Noninvasive ROS imaging and drug delivery monitoring in the tumor microenvironment.

Reactive oxygen species (ROS) that are overproduced in certain tumors can be considered an indicator of oxidative stress levels in the tissue. Here, we report a magnetic resonance imaging (MRI)-based probe capable of detecting ROS levels in the tumor microenvironment (TME) using ROS-responsive manganese ion (Mn2+)-chelated, biotinylated bilirubin nanoparticles (Mn@bt-BRNPs). These nanoparticles are disrupted in the presence of ROS, resulting in the release of free Mn2+, which induces T1-weighted MRI signal enhancement. Mn@BRNPs show more rapid and greater MRI signal enhancement in high ROS-producing A549 lung carcinoma cells compared with low ROS-producing DU145 prostate cancer cells. A pseudo three-compartment model devised for the ROS-reactive MRI probe enables mapping of the distribution and concentration of ROS within the tumor. Furthermore, doxorubicin-loaded, cancer-targeting ligand biotin-conjugated Dox/Mn@bt-BRNPs show considerable accumulation in A549 tumors and also effectively inhibit tumor growth without causing body weight loss, suggesting their usefulness as a new theranostic agent. Collectively, these findings suggest that Mn@bt-BRNPs could be used as an imaging probe capable of detecting ROS levels and monitoring drug delivery in the TME with potential applicability to other inflammatory diseases.

Longitudinal Magnetic Resonance Imaging with ROS-Responsive Bilirubin Nanoparticles Enables Monitoring of Nonalcoholic Steatohepatitis Progression to Cirrhosis.

Despite the vital importance of monitoring the progression of nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH), an efficient imaging modality that is readily available at hospitals is currently lacking. Here, a new magnetic-resonance-imaging (MRI)-based imaging modality is presented that allows for efficient and longitudinal monitoring of NAFLD and NASH progression. The imaging modality uses manganese-ion (Mn2+)-chelated bilirubin nanoparticles (Mn@BRNPs) as a reactive-oxygen-species (ROS)-responsive MRI imaging probe. Longitudinal T1-weighted MR imaging of NASH model mice is performed after injecting Mn@BRNPs intravenously. The MR signal enhancement in the liver relative to muscle gradually increases up to 8 weeks of NASH progression, but decreases significantly as NASH progresses to the cirrhosis-like stage at weeks 10 and 12. A new dual input pseudo-three-compartment model is developed to provide information on NASH stage with a single MRI scan. It is also demonstrated that the ROS-responsive Mn@BRNPs can be used to monitor the efficacy of potential anti-NASH drugs with conventional MRI. The findings suggest that the ROS-responsive Mn@BRNPs have the potential to serve as an efficient MRI contrast for monitoring NASH progression and its transition to the cirrhosis-like stage.

Anti-inflammatory Glycocalyx-Mimicking Nanoparticles for Colitis Treatment: Construction and In Vivo Evaluation.

Common medications for treating inflammatory bowel disease (IBD) have limited therapeutic efficacy and severe adverse effects. This underscores the urgent need for novel therapeutic approaches that can effectively target inflamed sites in the gastrointestinal tract upon oral administration, exerting potent therapeutic efficacy while minimizing systemic effects. Here, we report the construction and in vivo therapeutic evaluation of a library of anti-inflammatory glycocalyx-mimicking nanoparticles (designated GlyNPs) in a mouse model of IBD. The anti-inflammatory GlyNP library was created by attaching bilirubin (BR) to a library of glycopolymers composed of random combinations of the five most naturally abundant sugars. Direct in vivo screening of 31 BR-attached anti-inflammatory GlyNPs via oral administration into mice with acute colitis led to identification of a candidate GlyNP capable of targeting macrophages in the inflamed colon and effectively alleviating colitis symptoms. These findings suggest that the BR-attached GlyNP library can be used as a platform to identify anti-inflammatory nanomedicines for various inflammatory diseases.

Bilirubin Nanomedicine Rescues Intestinal Barrier Destruction and Restores Mucosal Immunity in Colitis.

Inflammatory bowel disease (IBD) manifests as intestinal barrier destruction, mucosal immunity dysregulation, and disrupted gut microbiome homeostasis. Conventional anti-inflammatory medications for IBD therapy partially alleviate symptoms but are unable to restore normal barrier and immune function. Here, we report a nanomedicine comprising bilirubin (BR)-attached low-molecular-weight, water-soluble chitosan nanoparticles (LMWC-BRNPs), that promotes restoration of the intestinal barrier, mucosal immunity, and the gut microbiome, thereby exerting robust therapeutic efficacy. In a mouse model of dextran sulfate sodium salt (DSS)-induced colitis, orally administered LMWC-BRNPs were retained in the GI tract much longer than other nonmucoadhesive BRNPs owing to the mucoadhesiveness of LMWC via electrostatic interaction. Treatment with LMWC-BRNPs led to considerable recovery of the damaged intestinal barrier compared with the current IBD medication, 5-aminosalicylic acid (5-ASA). Orally administered LMWC-BRNPs were taken up by pro-inflammatory macrophages and inhibited their activity. They also concurrently increased the population of regulatory T cells, thereby leading to the recovery of dysregulated mucosal immunity. An analysis of the gut microbiome revealed that LMWC-BRNPs treatment significantly attenuated the increase Turicibacter, an inflammation-related microorganism, resulting in protection of gut microbiome homeostasis. Taken together, our findings indicate that LMWC-BRNPs restored normal functions of the intestine and have high potential for use as a nanomedicine for IBD therapy.

Post Transplantation Bilirubin Nanoparticles Ameliorate Murine Graft Versus Host Disease via a Reduction of Systemic and Local Inflammation.

Allogeneic stem cell transplantation is a curative immunotherapy where patients receive myeloablative chemotherapy and/or radiotherapy, followed by donor stem cell transplantation. Graft versus host disease (GVHD) is a major complication caused by dysregulated donor immune system, thus a novel strategy to modulate donor immunity is needed to mitigate GVHD. Tissue damage by conditioning regimen is thought to initiate the inflammatory milieu that recruits various donor immune cells for cross-priming of donor T cells against alloantigen and eventually promote strong Th1 cytokine storm escalating further tissue damage. Bilirubin nanoparticles (BRNP) are water-soluble conjugated of bilirubin and polyethylene glycol (PEG) with potent anti-inflammatory properties through its ability to scavenge reactive oxygen species generated at the site of inflammation. Here, we evaluated whether BRNP treatment post-transplantation can reduce initial inflammation and subsequently prevent GVHD in a major histocompatibility (MHC) mismatched murine GVHD model. After myeloablative irradiation, BALB/c mice received bone marrow and splenocytes isolated from C57BL/6 mice, with or without BRNP (10 mg/kg) daily on days 0 through 4 post-transplantation, and clinical GVHD and survival was monitored for 90 days. First, BRNP treatment significantly improved clinical GVHD score compared to untreated mice (3.4 vs 0.3, p=0.0003), and this translated into better overall survival (HR 0.0638, p=0.0003). Further, BRNPs showed a preferential accumulation in GVHD target organs leading to a reduced systemic and local inflammation evidenced by lower pathologic GVHD severity as well as circulating inflammatory cytokines such as IFN-γ. Lastly, BRNP treatment post-transplantation facilitated the reconstitution of CD4+ iNK T cells and reduced expansion of proinflammatory CD8α+ iNK T cells and neutrophils especially in GVHD organs. Lastly, BRNP treatment decreased ICOS+ or CTLA-4+ T cells but not PD-1+ T cells suggesting a decreased level of T cell activation but maintaining T cell tolerance. In conclusion, we demonstrated that BRNP treatment post-transplantation ameliorates murine GVHD via diminishing the initial tissue damage and subsequent inflammatory responses from immune subsets.

Photomedicine based on heme-derived compounds.

Photoimaging and phototherapy have become major platforms for the diagnosis and treatment of various health complications. These applications require a photosensitizer (PS) that is capable of absorbing light from a source and converting it into other energy forms for detection and therapy. While synthetic inorganic materials such as quantum dots and gold nanorods have been widely explored for their medical diagnosis and photodynamic (PDT) and photothermal (PTT) therapy capabilities, translation of these technologies has lagged, primarily owing to potential cytotoxicity and immunogenicity issues. Of the various photoreactive molecules, the naturally occurring endogenous compound heme, a constituent of red blood cells, and its derivatives, porphyrin, biliverdin and bilirubin, have shown immense potential as noteworthy candidates for clinically translatable photoreactive agents, as evidenced by previous reports. While porphyrin-based photomedicines have attracted significant attention and are well documented, research on photomedicines based on two other heme-derived compounds, biliverdin and bilirubin, has been relatively lacking. In this review, we summarize the unique photoproperties of heme-derived compounds and outline recent efforts to use them in biomedical imaging and phototherapy applications.

Bilirubin Nanoparticles Protect Against Cardiac Ischemia/Reperfusion Injury in Mice.

Background Ischemia/reperfusion (I/R) injury causes overproduction of reactive oxygen species, which are the major culprits of oxidative stress that leads to inflammation, apoptosis, myocardial damage, and dysfunction. Bilirubin acts as a potent endogenous antioxidant that is capable of scavenging various reactive oxygen species. We have previously generated bilirubin nanoparticles (BRNPs) consisting of polyethylene glycol-conjugated bilirubin. In this study, we examined the therapeutic effects of BRNPs on myocardial I/R injury in mice. Methods and Results In vivo imaging using fluorophore encapsulated BRNPs showed BRNPs preferentially targeted to the site of I/R injury in the heart. Cardiac I/R surgery was performed by first ligating the left anterior descending coronary artery. After 45 minutes, reperfusion was achieved by releasing the ligation. BRNPs were administered intraperitoneally at 5 minutes before and 24 hours after reperfusion. Mice that received BRNPs showed significant improvements in their cardiac output, assessed by echocardiogram and pressure volume loop measurements, compared with the ones that received vehicle treatment. BRNPs treatment also significantly reduced the myocardial infarct size in mice that underwent cardiac I/R, compared with the vehicle-treatment group. In addition, BRNPs effectively suppressed reactive oxygen species and proinflammatory factor levels, as well as the amount of cardiac apoptosis. Conclusions Taken together, BRNPs could exert their therapeutic effects on cardiac I/R injury through attenuation of oxidative stress, apoptosis, and inflammation, providing a novel therapeutic modality for myocardial I/R injury.

Biomimetic lipid Nanocomplexes incorporating STAT3-inhibiting peptides effectively infiltrate the lung barrier and ameliorate pulmonary fibrosis.

Activation of signal transducer and activator of transcription 3 (STAT3) under conditions of inflammation plays a crucial role in the pathogenesis of life-threatening pulmonary fibrosis (PF), initiating pro-fibrotic signaling following its phosphorylation. While there have been attempts to interfere with STAT3 activation and associated signaling as a strategy for ameliorating PF, potent inhibitors with minimal systemic toxicity have yet to be developed. Here, we assessed the in vitro and in vivo therapeutic effectiveness of a cell-permeable peptide inhibitor of STAT3 phosphorylation, designated APTstat3-9R, for ameliorating the indications of pulmonary fibrosis. Our results demonstrate that APTstat3-9R formulated with biomimetic disc-shaped lipid nanoparticles (DLNPs) markedly enhanced the penetration of pulmonary surfactant barrier and alleviated clinical symptoms of PF while causing negligible systemic cytotoxicity. Taken together, our findings suggest that biomimetic lipid nanoparticle-assisted pulmonary delivery of APTstat3-9R may be a feasible therapeutic option for PF in the clinic, and could be applied to treat other fibrotic diseases.

Glutathione-Responsive Gold Nanoparticles as Computed Tomography Contrast Agents for Hepatic Diseases.

Glutathione (GSH) is produced at high levels in the normal liver, but its production is considerably reduced under certain pathological conditions. Accordingly, an imaging probe capable of visualizing the altered GSH level in the liver would be a useful tool for monitoring hepatic functions or diseases. Here, we report a gold nanoparticle (AuNP)-based computed tomography (CT) contrast agent that undergoes a change in colloidal stability in response to GSH levels, resulting in differential CT signal intensity between normal (higher intensity) and pathological (lower intensity) livers, enabling imaging of hepatic function. This GSH-responsive CT contrast agent, prepared by coating AuNPs with PEGylated bilirubin (PEG-BR), shows serum stability and high sensitivity to GSH. The resulting poly(ethylene glycol) (PEG)-BR@AuNPs preferentially accumulate in the normal liver, as evidenced by strongly enhanced CT intensity, but fail to do so in a GSH-depleted mouse model, where the CT signal in the liver was substantially decreased. In addition, injection of PEG-BR@AuNPs caused a greater reduction in CT signals in the liver in a drug-induced acute liver failure model than in healthy normal mice. These findings suggest that GSH-responsive PEG-BR@AuNPs have the potential to be used as a CT contrast agent to detect various hepatic function-related diseases and liver-metastasized tumors.

Association between the Intake of Fermented Soy Products and Hypertension Risk in Postmenopausal Women and Men Aged 50 Years or Older: The Korea National Health and Nutrition Examination Survey 2013-2018.

Sodium intake is positively associated with hypertension risk; however, it is not clear whether there is an association between the intake of fermented soy products, a major source of salt, and blood pressure (BP). This study aimed to investigate the hypothesis that hypertension risk and BP were negatively associated with the intake of fermented soy products but not with the intake of sodium from fermented soy products. This cross-sectional study was performed using data from the Korea National Health and Nutrition Examination Survey (2013-2018). In total, 11,566 men and postmenopausal women aged ≥50 years were divided according to quintiles of sodium or fermented soy product intake. The intake of fermented soy products was negatively associated with hypertension risk (odds ratio: 0.81, 95% confidence interval: 0.66-0.98; p-trend = 0.023) and systolic BP (SBP; p-trend = 0.043) in postmenopausal women. Mediation analysis showed that the intake of fermented soy products had total and direct effects on SBP; however, there was no indirect effect because soy nutrients, such as protein, fiber, calcium, and potassium, had no significant effects on SBP. Among men, fermented soy product intake was not associated with hypertension risk and BP. Additionally, the intake of sodium from fermented soy products was not significantly associated with hypertension risk and BP in both postmenopausal women and men. This study suggests that hypertension risk and BP were not associated with the intake of sodium from fermented soy products; further, hypertension risk and BP were inversely associated with fermented soy product intake in postmenopausal women. Further clinical studies are needed to confirm the effect of fermented soy product intake on hypertension risk and BP.

Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse.

We describe a small lipid nanoparticle (SLNP)-based nanovaccine platform and a new combination treatment regimen. Tumor antigen-displaying, CpG adjuvant-embedded SLNPs (OVAPEP -SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD-L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti-PD-1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti-PD-1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood.

Rationale: Magnetic relaxation switching (MRSw) induced by target-triggered aggregation or dissociation of superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for detection of diverse biomarkers. However, an MRSw-based biosensor for reactive oxygen species (ROS) has never been documented. Methods: To this end, we constructed a biosensor for ROS detection based on PEGylated bilirubin (PEG-BR)-coated SPIONs (PEG-BR@SPIONs) that were prepared by simple sonication via ligand exchange. In addition, near infra-red (NIR) fluorescent dye was loaded onto PEG-BR@SPIONs as a secondary option for fluorescence-based ROS detection. Results: PEG-BR@SPIONs showed high colloidal stability under physiological conditions, but upon exposure to the model ROS, NaOCl, in vitro, they aggregated, causing a decrease in signal intensity in T2-weighted MR images. Furthermore, ROS-responsive PEG-BR@SPIONs were taken up by lipopolysaccharide (LPS)-activated macrophages to a much greater extent than ROS-unresponsive control nanoparticles (PEG-DSPE@SPIONs). In a sepsis-mimetic clinical setting, PEG-BR@SPIONs were able to directly detect the concentrations of ROS in whole blood samples through a clear change in T2 MR signals and a 'turn-on' signal of fluorescence. Conclusions: These findings suggest that PEG-BR@SPIONs have the potential as a new type of dual mode (MRSw-based and fluorescence-based) biosensors for ROS detection and could be used to diagnose many diseases associated with ROS overproduction.

Dll4 Suppresses Transcytosis for Arterial Blood-Retinal Barrier Homeostasis.

RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.