SUMMARY Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short- term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T cell (Treg) expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6 specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimunnotherapy resulted in indefinite allograft survival. Together, we show that HDL- based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe–/– mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe–/– mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP–immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.

Highlights d We have developed a trained immunity-inducing nanobiologic therapeutic named MTP-HDL d MTP-HDL favorably accumulates in hematopoietic organs of mice and non-human primates d MTP-HDL nanotherapy induces trained immunity through bone marrow progenitors in vivo d MTP-HDL nanotherapy inhibits tumor growth and potentiates immune checkpoint inhibition

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in Apoe–/– mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated in vitro. Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis.

Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.

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Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell–specific nanobiologics in apolipoprotein E–deficient (Apoe−/−) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages’ inflammatory activity. By investigating transcriptome modifications, we identified Psap, a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap−/− bone marrow to low-density lipoprotein receptor knockout (Ldlr−/−) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.