Metabolic Modulation of Kynurenine Based on Kynureninase-Loaded Nanoparticle Depot Overcomes Tumor Immune Evasion in Cancer Immunotherapy.

Evading recognition of immune cells is a well-known strategy of tumors used for their survival. One of the immune evasion mechanisms is the synthesis of kynurenine (KYN), a metabolite of tryptophan, which suppresses the effector T cells. Therefore, lowering the KYN concentration can be an efficient antitumor therapy by restoring the activity of immune cells. Recently, kynureninase (KYNase), which is an enzyme transforming KYN into anthranilate, was demonstrated to show the potential to decrease KYN concentration and inhibit tumor growth. However, due to the limited bioavailability and instability of proteins in vivo, it has been challenging to maintain the KYNase concentration sufficiently high in the tumor microenvironment (TME). Here, we developed a nanoparticle system loaded with KYNase, which formed a Biodegradable and Implantable Nanoparticle Depot named 'BIND' following subcutaneous injection. The BIND sustainably supplied KYNase around the TME while located around the tumor, until it eventually degraded and disappeared. As a result, the BIND system enhanced the proliferation and cytokine production of effector T cells in the TME, followed by tumor growth inhibition and increased mean survival. Finally, we showed that the BIND carrying KYNase significantly synergized with PD-1 blockade in three mouse models of colon cancer, breast cancer, and melanoma.

Enhanced Local Delivery of Engineered IL-2 mRNA by Porous Silica Nanoparticles to Promote Effective Antitumor Immunity.

Localized expression of immunomodulatory molecules can stimulate immune responses against tumors in the tumor microenvironment while avoiding toxicities associated with systemic administration. In this study, we developed a polyethylenimine-modified porous silica nanoparticle (PPSN)-based delivery platform carrying cytokine mRNA for local immunotherapy in vivo. Our delivery platform was significantly more efficient than FDA-approved lipid nanoparticles for localized mRNA translation. We observed no off-target translation of mRNA in any organs and no evidence of systemic toxicity. Intratumoral injection of cytokine mRNA-loaded PPSNs led to high-level expression of protein within the tumor and stimulated immunogenic cancer cell death. Additionally, combining cytokine mRNA with an immune checkpoint inhibitor enhanced anticancer responses in several murine cancer models and enabled the inhibition of distant metastatic tumors. Our results demonstrate the potential of PPSNs-mediated mRNA delivery as a specific, effective, and safe platform for mRNA-based therapeutics in cancer immunotherapy.

Rod-to-sphere elemental reconstruction of biocompatible Ag2Te-Ag4.53-Te3 nanoparticles for triple negative breast cancer photo-nano-therapy.

Silver nanoparticles (AgNPs) continue to be applied to agricultural and medical applications because of their antibacterial and antifungal effects. However, AgNPs are vulnerable to poisoning by oxidation or sulfidation, and unintentional toxicity can occur via leaching. Therefore, ensuring the stability of AgNPs for practical applications is considered an important requirement. In this study, we propose the solvothermal galvanic replacement of a Te nanorod (TeNR) template with a Ag precursor to manufacture highly stable and biocompatible Ag-Te nanoparticles (AgTeNPs). In addition to their high stability, AgTeNPs composed of Ag2Te-Ag4.53Te3 were evaluated as a nanotherapeutic agent enabled by their selective toxicity through metabolic degradation in breast cancer cells. It has been demonstrated that combinatorial treatment with hyperthermic cancer-cell ablation through photothermal conversion provides an effective cancer treatment in vitro and in vivo. The discovered new biocompatible Ag nanomaterials with innate anticancer effects are expected to be applied to various application fields.

Development of a Cancer Nanovaccine to Induce Antigen-specific Immune Responses Based on Large-Sized Porous Silica Nanoparticles.

Cancer vaccine is one of the immunotherapeutic strategies aiming to effectively deliver cancer antigens to professional antigen-presenting cells such as dendritic cells (DCs), macrophages, and B cells to elicit a cancer-specific immune response. Despite the advantages of the cancer vaccine that can be applied to various cancer types, the clinical approach is limited due to the non-specific or adverse immune responses, stability, and safety issues. In this study, we report an injectable nanovaccine platform based on large-sized (∼350 nm) porous silica nanoparticles (PSNs). We found that large-sized PSNs, called PS3, facilitated the formation of an antigen supply depot at the site of injection so that a single injection of PSN-based nanovaccine elicited sufficient tumor-specific cell-mediated and humoral immune response. As a result, antigen-loaded PS3 induced successful tumor regression in prophylactic and therapeutic vaccination.

3D microengineered vascularized tumor spheroids for drug delivery and efficacy testing.

Tumor angiogenesis is regarded as a promising target for limiting cancer progression because tumor-associated vasculature supplies blood and provides a path for metastasis. Thus, in vitro recapitulation of vascularized tumors is critical to understand the pathology of cancer and identify the mechanisms by which tumor cells proliferate, metastasize, and respond to drugs. In this study, we microengineered a vascularized tumor spheroid (VTS) model to reproduce the pathological features of solid tumors. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Notably, the hybrid spheroids also exhibited expression profiles associated with aggressive behavior. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. With the VTS chip showing a progressive tumor phenotype, we validated the suppressive effects of axitinib on tumor growth and angiogenesis, which depended on exposure dose and time, highlighting the significance of tumor vascularization to predict the efficacy of anticancer drugs. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow. Thus, our VTS model is a valuable platform with which to investigate the interactions between tumor microenvironments and explore therapeutic strategies in cancer. STATEMENT OF SIGNIFICANCE: We conducted an integrative study within a vascularized tumor spheroid (VTS) model. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Through RNA sequencing, we elucidated that the tumor-EC hybrid spheroids exhibited expression profiles associated with aggressive behavior such as cancer progression, invasion and metastasis. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. We further validated the suppressive effects of axitinib on tumor growth and angiogenesis, depending on exposure dose and time. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow.

Rhodium-Tellurium Nanorod Synthesis Using Galvanic Replacement-Polyol Regrowth for Thermo-Dynamic Dual-Modal Cancer Phototherapy.

Rh is a noble metal introduced in bioapplications, including diagnosis and therapy, in addition to its consolidated utilization in organic catalysis and electrocatalysis. Herein, we designed the synthesis of highly crystalline Rh nanocrystal-decorated Rh-Te nanorods (RhTeNRs) through galvanic replacement of sacrificial Te nanorod (TeNR) templates and subsequent polyol regrowth. The obtained RhTeNRs showed excellent colloidal stability and efficient heat dissipation and photocatalytic activity under various laser irradiation wavelengths. Based on the confirmed biocompatibility, RhTeNRs were introduced into in vitro and in vivo cancer phototherapies. The results confirmed the selective physical death of cancer cells in the local area through laser irradiation. While chemotherapy does not guarantee successful treatment due to side effects and resistance, phototherapy using heat and reactive oxygen species generation of RhTeNRs induces physical death.

Multifunctional synthetic nano-chaperone for peptide folding and intracellular delivery.

Artificial, synthetic chaperones have attracted much attention in biomedical research due to their ability to control the folding of proteins and peptides. Here, we report bio-inspired multifunctional porous nanoparticles to modulate proper folding and intracellular delivery of therapeutic α-helical peptide. The Synthetic Nano-Chaperone for Peptide (SNCP) based on porous nanoparticles provides an internal hydrophobic environment which contributes in stabilizing secondary structure of encapsulated α-helical peptides due to the hydrophobic internal environments. In addition, SNCP with optimized inner surface modification not only improves thermal stability for α-helical peptide but also supports the peptide stapling methods in situ, serving as a nanoreactor. Then, SNCP subsequently delivers the stabilized therapeutic α-helical peptides into cancer cells, resulting in high therapeutic efficacy. SNCP improves cellular uptake and bioavailability of the anti-cancer peptide, so the cancer growth is effectively inhibited in vivo. These data indicate that the bio-inspired SNCP system combining nanoreactor and delivery carrier could provide a strategy to expedite the development of peptide therapeutics by overcoming existing drawbacks of α-helical peptides as drug candidates.

Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.

Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.

Precursor Heterogeneity Driven Mo-Te Nanoparticle Structural Diversification for Cancer Photo-Theranostics.

Chemical reactions between homogeneous precursors are typically used to synthesize monodisperse nanoparticles with well-controlled size and morphology. It is difficult to predict the evolved nanostructures when using two heterogeneous precursors. In this study, three types of Mo-Te nanoparticles shaped like leaves, spindles, and rice grains (denoted respectively as nanoleaf, nanospindle, and nanorice) were obtained from dextrose-mediated proton-coupled electron transfer reaction between the solid polyoxomolybdate (POM) and the ionic tellurite anion as precursors. All produced nanoparticles had excellent optical absorption in the ultraviolet(UV)-visible(Vis)-near-infrared(NIR) regions, with only slight deviations among them. After confirming nanoparticles' photothermal conversion and photocatalytic activity at multiple wavelengths, the Mo-Te nanorice was tested as a potential agent for cancer treatment due to its minimum toxicity, excellent colloidal stability, and intrinsic anticancer effect. Excellent treatment efficacy and clearance were confirmed in vitro and in vivo. Due to their photoacoustic imaging capability, the injection of pristine nanoparticles could also realize phototheranostics without using additional drugs, probes, or photosensitizers.

Synthesis of gold nano-mushrooms via solvent-controlled galvanic replacement to enhance phototherapeutic efficiency.

In advanced galvanic replacement, variable factors such as the combination of two elements where actual redox reaction and post-synthetic structural transformation take place. Research on manufacturing distinctive nanostructures has mainly focused on the shape of the sacrificial nanotemplate, the presence or absence of additives, and the reaction temperature. Here, we have attempted to confirm the dependency on the solvent, which was considered to simply serve as a medium for a homogeneous chemical reaction to proceed by aiding the dispersion of the nanotemplate and reactants. Thus, we obtained mushroom-like Au nanoplates (mAuNPs) by comprehensive galvanic replacement reaction between solvents, additives, and adsorbents. The mAuNPs with a porous Au nanoplate head and a hollow nanotube tail structure were formed via an optimization process in a 50 v/v% solvent comprising water and ethylene glycol. As a result of confirming the galvanic replacement in co-solvent conditions, in which various types of water miscible solvents were introduced, it was revealed that the most critical factors for regulating the surface polymeric environment of the nanoplate were the relative polarity index of the co-solvent and the hydrogen bonding type. These depend on the molecular structure of the solvent. The manufactured mAuNPs exhibited excellent absorbance in the near-infrared region, and efficient photothermal (PT) conversion-mediated heat dissipation under local laser irradiation. These results confirm the viability of the gene-thermo dual-modal combinatorial cancer therapy based on the surface loading of oligonucleotides and peptides, and the PT therapeutic approach in vitro and in vivo.

Nanoparticle delivery of recombinant IL-2 (BALLkine-2) achieves durable tumor control with less systemic adverse effects in cancer immunotherapy.

Recent strategies in cancer immunotherapy based on interleukin-2 (IL-2) are generally focused on reducing regulatory T cell (Treg) development by modifying IL-2 receptor alpha (IL-2Rα) domain. However, the clinical utility of high-dose IL-2 treatment is mainly limited by severe systemic toxicity. We find that peritumorally injectable 'BALLkine-2', recombinant human IL-2 (rIL-2) loaded porous nanoparticle, dramatically reduces systemic side effects of rIL-2 by minimizing systemic IL-2 exposure. Notably, in cynomolgus monkeys, subcutaneous (SC)-injection of BALLkine-2 not only dramatically reduces systemic circulation of rIL-2 in the blood, but also increases half-life of IL-2 compared to IV- or SC-injection of free rIL-2. Peritumorally-injected BALLkine-2 enhances intratumoral lymphocyte infiltration without inducing Treg development and more effectively synergizes with PD-1 blockade than high-dose rIL-2 administration in B16F10 melanoma model. BALLkine-2 could be a highly potent therapeutic option due to higher anti-tumor efficacy with lower and fewer doses and reduced systemic toxicity compared to systemic rIL-2.

Osmium-Tellurium Nanozymes for Pentamodal Combinatorial Cancer Therapy.

Although nanoparticles based on Group 8 elements such as Fe and Ru have been developed, not much is known about Os nanoparticles. However, Os-based nanostructures might have potential in various applications including biomedical fields. Therefore, in this study, we synthesized Os-Te nanorods (OsTeNRs) by solvothermal galvanic replacement with Te nanotemplates. We explored the nanozymatic activity of the synthesized OsTeNRs and found that they exhibited superior photothermal conversion and photocatalytic activity. Along with chemotherapy (regorafenib) and immunotherapy, the nanozymatic, photothermal, and photodynamic activities of OsTeNRs were harnessed to develop a pentamodal treatment for hepatocellular carcinoma (HCC); in vitro and in vivo studies demonstrated that the pentamodal therapy could alleviate hypoxia in HCC cells by generating oxygen and reduced unintended drug accumulation in organs. Moreover, bone-marrow toxicity due to regorafenib could be reduced as the drug was released in a sustained manner. Thus, OsTeNRs can be considered as suitable nanotemplates for combinatorial cancer therapy.

3D Microfluidic Platform and Tumor Vascular Mapping for Evaluating Anti-Angiogenic RNAi-Based Nanomedicine.

Three-dimensional (3D) visualization of tumor vasculature is a key factor in accurate evaluation of RNA interference (RNAi)-based antiangiogenic nanomedicine, a promising approach for cancer therapeutics. However, this remains challenging because there is not a physiologically relevant in vitro model or precise analytic methodology. To address this limitation, a strategy based on 3D microfluidic angiogenesis-on-a-chip and 3D tumor vascular mapping was developed for evaluating RNAi-based antiangiogenic nanomedicine. We developed a microfluidic model to recapitulate functional 3D angiogenic sprouting when co-cultured with various cancer cell types. This model enabled efficient and rapid assessment of antiangiogenic nanomedicine in treatment of hyper-angiogenic cancer. In addition, tissue-clearing-based whole vascular mapping of tumor xenograft allowed extraction of complex 3D morphological information in diverse quantitative parameters. Using this 3D imaging-based analysis, we observed tumor sub-regional differences in the antiangiogenic effect. Our systematic strategy can help in narrowing down the promising targets of antiangiogenic nanomedicine and then enables deep analysis of complex morphological changes in tumor vasculature, providing a powerful platform for the development of safe and effective nanomedicine for cancer therapeutics.

Intrinsic Peroxidase-Mimicking Ir Nanoplates for Nanozymatic Anticancer and Antibacterial Treatment.

The study of inorganic nanozymes to overcome the disadvantages of bio-enzymes, such as the requirement of optimized reaction conditions and lack of durability against environmental factors, is one of the most significant research topics at present. In this work, we comprehensively analyzed the intrinsic peroxidase-like activity of Ir-based nanoparticles, the biological and nanozymatic potentials of which have not yet been explored. These particles were synthesized by the galvanic replacement of Ag nanoplates with Ir. Through the confirmed peroxidase-like activity and hydrogen peroxide decomposition with free radical generation facilitated by these particles, the antibacterial and anticancer effects were successfully verified in vitro. The nanozyme-based therapeutic effect observed at concentrations at which these nanoparticles do not show cytotoxicity suggests that it is possible to achieve more precise and selective local treatment with these particles. The observed highly efficient peroxidase-like activity of these nanoparticles is attributed to the partially mixed composition of Ir-Ag-IrO2 formed through the galvanic replacement reaction in the synthetic process.

Large-Scale 3D Optical Mapping and Quantitative Analysis of Nanoparticle Distribution in Tumor Vascular Microenvironment.

Nanoparticles (NPs) are a promising carrier for cancer therapeutics. Systemically administered NPs are transported to tumor tissues via the bloodstream, extravasated from microvessels, and delivered to cancer cells. The distribution of NPs in the tumor vascular microenvironment critically determines the therapeutic efficacy of NP-delivered drugs, but its precise assessment in 3D across a large volume remains challenging. Here, an analytical platform-termed OMNIA (for Optical Mapping of Nanoparticles and Image Analysis)-integrating tissue clearing, high-resolution optical imaging, and semiautomated image analysis is presented, which enables accurate, unbiased, and quantitative analysis of the distribution of NPs in relation to the vasculature across a large 3D volume. Application of OMNIA to tumor tissues revealed higher accumulation and more efficient extravasation of NPs in the tumor periphery than the core. Time-course analysis demonstrated that the accumulation of NPs in tumor peaked at 24 h after injection, but the relative distribution of NPs from the vasculature remained remarkably stable over time. Comparisons between 45- and 200-nm-sized NPs showed a lower accumulation of smaller NPs in tumors relative to the liver, yet better vessel permeation. Together, our results demonstrate that OMNIA facilitates precise and reliable evaluation of NP biodistribution, and mechanistic investigations on NP delivery to tumor tissues.

A fluorescent nanobiosensor for the facile analysis of m6A RNA demethylase activity.

RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.

Nonrecurring Circuit Nanozymatic Enhancement of Hypoxic Pancreatic Cancer Phototherapy Using Speckled Ru-Te Hollow Nanorods.

Nanozymatic reactions that produce or consume oxygen (O2) or reactive oxygen species (ROS) consist of oxidase, peroxidase, superoxide dismutase (SOD), and catalase-type activity. Although extensive studies were conducted to overcome hypoxia through nanozymatic reactions, the construction of an ideal system is challenging, given that the reactants and products are arranged in a recurring structure for continuous consumption in a full cycle. In this study, speckled Ru-Te hollow nanorods were prepared through solvothermal galvanic replacement against Te nanorod templates with high yield and robustness. From their multicompositional characteristics, nonrecurring peroxidase-SOD-catalase-type nanozymatic properties were identified with photothermal and photodynamic feasibility over a wide range of laser irradiation wavelengths. Owing to the excellent colloidal stability and biocompatibility, the proposed Ru-Te-based nanozymatic platform was highly effective in hypoxic pancreatic cancer phototherapy in vitro and in vivo by near-infrared laser irradiation mediated photothermal and photodynamic combination treatment.

Environmentally Friendly Synthesis of Au-Te-Clustered Nanoworms via Galvanic Replacement for Wavelength-Selective Combination Cancer Therapy.

Au-Te-clustered nanoworms (AuTeNWs) were successfully synthesized under ambient conditions by spontaneous galvanic replacement using Te nanorods as a sacrificial nanotemplate. Along with the gradual replacement and on-surface crystalline Au cluster formation, Te nanotemplates were transformed into a serpentine nanoworm-like morphology. The present strategy was an environmentally friendly method that did not use surfactants to control the surface structure. The synthesized nanoworms exhibited excellent photothermal conversion, photocatalytic efficiencies, and high payloads for thiolated genes and cell-penetrating peptides. According to the visible and near-infrared wavelengths of light, the photodynamic and photothermal therapeutic pathways were dominantly acting, respectively. From this, wavelength-selective combination treatment with gene therapy was successfully accomplished. Taken together, excellent therapeutic effects for in vitro and in vivo mouse models against hepatitis C replicon human hepatocarcinoma were clearly identified by using the present AuTeNWs as a phototherapeutic nanocarrier.

Plant-Derived Purification, Chemical Synthesis, and In Vitro/In Vivo Evaluation of a Resveratrol Dimer, Viniferin, as an HCV Replication Inhibitor.

Oligostilbenoid compounds, a group of resveratrol multimers, display several anti-microbial activities through the neutralization of cytotoxic oxidants, and by inhibiting essential host and viral enzymes. In our previous study, we identified a series of oligostilbenoid compounds as potent hepatitis C virus (HCV) replication inhibitors. In particular, vitisin B, a resveratrol tetramer, exhibited the most dramatic anti-HCV activity (EC50 = 6 nM and CC50 > 10 µM) via the disruption of the viral helicase NS3 (IC50 = 3 nM). However, its further development as an HCV drug candidate was halted due to its intrinsic drawbacks, such as poor stability, low water solubility, and restricted in vivo absorption. In order to overcome these limitations, we focused on (+)-ε-viniferin, a resveratrol dimer, as an alternative. We prepared three different versions of (+)-ε-viniferin, including one which was extracted from the grapevine root (EVF) and two which were chemically synthesized with either penta-acetylation (SVF-5Ac) or no acetylation (SVF) using a newly established synthesis method. We confirmed their anti-HCV replication activities and minimal cytotoxicity by using genotype 1b and 2a HCV replicon cells. Their anti-HCV replication action also translated into a significant reduction of viral protein expression. Anti-HCV NS3 helicase activity by EVF was also verified in vitro. Finally, we demonstrated that SVF has improved pharmacokinetic properties over vitisin B. Overall, the favorable antiviral and pharmacokinetic properties of these three versions of viniferin warrant their further study as members of a promising new class of anti-HCV therapeutics.