A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers.

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.

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.

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.

Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System.

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-ß-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.

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.

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.

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.

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.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BACKGROUND: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder. RESULTS: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01). CONCLUSION: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.

Expansion of effective wet bulb globe temperature for vapor impermeable protective clothing.

The wet bulb globe temperature (WBGT) is an effective measure for risk screening to prevent heat dISOrders. However, a heat risk evaluation by WBGT requires adjustments depending on the clothing. In this study, we proposed a new effective WBGT (WBGTeff*) for general vapor permeable clothing ensembles and vapor impermeable protective clothing that is applicable to occupants engaged in moderate intensity work with a metabolic heat production value of around 174W/m2. WBGTeff* enables the conversion of heat stress into the scale experienced by the occupant dressed in the basic clothing ensemble (work clothes) based on the heat balances for a human body. We confirmed that WBGTeff* was effective for expressing the critical thermal environments for the prescriptive zones for occupants wearing vapor impermeable protective clothing. Based on WBGTeff*, we succeeded in clarifying how the weights for natural wet bulb, globe, and air temperatures and the intercept changed depending on clothing properties and the surrounding environmental factors when heat stress is expressed by the weighted sum of natural wet bulb, globe, and air temperatures and the intercept. The weight of environmental temperatures (globe and air temperatures) for WBGTeff* for vapor impermeable protective clothing increased compared with that for general vapor permeable clothing, whereas that of the natural wet bulb temperature decreased. For WBGTeff* in outdoor conditions with a solar load, the weighting ratio of globe temperature increased and that of air temperature decreased with air velocity. Approximation equations of WBGTeff* were proposed for both general vapor permeable clothing ensembles and for vapor impermeable protective clothing.

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.

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.

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.

Self-Assembled Nanogels of Cholesterol-Bearing Hydroxypropyl Cellulose: A Thermoresponsive Building Block for Nanogel Tectonic Materials.

Hydroxypropyl cellulose (HPC) is a fascinating polysaccharide to use in developing a nanogel to be a thermoresponsive building unit for nanogel tectonic materials. Cholesterol-bearing HPC (Ch-HPC) self-assembled to form nanogels through hydrophobic interactions of the cholesteryl groups in water. Ch-HPC nanogels had a lower critical solution temperature in line with that of native HPC. The particle size of Ch-HPC nanogels was reversibly controlled by the temperature and salting-out effect. The thermoresponsive property was also observed in Ch-HPC nanogel-cross-linked macrogels. These results suggest that a Ch-HPC nanogel is an attractive building block for thermoresponsive nanogel tectonic materials.

Magnetically Guided Protein Transduction by Hybrid Nanogel Chaperones with Iron Oxide Nanoparticles.

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone-like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered ß-galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins.

Effects of partial sleep restriction and subsequent daytime napping on prolonged exertional heat strain.

OBJECTIVES: It is considered that sleep restriction is one of the risk factors for the development of exertional heat stroke and illness. However, how sleep restriction affects exertional heat strain and the nature of the coping strategy involved in this phenomenon remain unclear. METHODS: Fourteen healthy subjects were studied on four occasions: after a night of normal sleep (NS, 7-8 h) and after a night of partial sleep restriction (PSR, 4 h), each with or without taking a daytime nap during the subsequent experimental day. The laboratory test consisted of two 40 min periods of moderate walking in a hot room in the morning and the afternoon. RESULTS: The increase in rectal temperature during walking was significantly greater in PSR than in NS in the afternoon. The rating scores for physical and psychological fatigue and sleepiness were significantly greater in PSR than in NS, both in the morning and in the afternoon. The reaction times and lapses in the psychomotor vigilance task (PVT) after walking were significantly worse in PSR than in NS in the morning and after lunch. The nap intervention attenuated significantly the scores for fatigue and sleepiness in PSR. Furthermore, the decreased PVT response in PSR was significantly reversed by the nap. CONCLUSIONS: These results suggest that PSR augments physiological and psychological strain and reduces vigilance in the heat. Taking a nap seemed to be effective in reducing psychological strain and inhibiting the decrease in vigilance.

Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment.

RNAi enables potent and specific gene silencing, potentially offering useful means for treatment of cancers. However, safe and efficient drug delivery systems (DDS) that are appropriate for intra-tumor delivery of siRNA or shRNA have rarely been established, hindering clinical application of RNAi technology to cancer therapy. We have devised hydrogel polymer nanoparticles, or nanogel, and shown its validity as a novel DDS for various molecules. Here we examined the potential of self-assembled nanogel of cholesterol-bearing cycloamylose with spermine group (CH-CA-Spe) to deliver vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF) into tumor cells. The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF. Intra-tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice. The treatment also inhibited induction of myeloid-derived suppressor cells, while it decreased interleukin-17A production. Therefore, the CH-CA-Spe nanogel may be a feasible DDS for intra-tumor delivery of therapeutic siRNA. The results also suggest that local suppression of VEGF may have a positive impact on systemic immune responses against malignancies.

Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.

Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from ß-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.

Cationic Glucan Dendrimer Gel-Mediated Local Delivery of Anti-OC-STAMP-siRNA for Treatment of Pathogenic Bone Resorption.

Osteoclast stimulatory transmembrane protein (OC-STAMP) plays a pivotal role in the promotion of cell fusion during osteoclast differentiation (osteoclastogenesis) in the context of pathogenic bone resorption. Thus, it is plausible that the suppression of OC-STAMP through a bioengineering approach could lead to the development of an effective treatment for inflammatory bone resorptive diseases with minimum side effects. Here, we synthesized two types of spermine-bearing (Spe) cationic glucan dendrimer (GD) gels (with or without C12) as carriers of short interfering RNA (siRNA) to silence OC-STAMP. The results showed that amphiphilic C12-GD-Spe gel was more efficient in silencing OC-STAMP than GD-Spe gel and that the mixture of anti-OC-STAMP siRNA/C12-GD-Spe significantly downregulated RANKL-induced osteoclastogenesis. Also, local injection of anti-OC-STAMP-siRNA/C12-GD-Spe could attenuate bone resorption induced in a mouse model of periodontitis. These results suggest that OC-STAMP is a promising target for the development of a novel bone regenerative therapy and that C12-GD-Spe gel provides a new nanocarrier platform of gene therapies for osteolytic disease.

Low-frequency CD8+ T cells induced by SIGN-R1+ macrophage-targeted vaccine confer SARS-CoV-2 clearance in mice.

Vaccine-induced T cells and neutralizing antibodies are essential for protection against SARS-CoV-2. Previously, we demonstrated that an antigen delivery system, pullulan nanogel (PNG), delivers vaccine antigen to lymph node medullary macrophages and thereby enhances the induction of specific CD8+ T cells. In this study, we revealed that medullary macrophage-selective delivery by PNG depends on its binding to a C-type lectin SIGN-R1. In a K18-hACE2 mouse model of SARS-CoV-2 infection, vaccination with a PNG-encapsulated receptor-binding domain of spike protein decreased the viral load and prolonged the survival in the CD8+ T cell- and B cell-dependent manners. T cell receptor repertoire analysis revealed that although the vaccine induced T cells at various frequencies, low-frequency specific T cells mainly promoted virus clearance. Thus, the induction of specific CD8+ T cells that respond quickly to viral infection, even at low frequencies, is important for vaccine efficacy and can be achieved by SIGN-R1+ medullary macrophage-targeted antigen delivery.