Effect of Anti-PEG Antibody on Immune Response of mRNA-Loaded Lipid Nanoparticles.

Lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccines have been approved for use to combat coronavirus disease 2019 (COVID-19). The mRNA-LNPs contain PEG-conjugated lipids. Clinical studies have reported that mRNA-LNPs induce the production of anti-PEG antibodies, but the anti-PEG antibodies do not affect the production of neutralizing antibodies. However, the detailed influence of anti-PEG antibodies on mRNA-LNP vaccines remains unclear. Therefore, in this study, we prepared ovalbumin (OVA) as a model antigen-encoding mRNA-loaded LNP (mRNA-OVA-LNP), and we determined whether anti-PEG antibodies could affect the antigen-specific immune response of mRNA-OVA-LNP vaccination in mice pretreated with PEG-modified liposomes to induce the production of anti-PEG antibodies. After intramuscular (i.m.) injection of the mRNA-LNP, the anti-PEG antibodies did not change the expression of protein or induction of cytokine and cellular immune response but did slightly increase the induction of antigen-specific antibodies. Furthermore, repeated mRNA-LNP i.m. injection induced the production of anti-PEG IgM and anti-PEG IgG. Our results suggest that mRNA-LNP induces the production of anti-PEG antibodies, but the priming of the antigen-specific immune response of mRNA-LNP vaccination is not notably affected by anti-PEG antibodies.

NMR-based analysis of impact of siRNA mixing conditions on internal structure of siRNA-loaded LNP.

This study aimed to assess the applicability of solution-state 1H NMR for molecular-level characterization of siRNA-loaded lipid nanoparticles (LNP). Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, MC3) was used as an ionizable lipid, and siRNA-loaded LNPs were prepared by pre-mixing and post-mixing methods. The pre-mixing method involved mixing an acidic solution containing siRNA with an ethanolic lipid solution using a microfluidic mixer. The pre-mixed LNP was prepared by dialyzing the mixed solution into the phosphate buffered saline (PBS, pH 7.4). The post-mixed LNP was prepared by mixing the siRNA solution with empty LNP in an acidic condition with and without ethanol, resulting in post-mixed LNP (A) and (B), respectively. Both pre-mixed and post-mixed LNPs formed LNP particles with an average diameter of approximately 50 nm. Moreover, the ratio of encapsulated siRNA to lipid content in each LNP particle remained constant regardless of the preparation method. However, small-angle X-ray scattering measurements indicated structural variations in the siRNA-MC3 stacked bilayer structure formed in the LNPs, depending on the preparation method. Solution-state 1H NMR analysis suggested that the siRNA was incorporated uniformly into the LNP core for pre-mixed LNP compared to post-mixed LNPs. In contrast, the post-mixed LNPs contained siRNA-empty regions with local enrichment of siRNA in the LNP core. This heterogeneity was more pronounced in post-mixed LNP (B) than in post-mixed LNP (A), suggesting that ethanol facilitated the homogeneous mixing of siRNA with LNP lipids. The silencing effect of each siRNA-loaded LNP was reduced in the order of pre-mixed LNP, post-mixed LNP (A), and post-mixed LNP (B). This suggested that the heterogeneity of the siRNA-loaded LNP could cause a reduction in the silencing effect of the incorporated siRNA inside LNPs. The present study highlighted that NMR-based characterization of siRNA-loaded LNP can reveal the molecular-level heterogeneity of siRNA-loaded LNP, which helps to optimize the preparation conditions of siRNA-loaded LNP formulations.

EVs-miR-17-5p attenuates the osteogenic differentiation of vascular smooth muscle cells potentially via inhibition of TGF-ß signaling under high glucose conditions.

Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor ß receptor type II (TGF-ß RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-ß RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.

The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells.

Diesel exhaust particles (DEPs) are very small (typically < 0.2 µm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs.

Lymphatic Endothelial Cells Produce Chemokines in Response to the Lipid Nanoparticles Used in RNA Vaccines.

RNA vaccines based on Lipid nanoparticles (LNP) were put into practical use within only one year after the global outbreak of the coronavirus disease 2019 (COVID-19). This success of RNA vaccine highlights the utility of an mRNA delivery system as a vaccination strategy. Potent immunostimulatory activity of LNPs (i.e., inflammation occurring at the injection site and the production of inflammatory cytokines) have recently been reported. However, we have only limited knowledge concerning which cells are responsible for responding to the LNPs. We report herein on in vitro chemokine production from non-immune cells in response to exposure to LNPs. In this study, SM-102, an ionizable lipid that is used in the approved RNA vaccine for the clinical usage of COVID-19 mRNA vaccine, was used. Immortalized mouse lymphatic endothelial cells (mLECs) or professional antigen presenting cells (APCs) such as RAW 264.7 monocyte/macrophage cells were incubated with LNPs that contained no mRNA. As a result, chemokines involved in the recruitment of monocytes/neutrophils were produced only by the mLECs following the LNP treatment. These findings indicate that LEC appear to serve as the cell that sends out initial signals to response LNPs.

The Effect of Cholesterol Content on the Adjuvant Activity of Nucleic-Acid-Free Lipid Nanoparticles.

RNA vaccines are applicable to the treatment of various infectious diseases via the inducement of robust immune responses against target antigens by expressing antigen proteins in the human body. The delivery of messenger RNA by lipid nanoparticles (LNPs) has become a versatile drug delivery system used in the administration of RNA vaccines. LNPs are widely considered to possess adjuvant activity that induces a strong immune response. However, the properties of LNPs that contribute to their adjuvant activity continue to require clarification. To characterize the relationships between the lipid composition, particle morphology, and adjuvant activity of LNPs, the nanostructures of LNPs and their antibody production were evaluated. To simply compare the adjuvant activity of LNPs, empty LNPs were subcutaneously injected with recombinant proteins. Consistent with previous research, the presence of ionizable lipids was one of the determinant factors. Adjuvant activity was induced when a tiny cholesterol assembly (cholesterol-induced phase, ChiP) was formed according to the amount of cholesterol present. Moreover, adjuvant activity was diminished when the content of cholesterol was excessive. Thus, it is plausible that an intermediate structure of cholesterol (not in a crystalline-like state) in an intra-particle space could be closely related to the immunogenicity of LNPs.

Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses.

Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS-CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor-binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV-infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and CD4+ T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines.

Development of a Ready-to-Use-Type RNA Vaccine Carrier Based on an Intracellular Environment-Responsive Lipid-like Material with Immune-Activating Vitamin E Scaffolds.

Because of its efficient and robust gene transfer capability, messenger RNA (mRNA) has become a promising tool in various research fields. The lipid nanoparticle (LNP) is considered to be a fundamental technology for an mRNA delivery system and has been used extensively for the development of RNA vaccines against SARS-CoV-2. We recently developed ssPalm, an environmentally responsive lipid-like material, as a component of LNP for mRNA delivery. In this study, a self-degradable unit (phenyl ester) that confers high transfection activity and an immune stimulating unit (vitamin E scaffold) for high immune activation were combined to design a material, namely, ssPalmE-Phe-P4C2, for vaccine use. To design a simple and user-friendly form of an RNA vaccine based on this material, a freeze-drying-based preparation method for producing a ready-to-use-type LNP (LNP(RtoU)) was used to prepare the LNPssPalmE-Phe. The optimization of the preparation method and the lipid composition of the LNPssPalmE-Phe(RtoU) revealed that dioleoyl-sn-glycero phosphatidylethanolamine (DOPE) was a suitable helper lipid for achieving a high vaccination activity of the LNPssPalmE-Phe(RtoU). Other findings indicated that to maintain particle properties and vaccination activity, a 40% cholesterol content was necessary. A single administration of the LNPssPalmE-Phe(RtoU) that contained mRNA-encoding Ovalbumin (mOVA-LNPssPalmE-Phe(RtoU)) demonstrated a significant suppression of tumor progression in a tumor-bearing mouse OVA-expressing cell line (E.G7-OVA). In summary, the LNPssPalmE-Phe(RtoU) is an easy-to-handle drug delivery system (DDS) for delivering mRNA antigens in immunotherapy.

An Ionizable Lipid Material with a Vitamin E Scaffold as an mRNA Vaccine Platform for Efficient Cytotoxic T Cell Responses.

RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.

A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice.

Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.

Tolerogenic Lipid Nanoparticles for Delivering Self-Antigen mRNA for the Treatment of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis is a disease caused by autoantigen-responsive immune cells that disrupt the myelin in the central nervous system (CNS). Although immunosuppressive drugs are used to suppress symptoms, no definitive therapy exists. As in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis, a partial sequence of the myelin oligodendrocyte glycoprotein (MOG35-55) was identified as a causative autoantigen. This suggests that the induction of immune tolerance that is specific to MOG35-55 would be a fundamental treatment for EAE. We previously reported that lipid nanoparticles (LNPs) containing an anionic phospholipid, phosphatidylserine (PS), in their lipid composition, can be used to deliver mRNA and that this leads to proteins of interest to be expressed in the spleen. In addition to the targeting capability of PS, PS molecules avoid activating the immune system. Physiologically, the recognition of PS on apoptotic cells suppresses immune activation against these cells by releasing cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF)-ß that negatively regulate immunity. In this study, we tested whether mRNA delivery of autoantigens to the spleen by PS-LNPs causes the expression of MOG35-55 antigens with minimal immune stimulation and whether this could be used to treat an EAE model by inducing immune tolerance.

Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery.

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.

A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice.

Introduction: Vaccinations are ideal for reducing the severity of clinical manifestations and secondary complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, SARS-CoV-2 continues to cause morbidity and mortality worldwide. In contrast to parenteral vaccines such as messenger RNA vaccines, nasal vaccines are expected to be more effective in preventing viral infections in the upper respiratory tract, the primary locus for viral infection and transmission. In this study, we examined the prospects of an inactivated whole-virion (WV) vaccine administered intranasally against SARS-CoV-2. Methods: Mice were immunized subcutaneously (subcutaneous vaccine) or intranasally (nasal vaccine) with the inactivated WV of SARS-CoV-2 as the antigen. Results: The spike protein (S)-specific IgA level was found to be higher upon nasal vaccination than after subcutaneous vaccination. The level of S-specific IgG in the serum was also increased by the nasal vaccine, although it was lower than that induced by the subcutaneous vaccine. The nasal vaccine exhibited a stronger defense against viral invasion in the upper respiratory tract than the subcutaneous vaccine and unimmunized control; however, both subcutaneous and nasal vaccines provided protection in the lower respiratory tract. Furthermore, we found that intranasally administered inactivated WV elicited robust production of S-specific IgA in the nasal mucosa and IgG in the blood of mice previously vaccinated with messenger RNA encoding the S protein. Discussion: Overall, these results suggest that a nasal vaccine containing inactivated WV can be a highly effective means of protection against SARS-CoV-2 infection.

Logistics and distribution of small extracellular vesicles from the subcutaneous space to the lymphatic system.

Small extracellular vesicles (sEVs) are small, cell-derived particles with sizes of approximately 100 nm. Since these particles include cargos such as host cell-derived proteins, messenger RNAs, and micro RNAs, they serve as mediators of cell-cell communication. While the analysis of the pharmacokinetic of sEVs after the intravenous injection have been reported, the lymphatic transport of sEVs remains unclear. The objective of this study was to provide insights into the intra-lymphatic trafficking and distribution of sEVs when they are injected into an interstitial space both in normal skin tissue and in cancerous tissue. When sEVs were Subcutaneously administered into the tail base and the tumor tissue, they preferably accumulated in the lymph nodes (LNs), rather than in the liver and the spleen. The findings reported herein show that the lymphatic transport of sEVs was drastically changed in model mice, in which a surgical treatment was used to modify to allow the dominant lymphatic flow from the footpad directly to the axillary LN via the inguinal LN. Based on the results, we conclude that when sEVs are injected into the subcutis space, they are preferably delivered to the LN via the lymphatic system. Further, the extent of accumulation of sEVs in the LN after subcutaneous injection was reduced when they were preliminarily incubated with Proteinase K. These results suggest that the lymphatic drainage of sEVs in normal skin tissue is regulated by membrane proteins on their surface. This reduction, however, was not observed in the case of cancer tissue. This discrepancy can be attributed to the presence of highly permeable lymphatic vessels in the tumor tissue. Further, the major cell subtypes that captured sEVs in the LN were LN-resident medullary sinus macrophages. These collective findings indicate that the lymphatic drainage of sEVs are mediated by proteins and, that they may appear to contribute to the control of the function of immune-responsive cells in the LNs.

Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability.

The lipid nanoparticle (LNP) is one of the promising nanotechnologies for the delivery of RNA molecules, such as small interfering RNA (siRNA) and messenger RNA (mRNA). A series of LNPs that contain an mRNA encoding the antigen protein of SARS-CoV-2 were already approved as RNA vaccines against this infectious disease. Since LNP formulations are generally metastable, their physicochemical properties are expected to shift toward a more stable state during the long-time storage of suspensions. The current mRNA vaccines are supplied in the form of frozen formulations with a cryoprotectant for preventing deterioration. They must be stored in a freezer at temperatures from -80 °C to -15 °C. It is thought that therapeutic applications of this mRNA-LNP technology could be accelerated if a new formulation that permits mRNA-LNPs to be stored under milder conditions were available. We previously reported on a one-pot method for producing siRNA-encapsulated LNPs by combining freeze-drying technology with the conventional alcohol dilution method (referred to herein as the "alcohol dilution-lyophilization method"). In this study, this method was applied to the preparation of mRNA-LNPs to provide a freeze-dried formulation of mRNA LNPs. The resulting formulation can be stored at 4 °C for at least 4 months.

Retiform hemangioendothelioma of the breast in a man with 18F-flurodeoxyglucose accumulation on positron emission tomography: a case report.

BACKGROUND: Retiform hemangioendothelioma (RH) is a rare, intermediate-grade vascular tumor that often arises in the trunk and extremities. The clinical and radiological features of RH remain largely unknown. CASE PRESENTATION: A male patient in his 70s presented with shortness of breath on exertion, and computed tomography incidentally revealed a tumor in his right breast. Positron emission tomography (PET) revealed moderate 18F-fluorodeoxyglucose (FDG) uptake in the tumor. RH was observed in the resected specimens. Three months after surgery, the patient was free of local recurrence and distant metastasis. CONCLUSIONS: RH was found in the male breast and was accompanied by FDG uptake on PET. PET may be useful in diagnosing RH. Although metastasis is rare in RH, local recurrence may occur, and careful follow-up is required.

Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA.

Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.

Reactivation of Anticancer Immunity by Resetting Interorgan Crosstalk in Immune-Suppressive Cells with a Nanoparticulated Anti-Inflammatory Drug.

The reactivation of anticancer immunity is a fundamental principle in cancer immunotherapy as evidenced by the use of immune checkpoint inhibitors (ICIs). While treatment with the ICIs is shown to have remarkable and durable therapeutic effects in the responders, the low objective response rate (<40%) continues to be a major problem. Since myeloid-derived suppressor cells (MDSCs), heterogenous cells with strong immunosuppressive activity that originate in the hematopoietic system, suppress the anticancer immunity via parallel immune checkpoint-dependent and independent pathways, these cells are potential targets for improving the efficacy of cancer immunotherapy. In this study, it is demonstrated that MDSCs can be depleted by delivering synthetic glucocorticoid dexamethasone to phagocytic cells in the spleen using a lipid nanoparticle. Since the interaction of nanoparticles with T cells is intrinsically poor, this strategy also enables the "detargeting" from T cells, thus avoiding the nonspecific suppression of cytotoxic immune responses against cancer cells. In addition to the direct anticancer effect of the nanoparticulated dexamethasone, their synergistic anticancer effect with ICIs is also reported.

Delivering mRNA to Secondary Lymphoid Tissues by Phosphatidylserine-Loaded Lipid Nanoparticles.

Lipid nanoparticles (LNPs) are one of the most successful technologies in messenger RNA (mRNA) delivery. While the liver is the most frequent target for LNP delivery of mRNA, technologies for delivering mRNA molecules to extrahepatic tissues are also important. Herein, it is reported on the development of an LNP that targets secondary lymphoid tissues. New types of alcohol-soluble phosphatidylserine (PS) derivatives are designed as materials that target immune cells and then incorporated into LNPs using a microfluidic technique with a high degree of scalability and reproducibility. The resulting LNP that contained the synthesized PS delivered mRNA to the spleen much more efficiently compared to a control LNP. A sub-organ analysis revealed that the PS-loaded LNP is extensively taken up by tissue-resident macrophages in the red pulp and the marginal zone of the spleen. Thus, the PS-loaded LNP reported in this study will be a promising strategy for clinical applications that involve delivering mRNA to the spleen.