipramine and fluoxetine also decreased the immobility of saline-treated rats; however, the extensive DG damage induced by colchicine had no effect on this decrease. In the novelty-suppressed feeding test, chronic treatment with desipramine and fluoxetine decreased the latency to feed in saline-treated rats while, once again, the extensive DG damage caused by colchicine had no effect on this decrease. Thus, we concluded that DG integrity was required for the behavioural effects of acute but not chronic antidepressant treatment; this disparity was not due to the time interval between surgery and behavioural tests. These findings indicate that treatment duration determines the influence of DG integrity on antidepressant effects.

Enzymatic synthesis of poly(α-ethyl ß-aspartate) by poly(ethylene glycol) modified poly(aspartate) hydrolase-1.

We recently discovered that poly(aspartate) (PAA) hydrolase-1 from Pedobacter sp. KP-2 has a unique property of specifically cleaving the amide bond between ß-aspartate units in thermally synthesized PAA (tPAA). In the present study, the enzymatic synthesis of poly(α-ethyl ß-aspartate) (ß-PAA) was performed by taking advantage of the substrate specificity of PAA hydrolase-1. No polymerization of diethyl L-aspartate by native PAA hydrolase-1 occurred because of the low dispersibility of the enzyme in organic solvent. Poly(ethylene glycol) (PEG) modification of the enzyme improved its dispersibility and enabled it to polymerize the monomer substrate. MALDI-TOF MS analysis showed that the synthesized polymer was observed in the range of m/z = 750-2 500. This analysis also revealed that the polymer was composed of ethyl aspartate units, containing either an ethyl ester or a free carboxyl end group at its carboxyl terminus. (1) H NMR analysis demonstrated that the synthesized polymer consisted of only ß-amide linkages. Thus, the present results indicate that PAA hydrolase-1 modified with PEG is useful for the synthesis of ß-PAA due to its unique substrate specificity and good dispersibility in organic solvent.

Design of poly(ethylene glycol)/streptavidin coimmobilized upconversion nanophosphors and their application to fluorescence biolabeling.

Infrared-to-visible upconversion phosphors (i.e., rare earth ion-doped Y2O3 nanoparticles (UNPs)) were synthesized by the homogeneous precipitation method. Because the charge on the erbium (Er) ion-doped Y2O3 (Y2O3:Er) NP (UNP1) surface is positive under neutral conditions, the UNP1 surface was electrostatically PEGylated using negatively charged poly(ethylene glycol)- b-poly(acrylic acid) (PEG- b-PAAc). The adsorption of PEG- b-PAAc was confirmed by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The surface charge of the PEGylated UNP1s (PEG-UNP1s) was effectively shielded by the PEGylation. The dispersion stability of the UNP1s was also significantly improved by the PEGylation. The PEG-UNP1s were dispersed over 1 week under physiological conditions as a result of the steric repulsion between the PEG chains on the UNP1 surface. The upconversion emission spectrum of PEG-UNP1s was observed under physiological conditions and was confirmed by near-infrared excited fluorescence microscope observation. Streptavidin (SA)-installed ytterbium (Yb) and Er ion-codoped Y2O3 (Y2O3:Yb,Er) NPs (UNP2s) were prepared by the coimmobilization of PEG- b-PAAc and streptavidin. The PEG/SA coimmobilized UNP2s (PEG/SA-UNP2s) specifically recognized biotinylated antibodies and emitted strong upconversion luminescence upon near-infrared excitation. The obtained PEG/streptavidin coimmobilized UNPs are promising as high-performance near-infrared biolabeling materials.