Rationale: Oral squamous cell carcinoma (OSCC) carries a poor prognosis despite advances in multimodal therapy. Nanomedicine represents a compelling strategy to enhance targeted drug delivery and improve therapeutic outcomes. Here, we investigated sEV-mediated delivery of curcumin as a novel therapeutic approach for OSCC. Methods: Small extracellular vesicles (sEVs) were isolated from Jurkat cells by size-exclusion chromatography and loaded with curcumin via sonication to generate JCsEV. Functional effects were assessed in vitro using wound healing, transwell invasion, and metabolic activity assays across multiple cancer cell lines. Therapeutic efficacy in vivo was evaluated in the 4-nitroquinoline 1-oxide (4-NQO) immunocompetent murine model of oral carcinogenesis. Female C57BL/6J mice received intraperitoneal treatment for four weeks with PBS, free curcumin, unloaded JsEV, or JCsEV. Tumor number, tumor burden, and body weight changes were assessed at the experimental endpoint. Results: In vitro, JCsEV significantly inhibited tumor cell migration, invasion, and metabolic activity compared with controls (p < 0.05). In vivo, treatment with JCsEV significantly reduced tumor number and tumor burden in the 4-NQO model (p < 0.01). In addition, body weight loss was reduced in JCsEV-treated mice compared with controls. Conclusion: sEV-mediated delivery of curcumin effectively suppresses tumor progression in experimental OSCC. These findings establish proof-of-concept for sEV-based nanomedicine as a therapeutic strategy for OSCC and provide a compelling rationale for further translational investigation of sEVs as drug delivery platforms.

Lipid nanoparticles (LNPs) are clinically proven mRNA delivery vehicles, and repeated administration is often required to maintain therapeutic or prophylactic benefit. However, a single dose can provoke adaptive immune responses against polyethylene glycol (PEG), a polymeric excipient on the LNP surface. In this event, resultant anti-PEG antibodies neutralize subsequent LNP doses, causing efficacy loss and limiting therapeutic utility. Here, we investigated whether altering the LNP formulation between initial and subsequent doses could mitigate PEG-associated efficacy loss. Following the administration of an LNP known to elicit anti-PEG antibodies, we re-dosed with LNPs formulated with varied PEG-lipid chemistry, PEG concentration, mRNA cargo, and administration route. None of these adjustments prevented efficacy loss, which consistently correlated with elevated anti-PEG immunoglobulin M (IgM) and, in some cases, immunoglobulin G (IgG). Reduced PEG concentration diminished immunogenicity but also potency, and intraperitoneal dosing elicited the strongest responses upon repeat injection. Further, LNPs that elicit anti-PEG antibodies also impaired the subsequent delivery of an FDA-approved MC3 formulation, suggesting that anti-PEG immune responses can compromise any PEGylated LNP formulation. Together, these findings demonstrate that PEG-associated efficacy loss cannot be resolved through formulation optimization alone. Instead, they suggest that the ionizable lipid’s engagement of innate immune receptors is the primary determinant of repeat dosing success.

MALT1 protease is an intracellular signaling molecule that promotes tumor progression via cancer cell-intrinsic and cancer cell-extrinsic mechanisms. MALT1 has been mostly studied in lymphocytes, and little is known about its role in tumor-associated macrophages. We show that MALT1 is expressed in glioblastoma (GBM)-associated macrophages. Mechanistically, GBM tumor cells induce a MALT1-NF-κB signaling axis in macrophages, leading to enhanced macrophage migration and polarization toward an immunosuppressive ('M2-like') phenotype. Inactivation of MALT1 protease promotes transcriptional reprogramming that reduces migration and restores a macrophage anti-tumor 'M1-like' phenotype. Preclinical in vivo analysis shows that MALT1 inhibitor treatment results in immuno-reactivity of GBM-associated macrophages and reduced GBM tumor growth. The addition of MALT1 inhibitor to temozolomide reduces immunosuppression in the tumor microenvironment, indicating that pharmacological inhibition of MALT1 protease may enhance the efficacy of chemotherapeutic. Thus, our findings suggest that MALT1 protease inhibition represents a promising macrophage-targeted immunotherapeutic strategy for the treatment of GBM.

Milk is a multifaceted biofluid that is essential for infant nutrition and development, yet its cellular and bioactive components, particularly maternal milk cells, remain understudied. Early research on milk cells indicated that they cross the infant's intestinal barrier and accumulate within systemic organs. However, due to the absence of modern analytical techniques, these studies were limited in scope and mechanistic analysis. To overcome this knowledge gap, we have investigated the transintestinal transport of milk cells and components in pups over a 21-day period. Studies employed a mT/mG foster nursing model in which milk cells express a membrane-bound fluorophore, tdTomato. Using flow cytometry, we tracked the transport of milk cell-derived components across local and systemic tissues, including the intestines, blood, thymus, mesenteric lymph nodes, and liver. These experiments identified milk-derived fluorescent signals in intestinal epithelial and immune cells as well as liver macrophages in 7-day-old pups. However, the minute numbers of macrophages in mouse milk suggest that maternal cells are not systemically accumulating in the infant; instead, pup macrophages are consuming milk cell membrane components, such as apoptotic bodies or extracellular vesicles (EVs). Ex vivo experiments using primary macrophages support this hypothesis, showing that immune cells preferentially consumed EVs over milk cells. Together, these data suggest a more complex interplay between milk cells and the infant's immune and digestive systems than previously recognized and highlight the need for future research on the role of milk cells in infant health.

The hierarchical architecture of skeletal muscle spans from microscale sarcomeres to macroscale myofibers and is integral to its contractile functionality. Pathologies such as volumetric muscle loss (VML) compromise this structure and destroy the native extracellular matrix (ECM), exceeding the regenerative capacity of endogenous repair mechanisms. Here, we present a novel method for tissue engineering biomimetic three-dimensional (3D) skeletal muscle with complex architectures by leveraging freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting of the ECM. Collagen type I scaffolds mimicking diverse muscle architectures (including parallel, unipennate, bipennate, multipennate, and convergent) were designed, FRESH printed, and seeded with C2C12 myoblasts to guide myogenesis. Engineered muscle tissues demonstrated scaffold-mediated alignment and fusion into functional myotubes, exhibiting contractile responses to electrical stimulation with architecture-dependent specific force of ~1 kN/m2 and a positive force-frequency relationship. In vivo implantation further revealed scaffold-directed cellular and vascular organization, underscoring the translational potential of this approach. In summary, this study demonstrates the capability to use FRESH 3D bioprinting to engineer physiologically relevant muscle architectures, significantly advancing the design of functional muscle tissues for regenerative medicine and in vitro modeling applications.  ### Competing Interest Statement AWF is employed by and has an equity stake in FluidFormBio, Inc, which is a startup company commercializing FRESH 3D printing. FRESH 3D printing is the subject of patent protection including US Patents 10,150,258, 11,672,887 and others.

Diseases caused by Streptococcus pneumoniae (pneumococcus) produce a great impact on public health, killing about one million people annually despite available vaccines. Recent research has revealed that the pneumococcus produces extracellular vesicles (pEVs), which display selective cargo and hold potential for vaccine development. Here, we evaluated the immunogenicity and protective potential of pEVs derived from a non-encapsulated pneumococcal strain (R6) using murine models of pneumococcal colonization and invasive pneumonia. Characterization of the immune response revealed that while pEVs contain multiple virulence determinants, immunization with these nanoparticles only induces antibodies against a subset of them. Specifically, subcutaneous immunization elicits a high antibody response against PspA, a modest response against PrsA, and limited response against Ply, MalX and PsaA. The antibody response was further supported by agglutination studies, showing that sera from pEV immunized mice agglutinate pneumococci and that PspA contributes to this response in a strain-dependent manner. Subcutaneous immunization with pEVs provides protection in the invasive pneumonia model whereas nasal immunization results in one log reduction in pneumococcal colonization of the upper respiratory tract. Finally, PspA is a strong contributor to protection in the invasive model and provides a degree of protection even across heterologous families of PspA. We conclude that pEVs demonstrate potential for vaccine development, protecting across capsular types and providing some degree of protection across heterologous PspA variants.

Abstract Safe delivery of mRNA to the brain will revolutionize the treatment of brain tumors. While lipid nanoparticles (LNPs) are clinically most advanced non-viral delivery vehicles for therapeutic mRNA, LNP-mediated mRNA delivery to the brain remains challenging. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain cells without undue toxicity. We engineered LNPs consisting of four components similar to the formulation used in the mRNA COVID-19 vaccines (Moderna and Pfizer-BioNTech): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. We screened ten classes of helper lipids based on lipids enriched in extracellular vesicles to engineer biomimetic LNPs and tested their GFP mRNA delivery efficacy in SIM-A9 mouse microglia cell line. Several unique LNP formulations with potent delivery efficacy (>90% cells transfected) and stable GFP expression kinetics (5 days) were identified. LNP formulations with high transfection efficacy were then tested in vivo for luciferase mRNA delivery via intrathecal injection in C57BL/6 mice. Luciferase expression in vivo confirmed widespread mRNA delivery in the brain. We then tested Cre recombinase mRNA delivery in Ai9 mouse to identify LNP-targeted cells via flow cytometry and histology. Flow cytometry and expansion microscopy confirmed Cre recombinase mRNA delivery to a variety of brain cells, including microglia (75-90%), neurons (31-40%), neural stem cells (39-62%), oligodendrocytes (70-90%), and astrocytes (44-76%). LNPs were further evaluated for Cas9 mRNA and CD81 sgRNA delivery in C57BL/6 mouse brains to assess brain-targeted gene editing. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~40% of cells in the mouse brain. In summary, we engineered extracellular vesicle-based LNP library that can deliver RNA therapeutics to a variety of brain cells in vivo. With further development, this technology could potentially enable genetic and epigenetic therapies targeting drivers of brain tumors.

Abstract BACKGROUND Glioblastoma (GBM), one of the deadliest primary tumors of the central nervous system, is characterized by a highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAMs) are a dominant population of immune cells in the GBM TME and substantially contribute to immunosuppression, tumor progression, and treatment resistance. Elucidating the molecular mechanisms underlying TAM behavior is crucial to the development of effective immuno-therapeutic strategies for treating GBM. The adaptor protein, Caspase Recruitment Domain-containing protein 9 (CARD9) is primarily expressed in myeloid cells and its activation triggers assembly of the CARD9-BCL10-MALT1 (CBM) complex, which then promotes activation of NF-κB and/or MAPK pathways. The aim of this study was to investigate the role of the CBM complex in the GBM TME and assess its potential as a therapeutic target. METHODS/RESULTS In silico analyses revealed that GBM tumors demonstrate increased CARD9 and MALT1 expression in comparison to non-tumor brain tissue, with CARD9 expressed only within the myeloid compartment. Using co-culture, scratch wound, and transwell assays, we found that GBM tumor cells induce macrophage M2 polarization and migration via a mechanism that is dependent on CARD9/MALT1 signaling within macrophages. scRNA-Seq and flow-cytometric analysis of orthotopic syngeneic immunocompetent GBM tumor models revealed that the GBM TME is dominated by macrophages. Disruption of MALT1 protease activity within the TME leads to reduced tumor infiltration by immunosuppressive TAMs, remodeling of TAMs toward an immunoreactive anti-tumor phenotype, and improved survival of GBM-bearing mice. Additionally, we found that tumor growth was abrogated when GBM-bearing mice were treated with either a MALT1-protease inhibitor or a CARD9 inhibitor. CONCLUSIONS Our study suggests that blockade of the CARD9-MALT1 signaling axis impairs GBM-induced macrophage recruitment and M2 polarization and inhibits tumor progression. These findings nominate the CARD9-MALT1 signaling axis as a new target for TAM-directed immunotherapy for the treatment of GBM.

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria and, in doing so, may promote the spread of drug resistance genes in the population.IMPORTANCEThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here, we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface and that this DNA can be imported by the transformation machinery, facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders.

ADVERTISEMENT RETURN TO ARTICLES ASAPEditorialNEXTPrioritizing Mentorship as Scientific LeadersJacky M. Deng*Jacky M. Deng*Email: [email protected]More by Jacky M. Denghttps://orcid.org/0000-0002-2633-6624, Salma Elgaili AhmedSalma Elgaili AhmedMore by Salma Elgaili Ahmed, Ernest Awoonor-WilliamsErnest Awoonor-WilliamsMore by Ernest Awoonor-Williamshttps://orcid.org/0000-0002-9127-8539, Progna BanerjeeProgna BanerjeeMore by Progna Banerjeehttps://orcid.org/0000-0003-3257-7317, Magda H. BareckaMagda H. BareckaMore by Magda H. Bareckahttps://orcid.org/0000-0002-0772-164X, Laura E. BickertonLaura E. BickertonMore by Laura E. Bickerton, Silvina A. Di PietroSilvina A. Di PietroMore by Silvina A. Di Pietro, Stanna K. DornStanna K. DornMore by Stanna K. Dornhttps://orcid.org/0000-0002-8644-9962, Kevin Maik JablonkaKevin Maik JablonkaMore by Kevin Maik Jablonka, Gabriele LaudadioGabriele LaudadioMore by Gabriele Laudadiohttps://orcid.org/0000-0002-2749-8393, Elisabeth KreidtElisabeth KreidtMore by Elisabeth Kreidt, Helena Mannochio-RussoHelena Mannochio-RussoMore by Helena Mannochio-Russo, Júlio TerraJúlio TerraMore by Júlio Terra, Olivia Harper WilkinsOlivia Harper WilkinsMore by Olivia Harper Wilkins, Saigopalakrishna S. YerneniSaigopalakrishna S. YerneniMore by Saigopalakrishna S. Yerneni, and Maha YusufMaha YusufMore by Maha Yusufhttps://orcid.org/0000-0001-7908-2915Cite this: ACS Cent. Sci. 2024, XXXX, XXX, XXX-XXXPublication Date (Web):January 10, 2024Publication History Published online10 January 2024https://doi.org/10.1021/acscentsci.3c00500Published 2024 by American Chemical Society. This publication is licensed under CC-BY 4.0. License Summary*You are free to share (copy and redistribute) this article in any medium or format and to adapt (remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:Creative Commons (CC): This is a Creative Commons license.Attribution (BY): Credit must be given to the creator.View full license*DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. This publication is Open Access under the license indicated. Learn MoreArticle Views-Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Mathematical methods,Order,Post-secondary education,Power,Students Get e-Alerts

Recent data has characterized human milk cells with unprecedented detail and provided insight into cell populations. While such analysis of freshly expressed human milk has been possible, studies of cell functionality within the infant have been limited to animal models. One commonly used animal model for milk research is the mouse; however, limited data are available describing the composition of mouse milk. In particular, the maternal cells of mouse milk have not been previously characterized in detail, in part due to the difficulty in collecting sufficient volumes of mouse milk. In this study, we have established a method to collect high volumes of mouse milk, isolate cells, and compare the cell counts and types to human milk. Surprisingly, we found that mouse milk cell density is three orders of magnitude higher than human milk. The cell types present in the milk of mice and humans are similar, broadly consisting of mammary epithelial cells and immune cells. These results provide a basis of comparison for mouse and human milk cells and will inform the most appropriate uses of mouse models for the study of human phenomena.

Abstract Purpose: This study evaluates MALT1 as a potential therapeutic target in H3K27-altered diffuse midline glioma (DMG). Background: H3K27-altered DMG is a devastating pediatric brain tumor that affects 200-300 individuals in the US per year. Median survival is 9-11 months, and there are virtually no long-term survivors. Despite decades of clinical trials, radiation therapy remains standard of care, extending survival by 2-3 months. Development of effective therapies for H3K27-altered DMG is a critical unmet need. MALT1 is the effector molecule of the CARMA-BCL10-MALT1 (CBM) signalosome, a cytoplasmic protein complex that drives downstream signaling, including activation of the pro-survival NF-kB transcription factor. MALT1, which possesses distinct scaffolding and protease activities, has been best characterized as an oncogenic driver in multiple lymphomas. More recently, MALT1 has been shown to act in multiple solid tumor types (breast, lung, glioblastoma) to promote cell viability, proliferation, and migration/invasion. Based on these studies, we hypothesize that MALT1 promotes H3K27-altered DMG cancer cell proliferation and survival and that MALT1 inhibition will abrogate DMG tumor progression. Methods/Results: We screened a panel of patient-derived DMG cell lines by Western blot and found that the CBM signalosome components BCL10 and MALT1 are present in all cell lines tested. We next optimized a brain-penetrant lipid nanoparticle (LNP) delivery system to achieve efficient siRNA knockdown in non-adherent DMG cells and utilized this approach to knockdown MALT1 in DIPG07, BT245, and DIPG XIII FL cell lines. Preliminary findings indicate that loss of MALT1 leads to a reduction in DMG cell viability. To complement this RNAi-based approach, we utilized the blood brain barrier (BBB)-penetrant small molecule MALT1 inhibitor “M1i-124.” This compound, discovered in our laboratory, is a protein-protein interaction inhibitor that disrupts the binding of BCL10 and MALT1 and blocks both MALT1 scaffold and protease activities (manuscript in revision). M1i-124 treatment, at nanomolar-range doses, reduces the viability of both DIPG07 and BT245 DMG cell lines. Conclusions: We found that both siRNA-mediated MALT1 knockdown and the small molecule MALT1 inhibitor M1i-124 are toxic to DMG cells. These preliminary findings point to the potential for therapeutic targeting of MALT1 in H3K27-altered DMG. Future Directions: We will next evaluate the mechanism by which MALT1 knockdown or pharmacological inhibition impacts DMG cell survival using RNA-sequencing and proteomics approaches. We will also use H3K27-altered DMG xenograft models to evaluate the impact of MALT1 inhibition on H3K27-altered DMG tumor progression in vivo. Citation Format: Hannah Butterfield, Juliana Hofstatter Azambuja, Lisa Maurer, Saigopalakrishna S. Yerneni, Andrea Cruz, Matthew Halbert, Taylor Gatesman, Sameer Agnihotri, Peter C. Lucas, Linda M. McAllister-Lucas. MALT1 as a regulator of tumor progression in H3K27-altered diffuse midline glioma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A002.

MALT1 protease is an intracellular signaling molecule that promotes tumor progression via cancer cell-intrinsic and cancer cell-extrinsic mechanisms. MALT1 has been mostly studied in lymphocytes, and little is known about its role in tumor-associated macrophages. Here, we show that MALT1 plays a key role in glioblastoma (GBM)-associated macrophages. Mechanistically, GBM tumor cells induce a MALT1-NF-κB signaling axis within macrophages, leading to macrophage migration and polarization toward an immunosuppressive phenotype. Inactivation of MALT1 protease promotes transcriptional reprogramming that reduces migration and restores a macrophage "M1-like" phenotype. Preclinical in vivo analysis shows that MALT1 inhibitor treatment results in increased immuno-reactivity of GBM-associated macrophages and reduced GBM tumor growth. Further, the addition of MALT1 inhibitor to temozolomide reduces immunosuppression in the tumor microenvironment, which may enhance the efficacy of this standard-of-care chemotherapeutic. Together, our findings suggest that MALT1 protease inhibition represents a promising macrophage-targeted immunotherapeutic strategy for the treatment of GBM.

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1β-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicle for RNA therapeutics, partly because of established lipid structure-activity relationships focused on formulation potency. Yet such knowledge has not extended to LNP immunogenicity. Here we show that the innate and adaptive immune responses elicited by LNPs are linked to their ionizable lipid chemistry. Specifically, we show that the amine headgroups in ionizable lipids drive LNP immunogenicity by binding to Toll-like receptor 4 and CD1d and by promoting lipid-raft formation. Immunogenic LNPs favour a type-1 T-helper-cell-biased immune response marked by increases in the immunoglobulins IgG2c and IgG1 and in the pro-inflammatory cytokines tumour necrosis factor, interferon gamma and the interleukins IL-6 and IL-2. Notably, the inflammatory signals originating from these receptors inhibit the production of anti-poly(ethylene glycol) IgM antibodies, preventing the often-observed loss of efficacy in the LNP-mediated delivery of siRNA and mRNA. Moreover, we identified computational methods for the prediction of the structure-dependent innate and adaptive responses of LNPs. Our findings may help accelerate the discovery of well-tolerated ionizable lipids suitable for repeated dosing. Amine headgroups in ionizable lipids drive the immunogenicity of lipid nanoparticles by binding to Toll-like receptor 4 and CD1d and by promoting lipid-raft formation.

Figure S1. Characterization of TEX. Figure S2. HUVEC viability expressed in % in response to treatment with exosome uptake inhibitors in increasing concentrations as indicated. Movie S1. Confirmation of internalized exosomes using confocal microscopy. Table S1. Clinicopathological characteristics of the HNSCC patients included in this study.