Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.

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.

Perforated Hydrogels Consisting of Cholesterol-Bearing Pullulan (CHP) Nanogels: A Newly Designed Scaffold for Bone Regeneration Induced by RANKL-Binding Peptides and BMP-2.

The receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4, is known to stimulate bone morphogenetic protein (BMP)-2-induced bone formation, but peptides tend to aggregate and lose their bioactivity. Cholesterol-bearing pullulan (CHP) nanogel scaffold has been shown to prevent aggregation of peptides and to allow their sustained release and activity; however, the appropriate design of CHP nanogels to conduct local bone formation needs to be developed. In the present study, we investigated the osteoconductive capacity of a newly synthesized CHP nanogel, CHPA using OP3-4 and BMP-2. We also clarified the difference between perforated and nonperforated CHPA impregnated with the two signaling molecules. Thirty-six, five-week-old male BALB/c mice were used for the calvarial defect model. The mice were euthanized at 6 weeks postoperatively. A higher cortical bone mineral content and bone formation rate were observed in the perforated scaffold in comparison to the nonperforated scaffold, especially in the OP3-4/BMP-2 combination group. The degradation rate of scaffold material in the perforated OP3-4/BMP-2 combination group was lower than that in the nonperforated group. These data suggest that perforated CHPA nanogel could lead to local bone formation induced by OP3-4 and BMP-2 and clarified the appropriate degradation rate for inducing local bone formation when CHPA nanogels are designed to be perforated.

Fusogenic Hybrid Extracellular Vesicles with PD-1 Membrane Proteins for the Cytosolic Delivery of Cargos.

Extracellular vesicles (EVs) are cell-derived lipid membrane capsules that can deliver functional molecules, such as nucleic acids, to target cells. Currently, the application of EVs is limited because of the difficulty of loading cargo into EVs. We constructed hybrid EVs by the fusion of liposomes and insect cell-derived EVs expressing recombinant programmed cell death 1 (PD-1) protein and baculoviral fusogenic glycoprotein gp64, and evaluated delivery of the model cargo molecule, Texas Red-labeled dextran (TR-Dex), into the cytosol. When PD-1 hybrid EVs were added to HeLa cells, the intracellular uptake of the hybrid EVs was increased compared with hybrid EVs without PD-1. After cellular uptake, the PD-1 hybrid EVs were shown to be localized to late endosomes or lysosomes. The results of fluorescence resonance energy transfer (FRET) indicated that membrane fusion between the hybrid EVs and organelles had occurred in the acidic environment of the organelles. When TR-Dex-loaded liposomes were fused with the PD-1 EVs, confocal laser scanning microscopy indicated that TR-Dex was distributed throughout the cells, which suggested that endosomal escape of TR-Dex, through membrane fusion between the hybrid EVs and acidic organelles, had occurred. These engineered PD-1 hybrid EVs have potential as delivery carriers for biopharmaceuticals.

Theranostic Agent Combining Fullerene Nanocrystals and Gold Nanoparticles for Photoacoustic Imaging and Photothermal Therapy.

Developing photoactivatable theranostic platforms with integrated functionalities of biocompatibility, targeting, imaging contrast, and therapy is a promising approach for cancer diagnosis and therapy. Here, we report a theranostic agent based on a hybrid nanoparticle comprising fullerene nanocrystals and gold nanoparticles (FGNPs) for photoacoustic imaging and photothermal therapy. Compared to gold nanoparticles and fullerene crystals, FGNPs exhibited stronger photoacoustic signals and photothermal heating characteristics by irradiating light with an optimal wavelength. Our studies demonstrated that FGNPs could kill cancer cells due to their photothermal heating characteristics in vitro. Moreover, FGNPs that are accumulated in tumor tissue via the enhanced permeation and retention effect can visualize tumor tissue due to their photoacoustic signal in tumor xenograft model mice. The theranostic agent with FGNPs shows promise for cancer therapy.

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.

Radiation-Induced Asymmetric Grafting of Different Monomers into Base Films to Prepare Novel Bipolar Membranes.

We prepared novel bipolar membranes (BPMs) consisting of cation and anion exchange layers (CEL and AEL) using radiation-induced asymmetric graft polymerization (RIAGP). In this technique, graft polymers containing cation and anion exchange groups were introduced into a base film from each side. To create a clear CEL/AEL boundary, grafting reactions were performed from each surface side using two graft monomer solutions, which are immiscible in each other. Sodium p-styrenesulfonate (SSS) and acrylic acid (AA) in water were co-grafted from one side of the base ethylene-co-tetrafluoroethylene film, and chloromethyl styrene (CMS) in xylene was simultaneously grafted from the other side, and then the CMS units were quaternized to afford a BPM. The distinct SSS + AA- and CMS-grafted layers were formed owing to the immiscibility of hydrophilic SSS + AA and hydrophobic CMS monomer solutions. This is the first BPM with a clear CEL/AEL boundary prepared by RIAGP. However, in this BPM, the CEL was considerably thinner than the AEL, which may be a problem in practical applications. Then, by using different starting times of the first SSS+AA and second CMS grafting reactions, the CEL and AEL thicknesses was found to be controlled in RIAGP.

Magnetically Navigated Protein Transduction In Vivo using Iron Oxide-Nanogel Chaperone Hybrid.

Systems for "protein transduction," intracellular delivery of functional proteins, are needed to address deliverability challenges of protein therapeutics. However, in vivo protein transduction remains challenging because of instability in serum, extracellular protease digestion and rapid excretion from the bloodstream. Here, a magnetically guided in vivo protein transduction using magnetic nanogel chaperone (MC) composed of iron oxide nanoparticles and a polysaccharide nanogel, a protein carrier inspired by "catch and release" mechanisms of molecular chaperones is demonstrated. The MC system enables efficient delivery of anti-cancer proteins, saporin and RNaseA, into cultured tumor lines and inhibits cell proliferation, mainly via apoptosis. Magnetic in vivo protein transduction via intravenous whole body administration is demonstrated in a fibrosarcoma model. By in vivo optical imaging, MC accumulated in tumor tissues under magnetic field three times more than without irradiation. With subcutaneous injection, saporin is delivered by MC to the cytoplasm in magnetically targeted tissues. In an oral cancer model, MC-delivered magnetically targeted saporin decreased tumor volume without significant body weight changes and no regrowth of tumor at 3 months after complete regression. Protein transduction with MC shows promise for cancer therapeutics and, potentially, for regenerative medicine and other biomedical applications.

Identification of a dominant CD8+ CTL epitope in the SARS-associated coronavirus 2 spike protein.

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has been spreading throughout the world. To date, there are still no approved human vaccines for this disease. To develop an effective vaccine, the establishment of animal models for evaluating post-vaccination immune responses is necessary. In this study, we have identified a CTL epitope in the SARS-CoV-2 spike (S) protein that could be used to measure the cellular immune response against this protein. Potential predicted CTL epitopes of the SARS-CoV-2 S protein were investigated by immunizing BALB/c mice with a recombinant of the receptor-binding domain (RBD) of the S protein. Then, CD8+ T cells specific for S-RBD were detected by stimulating with potential epitope peptides and then measuring the interferon-gamma production. Truncation of this peptide revealed that S-RBD-specific CD8+ T cells recognized a H2-Dd-restricted S526-533 peptide. In conclusion, this animal model is suitable for evaluating the immunogenicity of SARS-CoV-2 vaccines.

Preparation of engineered extracellular vesicles with full-length functional PD-1 membrane proteins by baculovirus expression system.

Extracellular vesicles (EVs) facilitate intercellular communication by transporting functional molecules. The modification of EVs for clinical use as drug delivery systems is of considerable interest because of their biocompatibility and molecular transport ability. Programmed cell death ligand 1 (PD-L1) is an effective target molecule for drug delivery to cancer tissues and binds the single-transmembrane protein, Programmed cell death protein 1 (PD-1), an immune checkpoint that guards against autoimmunity. In this study, EVs were modified in a new surface engineering strategy to incorporate recombinant full-length functional PD-1 using a baculovirus system and newly designed PD-1 mutant with higher PD-L1 affinity. The insect cell line Spodoptera frugiperda 9 was infected with recombinant baculoviruses incorporating the PD-1 mutant gene to express the target membrane proteins. To ensure an effective insertion into the membrane, the native signal peptide of PD-1 was also replaced with that of the baculovirus envelope glycoprotein. Engineered EVs expressing the high-affinity PD-1 mutants (PD-1 EVs) were then isolated and characterized. Immunostaining and confocal laser scanning microscopy results confirmed the presence of full-length functional PD-1 mutants expressed by viral infection on both infected Spodoptera frugiperda 9 cell membrane surfaces and released EV membranes. Furthermore, the signal peptide substitution drastically increased the binding between PD-1 EVs and PD-L1. PD-1 EVs effectively bound PD-L1 and PD-L1-expressing cancer cells, showing potential as a candidate in new therapy approaches targeting PD-L1 EVs.

Crosslinked nanogel-based porous hydrogel as a functional scaffold for tongue muscle regeneration.

Surgical resection in tongue cancer can impair speech and swallowing, reducing quality of life. There is a need for biomaterials that can regenerate tongue muscle tissue defects. Ideally, such a biomaterial would allow controlled release of therapeutic proteins, support the survival and differentiation of therapeutic cells, and promote tongue muscle regeneration in vivo. The aim of the current study was to assess these factors in an acryloyl group-modified crosslinked nanogel, consisting of cholesterol-bearing pullulan hydrogel nanoparticles, to determine its potential as a regenerative therapeutic following tongue resection. The hydrogel demonstrated substantial porosity and underwent slow biodegradation. When loaded with a model protein, the gel enabled sustained protein release over two weeks in serum, with no initial burst release. Mouse myoblasts demonstrated adhesion to the hydrogel and cell survival was observed up to one week. Gel-encapsulated myoblasts demonstrated normal myotube differentiation. Myoblast-loaded gels were implanted in a tongue defect in mice, and there was a significant increase in newly-regenerated myofibers in gel-implanted animals. The developed biomaterial platform demonstrates significant potential as a regenerative treatment following tongue resection, as it facilitates both protein and cell-mediated therapy, and stimulates tongue muscle regeneration in vivo.

Antigen Delivery to Antigen-Presenting Cells for Adaptive Immune Response by Self-Assembled Anionic Polysaccharide Nanogel Vaccines.

Although current vaccine technology induces sufficient antibody responses to prophylactically ward off viral infections, an anticancer vaccine that directs the patient's immune system to directly fight extant malignant cells will require inducing Th1 and cytotoxic T lymphocyte responses in addition to antibody-mediated activities. Thus, new mechanisms are necessary to deliver antigen to cells in the lymphatic system that will induce these responses. To this end, we have developed a cholesterol-bearing pullulan (CHP) self-assembly nanogel of less than 100 nm, which we have now further modified to be anionic by carboxyl group substitution. Overall, the nanogel-protected antigens during transport to the lymphatic system and converting the vehicle to an anionic charge improved interactions with antigen-presenting cells. We further show that these modified nanogels are a more efficient system for delivering antigen to antigen-presenting cells, particularly langerin-expressing cells, and that this induced significant adaptive immunity. Therefore, we think that this technology could be used to improve anticancer immunotherapies.

Synergistic anti-tumor efficacy by combination therapy of a self-assembled nanogel vaccine with an immune checkpoint anti-PD-1 antibody.

Therapeutic strategies for cancer involving immune checkpoint inhibitors (ICIs) have been gaining widespread attention, but their efficacy remains limited. Thus, combination of ICI therapies with other therapeutic modalities may be required to improve their outcomes. In this study, we examined the improved efficacy of a CHP nanogel-based vaccine delivery system after combination with ICI therapy. For this, we evaluated the therapeutic efficacy of combining an anti-PD-1 antibody as an ICI with an OVA antigen-complexed CHP nanogel vaccine delivery system in a mouse E.G7-OVA tumor model. Mice were subcutaneously inoculated with E.G7-OVA tumor cells on one side of the back, and subcutaneously injected with OVA or the OVA/CHP nanogel vaccine on the other side of the back. Anti-PD-1 antibody was administered at defined intervals. Tumor volume, immune responses, and tumor-infiltrating cells were evaluated. Mice treated with OVA vaccine alone showed weak tumor suppression compared with untreated control mice. Mice receiving combined OVA/CHP nanogel vaccine and anti-PD-1 antibody therapy exhibited strong tumor growth suppression and markedly improved survival, suggesting that PD-1 signaling blockade by the anti-PD-1 antibody enhanced the anti-tumor efficacy of the OVA vaccine. Furthermore, tumor-infiltrating cells and immune responses were increased in the combined therapy group. No serious side effects were observed for any of the treatments. Taken together, the immune system activation induced by the CHP nanogel vaccine was synergistically enhanced by the anti-PD-1 antibody. The present findings suggest the potential for enhanced therapeutic efficacy by combining the CHP nanogel vaccine delivery system with ICI therapy for various cancer types.

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.

Magnetically Navigated Intracellular Delivery of Extracellular Vesicles Using Amphiphilic Nanogels.

Various cells in vivo secrete exosomes consisting of lipid bilayers. They carry mRNAs and miRNAs capable of controlling cellular functions and can be used as drug delivery system nanocarriers. There is the current need to further improve the efficiency of exosome uptake into target cells. In this study, we prepared a hybrid of exosomes and magnetic nanoparticles, which could be guided to target cells by a magnetic field for efficient uptake. Magnetic nanogels were prepared and hybridized to fluorescently labeled exosomes isolated from PC12 cells. By applying a magnetic field to a hybrid with magnetic nanogel, exosomes were efficiently transferred into target cells as confirmed by confocal laser microscopy. Finally, we found that differentiation of adipose-derived stem cells to neuron-like cells was enhanced by magnetic induction of the exosome-magnetic nanogel hybrid, indicating maintenance of the intrinsic functions of the exosomes in the differentiation of adipose-derived stem cells.

Antigen delivery targeted to tumor-associated macrophages overcomes tumor immune resistance.

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies.

Structural effects and lymphocyte activation properties of self-assembled polysaccharide nanogels for effective antigen delivery.

The success of immunotherapeutic vaccines is often limited by their inability to activate the cytotoxic T lymphocyte (CTL)-inducing Th1 pathway. We investigated the ability of self-assembled nanogels (CHP or CH-CDex) to activate this pathway, and characterised them chemically and biologically. Once loaded with antigen (ovalbumin, OVA) their OVA encapsulation and dissociation rates suggested the possibility of effective antigen delivery. The DC2.4 dendritic cell line took up either vaccine time-dependently, but both vaccines required CpG DNA for class I MHC presentation. The nanogel vaccines interacted with RAW264.7, a Balb/c mouse-derived macrophage cell line, and co-localised with lysosomes, suggesting their endocytotic internalization in RAW264.7. Both vaccines activated CTLs better than OVA alone. Unlike OVA alone, the nanogel vaccines induced IgG2a antibody production in mice, whereas the former induced IgG1 antibodies. OVA-nanogel delivery to the draining lymph nodes (DLNs) was higher than that for OVA alone, reaching a deeper medullary area. Furthermore, Langerin+ CD103+ DCs interacted with the nanogel vaccines effectively, which is a subset of cross-presentation DC, in the DLNs. The nanogel vaccines each had good anti-tumour efficacy in OVA tumour-bearing mice compared with the OVA alone. Thus, CHP and CH-CDex nanogels should be investigated further because of the great potential they offer for immunotherapy.

Heat balance model for a human body in the form of wet bulb globe temperature indices.

The purpose of this study is to expand the empirically derived wet bulb globe temperature (WBGT) index to a rational thermal index based on the heat balance for a human body. We derive the heat balance model in the same form as the WBGT for a human engaged in moderate intensity work with a metabolic heat production of 174W/m2 while wearing typical vapor-permeable clothing under shady and sunny conditions. Two important relationships are revealed based on this derivation: (1) the natural wet bulb and black globe temperature coefficients in the WBGT coincide with the heat balance equation for a human body with a fixed skin wettedness of approximately 0.45 at a fixed skin temperature; and (2) the WBGT can be interpreted as the environmental potential to increase skin temperature rather than the heat storage rate of a human body. We propose an adjustment factor calculation method that supports the application of WBGT for humans dressed in various clothing types and working under various air velocity conditions. Concurrently, we note difficulties in adjusting the WBGT by using a single factor for humans wearing vapor-impermeable protective clothing. The WBGT for shady conditions does not need adjustment depending on the positive radiant field (i.e., when a radiant heat source exists), whereas that for the sunny condition requires adjustments because it underestimates heat stress, which may result in insufficient human protection measures.

Self-Assembled Polypeptide Nanogels with Enzymatically Transformable Surface as a Small Interfering RNA Delivery Platform.

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.

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.