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In complex diseases such as cancer, modulating cytokine signatures of disease using innate immune agonists holds therapeutic promise. Novel multi-agonist treatments offer tunable control of the immune system because they are uniquely pathogen inspired, eliciting robust antitumor responses by promoting synergistic cytokine responses. However, the chief strategic hurdle is ensuring multi-agonist delivery to the same target cells, highlighting the importance of using nanomaterial-based carriers. Here, we place nanocarriers in center stage and review the delivery hurdles related to the varying extra- and intracellular localizations of innate immune receptors. We discuss a range of nanomaterials used for multi-agonist delivery, highlighting their respective benefits and drawbacks. Our overarching stance is that rational nanocarrier design is crucial for developing pathogen-inspired multi-agonist immunotherapies., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) has quickly risen to become the third leading cause of cancer-related death in the United States. This is in part because of its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here, we investigated an immunotherapy approach combining delivery of stimulator of interferon genes (STING) and Toll-like receptor 4 (TLR4) innate immune agonists by lipid-based nanoparticle (NP) coencapsulation with senescence-inducing RAS-targeted therapies, which can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other proinflammatory signaling pathways, increased antigen presentation by tumor cells and antigen-presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and type I interferon-mediated tumor regression and long-term survival in preclinical PDAC models dependent on both tumor and host STING activation. STING and TLR4-mediated type I interferon signaling was also associated with enhanced natural killer and CD8 + T cell immunity in human PDAC samples. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can orchestrate a coordinated type I interferon-driven innate and adaptive immune response with durable antitumor efficacy against PDAC.

Cancer immunotherapy offers transformative promise particularly for the treatment of lethal cancers, since a correctly trained immune system can comprehensively orchestrate tumor clearance with no need for continued therapeutic intervention. Historically, the majority of immunotherapies have been T cell-focused and have included immune checkpoint inhibitors, chimeric antigen receptor T cells, and T-cell vaccines. Unfortunately T-cell-focused therapies have failed to achieve optimal efficacy in most solid tumors largely because of a highly immunosuppressed 'cold' or immune-excluded tumor microenvironment (TME). Recently, a rapidly growing treatment paradigm has emerged that focuses on activation of tumor-resident innate antigen-presenting cells, such as dendritic cells and macrophages, which can drive a proinflammatory immune response to remodel the TME from 'cold' or immune-excluded to 'hot'. Early strategies for TME remodeling centered on free cytokines and agonists, but these approaches have faced significant hurdles in both delivery and efficacy. Systemic toxicity from off-target inflammation is a paramount concern in these therapies. To address this critical gap, engineering approaches have provided the opportunity to add 'built-in' capabilities to cytokines, agonists, and other therapeutic agents to mediate improved delivery and efficacy. Such capabilities have included protective encapsulation to shield them from degradation, targeting to direct them with high specificity to tumors, and co-delivery strategies to harness synergistic proinflammatory pathways. Here, we review innate immune-mediated TME remodeling engineering approaches that focus on cytokines, innate immune agonists, immunogenic viruses, and cell-based methods, highlighting emerging preclinical approaches and strategies that are either being tested in clinical trials or already Food and Drug Administration approved., (© 2023 The Author(s).)

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention., Competing Interests: Declaration of interests A provisional patent on targeting SLC46s to inhibit inflammation in psoriasis and other auto-inflammatory diseases has been filed by some of the authors (N.S., R.B., and M.H.O.). T.-D.K. consults for Pfizer., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lethal cancer is characterized by drug-resistant relapse and metastasis. Here, we evaluate the efficacy of a neoadjuvant therapeutic strategy prior to surgery that combines the immune checkpoint inhibitor anti-PD1 with a powerful immunostimulatory nanoparticle (immuno-NP). Lipid-based immuno-NPs are uniquely designed to co-encapsulate a STING and TLR4 agonist that are functionally synergistic. Efficacy of neoadjuvant combination immunotherapy was assessed in three aggressive murine tumor models, including B16F10 melanoma and 4T1 and D2.A1 breast cancer. Primary splenocytes treated with dual-agonist immuno-NPs produced a 75-fold increased production of interferon β compared to single-agonist treatments. Systemic delivery facilitated the widespread deposition of immuno-NPs in the perivascular space throughout the tumor mass and their preferential uptake by tumor-resident antigen-presenting cells. Our findings strongly suggested that immuno-NPs, when administered in combination with anti-PD1, harnessed and activated the otherwise "exhausted" CD8 + T cells as key mediators of tumor clearance. Neoadjuvant combination immunotherapy resulted in significant efficacy, curative responses, and protective immunological memory in 71% of good-responding mice bearing B16F10 melanoma tumors and showed similar trends in the two breast cancer models. Finally, this neoadjuvant combination immunotherapy drove the generation of B and T cell de novo epitopes for a comprehensive memory response.

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon β, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, α v β 3 integrin and P-selectin that are commonly used to direct nanoparticles to tumors. Using the 4T1 mouse model, we assessed the microdistribution of the four NP variants in the tumor immune microenvironment in three different breast cancer landscapes, including primary tumor, early metastasis, and late metastasis. The different NP variants resulted in variable uptake by immune cell subsets depending on the organ and tumor stage. Among the NP variants, therapeutic studies indicated that the untargeted NPs and the integrin-targeting NPs exhibited a remarkable short- and long-term immune response and long-lasting antitumor effect.

The efficacy of immunotherapies is often limited by the immunosuppressive tumor microenvironment, which is populated with dysfunctional innate immune cells. To reprogram the tumor-resident innate immune cells, we developed immunostimulatory silica mesoporous nanoparticles (immuno-MSN). The cargo of immuno-MSN is a Stimulator of Interferon Gene (STING) agonist, which activates innate immune cells leading to production of interferon (IFN) β. By proficiently trafficking its cargo into immune cells, the immuno-MSN induced a 9-fold increase of IFN-β secretion compared to free agonist. While an external PEG shield has historically been used to protect nanoparticles from immune recognition, a PEGylated immunostimulatory nanoparticle needs to strike a balance between immune evasion to avoid off-site accumulation and uptake by target immune cells in tumors. Using the 4T1 mouse model of metastatic breast cancer and flow cytometry, it was determined that the degree of PEGylation significantly influenced the uptake of 'empty' MSNs by tumor-resident innate immune cells. This was not the case for the agonist-loaded immuno-MSN variants. It should be noted the surface charge of the 'empty' MSNs was positive rather than neutral for the agonist-loaded immuno-MSNs. However, even though the cellular uptake was similar at 24 h after injection for the three immuno-MSN variants, we observed a significant beneficial effect on the activation and expansion of APCs especially in lung metastasis using the lightly PEGylated immuno-MSN variant., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Recent advancements in unravelling elements of cancer biology involved in disease progression and treatment resistance have highlighted the need for a holistic approach to effectively tackle cancer. Stimuli-responsive nanotheranostics based on iron oxide nanoparticles are an emerging class of versatile nanomedicines with powerful capabilities to "seek, sense, and attack" multiple components of solid tumors. In this work, the rationale for using iron oxide nanoparticles and the basic physical principles that impact their function in biomedical applications are reviewed. Subsequently, recent advances in the integration of iron oxide nanoparticles with various stimulus mechanisms to facilitate the development of stimuli-responsive nanotheranostics for application in cancer therapy are summarized. The integration of an iron oxide core with various surface coating mechanisms results in the generation of hybrid nanoconstructs with capabilities to codeliver a wide variety of highly potent anticancer therapeutics and immune modulators. Finally, emerging future directions and considerations for their clinical translation are touched upon., (© 2020 Wiley-VCH GmbH.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease, where even surgical resection and aggressive chemotherapy produce dismal outcomes. Immunotherapy is a promising alternative to conventional treatments, possessing the ability to elicit T cell-mediated killing of tumor cells and prevent disease recurrence. Immunotherapeutic approaches thus far have seen limited success in PDAC due to a poorly immunogenic and exceedingly immunosuppressive tumor microenvironment, which is enriched with dysfunctional and immunosuppressed antigen-presenting cells (APCs). We developed a highly potent immunostimulatory nanoparticle (immuno-NP) to activate and expand APCs in the tumor and induce local secretion of interferon β (IFNβ), which is a pro-inflammatory cytokine that plays a major role in APC recruitment. The effectiveness of the immuno-NP stems from its dual cargo of two synergistic immune modulators consisting of an agonist of the stimulator of interferon genes (STING) pathway and an agonist of the Toll-like receptor 4 (TLR4) pathway. We show the functional synergy of the dual-agonist cargo can be tweaked by adjusting the ratio of the two agonists loaded in the immuno-NP, leading to an increase in IFNβ production (11-fold) compared to any single agonist immuno-NP variant. Using the orthotopic murine Panc02 model of PDAC, we show that systemic administration allowed immuno-NPs to deposit into the perivascular regions of the tumor, which coincided with the APC-rich tumor areas leading to predominant uptake of immuno-NPs by APCs. The immuno-NPs were effectively taken up by a significant portion of dendritic cells in the tumor (>56%). This led to a significant expansion of APCs, resulting in an 11.5-fold increase of dendritic cells and infiltration of lymphocytes throughout the pancreatic tumor compared to untreated animals., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The high mortality associated with glioblastoma multiforme (GBM) is attributed to its invasive nature, hypoxic core, resistant cell subpopulations and a highly immunosuppressive tumor microenvironment (TME). To support adaptive immune function and establish a more robust antitumor immune response, we boosted the local innate immune compartment of GBM using an immunostimulatory mesoporous silica nanoparticle, termed immuno-MSN. The immuno-MSN was specifically designed for systemic and proficient delivery of a potent innate immune agonist to dysfunctional antigen-presenting cells (APCs) in the brain TME. The cargo of the immuno-MSN was cyclic diguanylate monophosphate (cdGMP), a Stimulator of Interferon Gene (STING) agonist. Studies showed the immuno-MSN promoted the uptake of STING agonist by APCs in vitro and the subsequent release of the pro-inflammatory cytokine interferon β, 6-fold greater than free agonist. In an orthotopic GBM mouse model, systemically administered immuno-MSN particles were taken up by APCs in the near-perivascular regions of the brain tumor with striking efficiency. The immuno-MSNs facilitated the recruitment of dendritic cells and macrophages to the TME while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8 + T cell activity (2.5-fold) and delayed GBM tumor growth. We show that an engineered immunostimulatory nanoparticle can support pro-inflammatory innate immune function in GBM and subsequently augment current immunotherapeutic interventions and improve their therapeutic outcome.

Effective cancer immunotherapy depends on the robust activation of tumor-specific antigen-presenting cells (APC). Immune agonists encapsulated within nanoparticles (NP) can be delivered to tumor sites to generate powerful antitumor immune responses with minimal off-target dissemination. Systemic delivery enables widespread access to the microvasculature and draining to the APC-rich perivasculature. We developed an immuno-nanoparticle (immuno-NP) coloaded with cyclic diguanylate monophosphate, an agonist of the stimulator of interferon genes pathway, and monophosphoryl lipid A, and a Toll-like receptor 4 agonist, which synergize to produce high levels of type I IFNβ. Using a murine model of metastatic triple-negative breast cancer, systemic delivery of these immuno-NPs resulted in significant therapeutic outcomes due to extensive upregulation of APCs and natural killer cells in the blood and tumor compared with control treatments. These results indicate that NPs can facilitate systemic delivery of multiple immune-potentiating cargoes for effective APC-driven local and systemic antitumor immunity. SIGNIFICANCE: Systemic administration of an immuno-nanoparticle in a murine breast tumor model drives a robust tumor site-specific APC response by delivering two synergistic immune-potentiating molecules, highlighting the potential of nanoparticles for immunotherapy., (©2019 American Association for Cancer Research.)

The development of synthetic nanomaterials that could embed within, penetrate, or induce fusion between membranes without permanent disruption would have great significance for biomedical applications. Here we describe structure-function relationships of highly water-soluble gold nanoparticles comprised of an ∼1.5-5 nm diameter metal core coated by an amphiphilic organic ligand shell, which exhibit membrane embedding and fusion activity mediated by the surface ligands. Using an environment-sensitive dye anchored within the ligand shell as a sensor of membrane embedding, we demonstrate that particles with core sizes of ∼2-3 nm are capable of embedding within and penetrating fluid bilayers. At the nanoscale, these particles also promote spontaneous fusion of liposomes or spontaneously embed within intact liposomal vesicles. These studies provide nanoparticle design and selection principles that could be used in drug delivery applications, as membrane stains, or for the creation of novel organic/inorganic nanomaterial self-assemblies.

This corrects the article DOI: 10.1038/ncomms14069.

This review seeks to highlight the enormous potential of targeted nanoparticles for molecular imaging applications. Being the closest point-of-contact, circulating nanoparticles can gain direct access to targetable molecular markers of disease that appear on the endothelium. Further, nanoparticles are ideally suitable to vascular targeting due to geometrically enhanced multivalent attachment on the vascular target. This natural synergy between nanoparticles, vascular targeting and molecular imaging can provide new avenues for diagnosis and prognosis of disease with quantitative precision. In addition to the obvious applications of targeting molecular signatures of vascular diseases (e.g., atherosclerosis), deep-tissue diseases often manifest themselves by continuously altering and remodeling their neighboring blood vessels (e.g., cancer). Thus, the remodeled endothelium provides a wide range of targets for nanoparticles and molecular imaging. To demonstrate the potential of molecular imaging, we present a variety of nanoparticles designed for molecular imaging of cancer or atherosclerosis using different imaging modalities., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Inorganic nanoparticles (NPs) are studied as drug carriers, radiosensitizers and imaging agents, and characterizing nanoparticle biodistribution is essential for evaluating their efficacy and safety. Tracking NPs at the single-cell level with current technologies is complicated by the lack of reliable methods to stably label particles over extended durations in vivo. Here we demonstrate that mass cytometry by time-of-flight provides a label-free approach for inorganic nanoparticle quantitation in cells. Furthermore, mass cytometry can enumerate AuNPs with a lower detection limit of ∼10 AuNPs (3 nm core size) in a single cell with tandem multiparameter cellular phenotyping. Using the cellular distribution insights, we selected an amphiphilic surface ligand-coated AuNP that targeted myeloid dendritic cells in lymph nodes as a peptide antigen carrier, substantially increasing the efficacy of a model vaccine in a B16-OVA melanoma mouse model. This technology provides a powerful new level of insight into nanoparticle fate in vivo.

Erythrocytes are attractive as potential cell-based drug carriers because of their abundance and long lifespan in vivo. Existing methods for loading drug cargos into erythrocytes include hypotonic treatments, electroporation, and covalent attachment onto the membrane, all of which require ex vivo manipulation. Here, we characterized the properties of amphiphilic gold nanoparticles (amph-AuNPs), comprised of a ∼2.3 nm gold core and an amphiphilic ligand shell, which are able to embed spontaneously within erythrocyte membranes and might provide a means to load drugs into red blood cells (RBCs) directly in vivo. Particle interaction with RBC membranes occurred rapidly at physiological temperature. We further show that amph-AuNP uptake by RBCs was limited by the glycocalyx and was particularly influenced by sialic acids on cell surface proteoglycans. Using a reductionist model membrane system with synthetic lipid vesicles, we confirmed the importance of membrane fluidity and the glycocalyx in regulating amph-AuNP/membrane interactions. These results thus provide evidence for the interaction of amph-AuNPs with erythrocyte membranes and identify key membrane components that govern this interaction, providing a framework for the development of amph-AuNP-carrying erythrocyte 'pharmacytes' in vivo.

Anionic, monolayer-protected gold nanoparticles (AuNPs) have been shown to nondisruptively penetrate cellular membranes. Here, we show that a critical first step in the penetration process is potentially the fusion of such AuNPs with lipid bilayers. Free energy calculations, experiments on unilamellar and multilamellar vesicles, and cell studies all support this hypothesis. Furthermore, we show that fusion is only favorable for AuNPs with core diameters below a critical size that depends on the monolayer composition.

We recently described a strategy for intracellular delivery of macromolecules, utilizing pH-responsive "core-shell" structured gel particles. These cross-linked hydrogel particles disrupt endosomes with low toxicity by virtue of physical sequestration of an endosome-disrupting "proton sponge" core inside a nontoxic hydrophilic shell. Here we tested the efficacy of this system for cytosolic delivery of a broad range of macromolecular cargos, and demonstrate the delivery of proteins, whole viral particles, or siRNA oligonucleotides into the cytosol of dendritic cells and epithelial cells via core-shell particles. We assessed the functional impact of particle delivery for vaccine applications and found that cytosolic delivery of protein antigens in dendritic cells via the core-shell particles promotes priming of CD8(+) T-cells at 100-fold lower doses than soluble protein. Functional gene knockdown following delivery of siRNA using the particles was demonstrated in epithelial cells. Based on these findings, these materials may be of interest for a broad range of biomedical applications.