Surface Glycan Profiling of Extracellular Vesicles by Lectin Microarray and Glycoengineering for Control of Cellular Interactions.

BACKGROUND: Extracellular vesicles (EVs) are a group of cell-derived membrane vesicles that carry a variety of cargo such as protein, nucleic acids, and lipids, and are secreted by almost all cell types. Functionally, EVs play important roles in physiological and pathological processes such as immune responses and tumor growth through intercellular communication by transferring this molecular information between cells. Therefore, they have potential versatile clinical applications as disease biomarkers and drug delivery carriers. PROBLEM: Notably, subpopulations of EVs exhibit distinct characteristics depending on their cell of origin, including the expression of surface glycans, which have been implicated in a variety of cellular processes such as field cancerization, cell recognition, and signal transduction. However, these are features have not been fully exploited because of the difficulty in analyzing these proteins. APPROACH: In this paper, we summarize the advancements in glycoengineering and high-performance lectin microarray for high-throughput analysis of EV glycans to generate an index of heterogeneity to identify disease biomarkers, and describe how understanding the function of EVs in disease can enhance their potential application in the clinic.

Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media.

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 µm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

CD63-positive extracellular vesicles are potential diagnostic biomarkers of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest gastrointestinal cancers with a 5-year survival rate of less than 10%. Biomarkers for early PDAC detection are useful in treating patients with PDAC. Extracellular vesicles (EVs) are lipid-bound vesicles that are potential biomarkers of various diseases such as PDAC. In this study, we quantitatively measured the serum levels of EVs (CD63+-EVs) or platelet-derived EVs (CD41+- and CD61+-EVs) and evaluated their potential use as biomarkers of PDAC. METHODS: We measured the serum levels of CD63+-, CD41+-, CD61+-EVs using sandwich enzyme-linked immunosorbent assay based on Tim4 with specificity for phosphatidylserine on EVs in age- and sex-matched healthy controls (HCs, n = 39) and patients with PDAC (n = 39). We also examined the effect of tumor burden on the serum EV levels after surgical resection (n = 28). CA19-9, a clinical PDAC biomarker, was also measured for comparison. RESULTS: Serum levels of CD63+-EVs, CD41+-EVs, and CD61+-EVs were significantly increased in patients with PDAC compared to HCs. Receiver operating characteristic analysis revealed that CD63+-EVs exhibited the highest diagnostic performance to discriminate patients with PDAC from HCs (area under the curve (AUC): 0.846), which was comparable to CA19-9 (AUC: 0.842). CA19-9 showed lower AUC values in early stages (I-II, AUC: 0.814) than in late stages (III-IV, AUC: 0.883) PDAC. Conversely, CD63+-EVs, CD41+-EVs, and CD61+-EVs showed comparable AUCs between early- and late-stage PDAC. The combined use of CA19-9 and CD63+-EVs showed a higher diagnostic performance for early-stage PDAC (AUC: 0.903) than CA19-9. The serum levels of CD63+-EVs, CD41+-EVs, CD61+-EVs, and CA19-9 decreased significantly after surgical resection, demonstrating that EVs are increased in sera of patients depending on the tumor burden. CONCLUSIONS: The serum levels of CD63+-EVs and platelet-derived EVs (CD41+-EVs, CD61+-EVs) are increased in patients with PDAC than HCs. Since CD63+-EVs showed a high AUC to discriminate patients with PDAC from HCs; they might be useful as potential biomarkers for PDAC.

Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

The development of a new large-scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high-performance exosomes (EXOs) by using an anion-exchange method. Cytotoxic T-lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut-off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M-0.3 M) and high (0.3 M-0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion-exchange column chromatography. The former are abundant in EXO proteins, including late endosome-associated proteins and rab-family and integrin-family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)-like particles including DNA, core histone and ribosomal proteins, and GC-rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion-exchange method is suitable for the large-scale separation of bioactive EXOs and MV-like EVs as a cargo for dangerous nucleic acids at high-purity.

Assessment of Surface Glycan Diversity on Extracellular Vesicles by Lectin Microarray and Glycoengineering Strategies for Drug Delivery Applications.

Extracellular vesicles (EVs) are released by all types of mammalian cells for cell-cell communication. In this study, surface glycans on EVs are compared in terms of their cell type, size, and isolation method to examine whether EV glycan profiles by lectin microarray can be used to define EV subpopulations. Moreover, EVs are glycoengineered with four distinctive surface glycan patterns and evaluated their cellular uptake efficiencies for potential drug delivery applications. Both similarities and differences in glycan patterns are identified on EVs obtained under each experimental condition. EV size- and isolation method-dependent lectin-binding patterns are observed. Moreover, cellular uptake behaviors of EVs are affected by EV glycan profiles and acceptor cells. The in vivo biodistribution of EVs is also dependent on their glycan profile. These results suggest that EV surface glycans are a potential novel indicator of EV heterogeneity, and glycoengineering is a useful approach to regulate cell-EV interactions for biomedical applications.

New Lipophilic Fluorescent Dyes for Labeling Extracellular Vesicles: Characterization and Monitoring of Cellular Uptake.

PKH dyes, which are currently the most widely used fluorescent probes for extracellular vesicle (EV) labeling, have some limitations. For example, these dyes tend to aggregate, leading to formation of EV-like nanoparticles that can be taken up by cells. Moreover, it has been suggested that PKH dyes trigger an enlargement of EVs because of membrane fusion or intercalation. To overcome these limitations, we developed three novel extracellular vesicular-membrane-binding fluorescent probes-Mem dye-Green, Mem dye-Red, and Mem dye-Deep Red-for monitoring EV uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold and amphiphilic moieties on the cyanine. The three dyes have different photophysical characteristics. To investigate the characteristics of the Mem dyes for EV labeling, we performed nanoparticle tracking, zeta potential measurements, and confocal microscopy. The dyes enable highly sensitive fluorescence imaging of EVs. They can also be used to observe EV dynamics in live cells. The Mem dyes show excellent EV labeling with no aggregation and less particle enlargement.

Platelet-derived extracellular vesicles are increased in sera of Alzheimer's disease patients, as revealed by Tim4-based assays.

Alzheimer's disease (AD) is the most common form of dementia, characterized by the accumulation of ß-amyloid plaques and the formation of neurofibrillary tangles. Extracellular vesicles (EVs) are small vesicles surrounded by a lipid bilayer membrane, which may be involved in the progression of AD. Glycans are essential building blocks of EVs, and we hypothesized that EV glycans may reflect pathological conditions of various diseases. Here, we performed glycan profiling of EVs prepared from sera of three AD patients (APs) compared to three healthy donors (HDs) using lectin microarray. Distinct glycan profiles were observed. Mannose-binding lectins exhibited significantly higher signals for AP-derived EVs than HD-derived EVs. Lectin blotting using mannose-binding lectin (rPALa) showed a single protein band at ~ 80 kDa exclusively in AP-derived EVs. LC-MS/MS analysis identified a protein band precipitated by rPALa as CD61, a marker of platelet-derived exosomes (P-Exo). Sandwich assays using Tim4 with specificity for phosphatidylserine on EVs and antibodies against P-Exo markers (CD61, CD41, CD63, and CD9) revealed that P-Exo is significantly elevated in sera of APs (n = 16) relative to age- and sex-matched HDs (n = 16). Tim4-αCD63 showed the highest value for the area under the curve (0.957) for discriminating APs from HDs, which should lead to a better understanding of AD pathology and may facilitate the development of a novel diagnostic method for AD.

Exosome surface glycans reflect osteogenic differentiation of mesenchymal stem cells: Profiling by an evanescent field fluorescence-assisted lectin array system.

Extracellular vesicles (EVs) carry information between cells in the form of biomolecules. Such molecules have been found to serve as biomarkers. Glycans attached to surface molecules on EVs are involved in their cellular uptake. In this study, we examined glycan profiles of small EVs which are generally termed exosomes before and after osteogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) by an evanescent field fluorescence-assisted (EFF)-lectin array system to discover glycan biomarkers for osteogenic differentiation. We found few differences between exosomes before and after osteogenic differentiation of MSCs in terms of fundamental characteristics such as size, morphology, and exosomal marker proteins. However, specific lectins bound strongly to exosomes from differentiated cells. Exosomes from osteogenically differentiated MSCs bound strongly to fucose- and mannose-binding lectins, especially at a high concentration of exosomes. In summary, we found that several lectins bound to exosomes from differentiated MSCs more strongly than to those from undifferentiated cells using an EFF-lectin array system, indicating that monitoring exosomal surface glycans may identify predictive indexes of osteogenic differentiation.

Glycan profiling analysis using evanescent-field fluorescence-assisted lectin array: Importance of sugar recognition for cellular uptake of exosomes from mesenchymal stem cells.

Studies involving the functional analysis of exosomal contents including proteins, DNA, and RNA have been reported. Most membrane proteins and lipids are glycosylated, which controls their physical properties and functions, but little is known about glycans on exosomes owing to the difficulty of analysing them. To shed light on these issues, we collected exosomes from mesenchymal stem cells (MSCs) derived from human adipose tissue for glycan profiling using evanescent-field fluorescence-assisted lectin array as well as analysis of their uptake in vivo. Initial analyses showed that the mean diameter of the collected exosomes was 178 nm and they presented with typical exosomal and MSC markers. Regarding the glycan profiling, exosomes interacted more strongly than the membrane of the original MSCs did with a range of lectins, especially sialic acid-binding lectins. The findings also showed that cellular exosome uptake involved recognition by HeLa cell-surface-bound sialic acid-binding immunoglobulin (Ig)-like lectins (siglecs). Confirming this siglec-related uptake, in vivo experiments involving subcutaneous injection of the fluorescently labelled exosomes into mice showed their transport into lymph nodes and internalization by antigen-presenting cells, particularly those expressing CD11b. Closer analysis revealed the colocalization of the exosomes with siglecs, indicating their involvement in the uptake. These findings provide us with an improved understanding of the importance of exosomal transport and targeting in relation to glycans on exosomal surfaces, potentially enabling us to standardize exosomes when using them for therapeutic purposes.

Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA.

CagA, encoded by cytotoxin-associated gene A (cagA), is a major virulence factor of Helicobacter pylori, a gastric pathogen involved in the development of upper gastrointestinal diseases. Infection with cagA-positive H. pylori may also be associated with diseases outside the stomach, although the mechanisms through which H. pylori infection promotes extragastric diseases remain unknown. Here, we report that CagA is present in serum-derived extracellular vesicles, known as exosomes, in patients infected with cagA-positive H. pylori (n = 4). We also found that gastric epithelial cells inducibly expressing CagA secrete exosomes containing CagA. Addition of purified CagA-containing exosomes to gastric epithelial cells induced an elongated cell shape, indicating that the exosomes deliver functional CagA into cells. These findings indicated that exosomes secreted from CagA-expressing gastric epithelial cells may enter into circulation, delivering CagA to distant organs and tissues. Thus, CagA-containing exosomes may be involved in the development of extragastric disorders associated with cagA-positive H. pylori infection.

Nanogel-crosslinked nanoparticles increase the inhibitory effects of W9 synthetic peptide on bone loss in a murine bone resorption model.

We investigated the biological activity of W9, a bone resorption inhibitor peptide, using NanoClik nanoparticles as an injectable carrier, where acryloyl group-modified cholesterol-bearing pullulan (CHPOA) nanogels were crosslinked by pentaerythritol tetra (mercaptoethyl) polyoxyethylene. Thirty 5-week-old male C57BL/6J mice were fed a low calcium diet and received once-daily subcutaneous injections of the carrier alone, W9 24 mg/kg/day alone, W9 24 mg/kg/day incorporated in cholesterol bearing pullulan (CHP) nanogels, or W9 (8 and 24 mg/kg/day) incorporated in NanoClik nanoparticles for 4 days (n=5). Mice that received a normal calcium diet with NanoClik nanoparticle injections without W9 were used as a control group. Radiological analyses showed that administration of W9 24 mg/kg/day significantly prevented low calcium-induced reduction of bone mineral density in the long bones and lumbar vertebrae, but only when the NanoClik nanoparticles were used as a carrier. Histomorphometric analyses of the proximal tibiae revealed that W9 24 mg/kg/day incorporated in NanoClik nanoparticles prevented the increase in bone resorption indices induced by a low calcium diet, which was confirmed by measurement of serum bone resorption markers. These data suggest that NanoClik nanoparticles could be a useful carrier for peptide therapeutics, and also demonstrate that daily subcutaneous injections of the W9 peptide with the nanoparticles were able to inhibit bone loss in vivo. An osteoclastogenesis inhibition assay performed in vitro confirmed a slower release profile of W9 from NanoClik nanoparticles compared with conventional CHP nanogels.

Cholesteryl group- and acryloyl group-bearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering.

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering.

Dual crosslinked hydrogel nanoparticles by nanogel bottom-up method for sustained-release delivery.

Polysaccharide-PEG hybrid nanogels (CHPOA-PEGSH) crosslinked by both covalent ester bonds and physical interactions were prepared by the reaction of a thiol-modified poly(ethylene glycol) (PEGSH) with acryloyl-modified cholesterol-bearing pullulan (CHPOA). Experimental parameters, including CHPOA concentration, the degree of acryloyl substitution of CHPOA, and the initial amounts of CHPOA and PEGSH, were modified in order to assess their effect on the size of the nanogels (50-150 nm) and on their degradation kinetics, monitored by dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) chromatography. Rhodamine-labeled nanogels were injected intravenously into mice and their concentration in blood was determined by a fluorescence assay as a function of post-injection time. The elimination half-life (t(1/2)) of CHPOA-PEGSH nanoparticles was about 15-fold longer (18 h) than that of CHP nanogels (1.2 h). The half-life enhancement of CHPOA-PEGSH was attributed to the presence of the crosslinker PEG chains, which prevent non-specific protein adsorption, and to the slow hydrolysis kinetics of the crosslinking esters in the biological milieu. The hybrid CHPOA-PEGSH nanogels are expected to be useful as injectable nanocarriers for drugs and proteins, in view of their low surface fouling and slow hydrolysis rate.

Exploitation of a novel polysaccharide nanogel cross-linking membrane for guided bone regeneration (GBR).

Cholesterol-bearing pullulan (CHP) nanogel is a synthetic degradable biomaterial for drug delivery with high biocompatibility. Guided bone regeneration (GBR) is a bone augmentation technique in which a membrane is used to create and keep a secluded regenerative space. The purpose of the present study was to evaluate the effects of the novel CHP nanogel membrane in GBR. Thirty-six adult Wistar rats were used and bilaterally symmetrical full-thickness parietal bone defects of 5 mm diameter were created with a bone trephine burr. Each defect was covered with the collagen membrane or the CHP nanogel membrane or untreated without any membrane. The animals were sacrificed at 2, 4 and 8 weeks and analysed radiologically and histologically. Furthermore, after incubating human serum with CHP nanogel or collagen, the amount of PDGF in the serum was measured using ELISA. New bone formation in terms of bone volume was higher in the nanogel group than in the control or collagen groups at 2 and 4 weeks. At 8 weeks, both membrane groups showed higher bone volumes than the control group. Notably, the newly-formed bone in the bone defect in the nanogel group was uniform and histologically indistinguishable from the original bone, whereas in the collagen group the new bone showed an irregular structure that was completely different from the original bone. After incubating with CHP nanogel, the amount of PDGF in the serum decreased significantly. CHP nanogel GBR membrane favourably stimulated bone regeneration, in which a unique characteristic of CHP nanogel, the storage of endogenous growth factors, was likely implicated.

The tumor necrosis factor type 2 receptor plays a protective role in tumor necrosis factor-α-induced bone resorption lacunae on mouse calvariae.

Tumor necrosis factor (TNF)-α exerts its biological function via TNF type 1 and type 2 receptors (TNFR1 and TNFR2). We have previously reported that bone resorption induced by lipopolysaccharide (LPS) in TNFR2-deficient mice is accelerated compared to that in wild-type (WT) mice. Although these results suggested that TNFR2 might have a protective role in bone resorption, we could not exclude the possibility that TNFR2 has no role in bone resorption. To clarify the role of TNFR2, we developed a TNF-α-induced bone resorption model using cholesterol-bearing pullulan nanogel as a TNF-α carrier to minimize the influence of inflammatory cytokines other than TNF-α. Injections of human TNF-α (hTNF), an agonist of mouse TNFR1, stimulated bone resorption lacunae on the calvariae in WT mice, but mouse TNF-α (mTNF), an agonist of both mouse TNFR1 and TNFR2, could not. To eliminate the possibility that the TNFR1 agonistic effects of hTNF were stronger than those of mTNF, we used the same model in TNFR2-deficient mice. Injection of mTNF resulted in clear bone resorption lacunae to the same extent observed after using hTNF in the TNFR2-deficient mice. Histomorphometric analysis of osteoclast number supported the observed changes in bone resorption lacunae. These data suggest that TNFR2 has a protective role in TNF-α-induced bone resorption.

Cell specific peptide-conjugated polysaccharide nanogels for protein delivery.

A cell specific peptide (Arg-Gly-Asp; RGD)-modified nanogel was prepared and evaluated for its potential to act as a protein delivery carrier. A bovine serum albumin (BSA)/RGD-modified nanogel complex was efficiently internalized into cells through integrin-mediated endocytosis. Endosomal escape of the RGD-modified nanogel was observed after 24 h incubation. The nanogel proved useful for targeted protein delivery.

Suppression of NF-kappaB increases bone formation and ameliorates osteopenia in ovariectomized mice.

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-κB reduces the osteoclast bone resorption. The role of NF-κB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-κB inhibitor named S1627, we demonstrated that inhibition of NF-κB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-κB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg·d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-κB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-κB inhibitor S1627. Taken collectively, our results suggest that NF-κB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.

Dietary protein modulates the effect of CLA on lipid metabolism in rats.

The effect of the interaction of CLA and type of dietary protein on lipid metabolism was studied in male rats by feeding diets containing casein (CAS) or soy protein (SOY) as dietary protein and either linoleic acid (LA, a control FA) or graded levels of CLA at 0, 0.1, 0.5, and 1.0% for 28 d. CLA reduced the weight of perirenal adipose tissue in a dose-dependent manner, but the magnitude of the reduction was greater when rats were fed SOY. Feeding SOY resulted in a significant reduction of the concentrations of serum total and HDL cholesterol, TG, glucose, and insulin irrespective of dietary CLA. The concentration of serum leptin tended to be lower on the SOY diet free of CLA than in the corresponding CAS diet, but it fell with an increasing dietary level of CLA in the CAS groups. In contrast, serum leptin tended to increase when CLA was added to SOY diets. The concentration of serum adiponectin was higher in the CAS than in the SOY groups, and it tended to increase in response to dietary CLA levels in the CAS-fed rats, whereas CLA showed no effect in SOY-fed rats. The activity of liver mitochondrial carnitine palmitoyltransferase was higher in the SOY than in the CAS groups, but it tended to increase with an increasing dietary level of CLA in both protein groups. Although the body fat-reducing activity of CLA was more effective when the protein source was SOY, rats fed CAS appeared to be more susceptible to CLA than in those fed SOY with respect to cytokines examined. These results suggest that the type of dietary protein may modify the antiobesity activity of CLA.