Identification of Lymphatic and Hematogenous Routes of Rapidly Labeled Radioactive and Fluorescent Exosomes through Highly Sensitive Multimodal Imaging.

There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.

In vivo imaging of activated macrophages by 18F-FEDAC, a TSPO targeting PET ligand, in the use of biologic disease-modifying anti-rheumatic drugs (bDMARDs).

Rheumatoid arthritis (RA) is a chronic disease with systemic inflammation resulting in destruction of multiple articular cartilages and bones. Activated macrophage plays a pivotal role during the disease course and has been one of main targets to inhibit inflammatory reaction of RA by using biological disease-modifying anti-rheumatic drugs (bDMARDs). 18F-FEDAC is one of PET imaging agents targeting TSPO, which is overexpressed in activated macrophages. The aim of this study was to evaluate the roles of 18F-FEDAC PET as an in vivo imaging of activated macrophages on etanercept (ETN), a TNF-antagonist as one of bDMARDs in collagen induced arthritis mice. In RAW 264.7 cells, the expressions of TSPO as well as iNOS and infiltrated nucleus of NF-κB were induced by activation with lipopolysaccharide and interferon-gamma. TSPO expression was slightly attenuated by ETN treatment, not by methotrexate (MTX) as a cytotoxic agent. However, cell uptake of 18F-FEDAC did not show significant changes according to both of the treatments. Similarly in CIA mice, 18F-FEDAC uptake in inflamed paws on PET imaging did not show significant changes during both of the treatments, contrary to the uptake decrease of 18F-FDG, a glucose analog to reflect metabolic or active inflammatory activity. Interestingly, when we divided joints according to the degree of 18F-FEDAC uptake before ETN treatment, the joints of high 18F-FEDAC uptake showed better response to ETN than the joints with low 18F-FEDAC uptakes. In case of 18F-FDG, there was no such kinds of patterns. We can speculate that 18F-FEDAC PET imaging may identify activated macrophage-induced arthritis because that 18F-FEDAC can reflect activated macrophages, which is the therapeutic target of ETN by TNF antagonistic effect. Thus, in vivo imaging using 18F-FEDAC may be used as a predictor of therapeutic effects among those kinds of bDMARDs having anti-inflammatory actions to inhibit activated macrophage.

Targeted Biodegradable Near-Infrared Fluorescent Nanoparticles for Colorectal Cancer Imaging.

Colorectal cancer (CRC) is the third leading cause of cancer death in the U.S., and early detection and diagnosis are essential for effective treatment. Current methods are inadequate for rapid detection of early disease, revealing flat lesions, and delineating tumor margins with accuracy and molecular specificity. Fluorescence endoscopy can generate wide field-of-view images enabling detection of CRC lesions and margins; increased signal intensity and improved signal-to-noise ratios can increase both speed and sensitivity of cancer detection. For this purpose, we developed targeted near-infrared (NIR) fluorescent silica nanoparticles (FSNs). We tuned their size to 50-200 nm and conjugated their surface with an antibody to carcinoembryonic antigen (CEA) to prepare CEA-FSNs. The physicochemical properties and biodegradable profiles of CEA-FSN were characterized, and molecular targeting was verified in culture using HT29 (CEA positive) and HCT116 (CEA negative) cells. CEA-FSNs bound to the HT29 cells to a greater extent than to the HCT116 cells, and smaller CEA-FSNs were internalized into HT29 cells more efficiently than larger CEA-FSNs. After intravenous administration of CEA-FSNs, a significantly greater signal was observed from the CEA-positive HT29 than the CEA-negative HCT116 tumors in xenografted mice. In F344-PIRC rats, polyps in the intestine were detected by white-light endoscopy, and NIR fluorescent signals were found in the excised intestinal tissue after topical application of CEA-FSNs. Immunofluorescence imaging of excised tissue sections demonstrated that the particle signals coregistered with signals for both CRC and CEA. These results indicate that CEA-FSNs have potential as a molecular imaging marker for early diagnosis of CRC.

Exosome-Coated Prussian Blue Nanoparticles for Specific Targeting and Treatment of Glioblastoma.

Glioblastoma is one of the most aggressive and invasive types of brain cancer with a 5-year survival rate of 6.8%. With limited options, patients often have poor quality of life and are moved to palliative care after diagnosis. As a result, there is an extreme need for a novel theranostic method that allows for early diagnosis and noninvasive treatment as current peptide-based delivery standards may have off-target effects. Prussian Blue nanoparticles (PBNPs) have recently been investigated as photoacoustic imaging (PAI) and photothermal ablation agents. However, due to their inability to cross the blood-brain barrier (BBB), their use in glioblastoma treatment is limited. By utilizing a hybrid, biomimetic nanoparticle composed of a PBNP interior and a U-87 cancer cell-derived exosome coating (Exo:PB), we show tumor-specific targeting within the brain and selective thermal therapy potential due to the strong photoconversion abilities. Particle characterization was carried out and showed a complete coating around the PBNPs that contains exosome markers. In vitro cellular uptake patterns are similar to native U-87 exosomes and when exposed to an 808 nm laser, show localized cell death within the specified region. After intravenous injection of Exo:PB into subcutaneously implanted glioblastoma mice, they have shown effective targeting and eradication of tumor volume compared to PEG-coated PBNPs (PEG:PB). Through systemic administration of Exo:PB particles into orthotopic glioblastoma-bearing mice, the PBNP signal was detected in the brain tumor region through PAI. It was seen that Exo:PB had preferential tumor accumulation with less off-targeting compared to the RGD:PB control. Ex vivo analysis validated specific targeting with a direct overlay of Exo:PB with the tumor by both H&E staining and Ki67 labeling. Overall, we have developed a novel biomimetic material that can naturally cross the BBB and act as a theranostic agent for systemic targeting of glioblastoma tissue and photothermal therapeutic effect.

Europium-Doped Calcium Silicate Nanoparticles as High-Quantum-Yield Red-Emitting Phosphors.

Europium ion-activated calcium silicate phosphors (Ca2SiO4:Eu3+) with sharp red-light emission were fabricated via the hydrothermal method. The size of Ca2SiO4:Eu3+ phosphors was controlled between 20 and 200 nm by precursor silicate particle sizes. Systematic studies to determine morphology, crystal phase, and photoluminescence (PL) were carried out for all the phosphors, and their optical efficiencies were compared. We found that the luminescence intensity and emission wavelength of Ca2SiO4:Eu3+ phosphors depend on their particle sizes. Particularly, the Ca2SiO4:Eu3+ synthesized with 20 nm silica seed contains the most intense red emission, high color purity, and high PL quantum yield. For the 20 nm-sized Ca2SiO4:Eu3+ phosphor, PL quantum yields are measured to be above 87.95% and high color purity of 99.8%. The unusually high intensity of 5D0 → 7F4 emission (712 nm) is explained by structural distortion arising from silicate particle size reductions. We show that the obtained phosphor is a suitable candidate for solid-state lighting as a red component through CIE chromaticity coordinate and color purity measurements. Furthermore, the Ca2SiO4:Eu3+ particles are examined for their validity as promising bio-imaging probes through cell labeling and imaging experiments and biodegradability studies.

Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers.

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lie at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers.

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming.

Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, delayed cellular changes were observed that are not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric l-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. The results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

A Mouse Model of Endometriosis with Nanoparticle Labeling for In Vivo Photoacoustic Imaging.

Endometriosis is a condition of the female reproductive tract characterized by endometrium-like tissue growing outside the uterus. Though it is a common cause of pelvic pain and infertility, there is currently no reliable noninvasive method to diagnose the presence of endometriosis without surgery, and the pathophysiological mechanisms that lead to the occurrence of symptoms require further inquiry. Due to patient heterogeneity and delayed diagnosis, animal models are commonly used to study the development of endometriosis, but these are costly due to the large number of animals needed to test various treatments and experimental conditions at multiple endpoints. Here, we describe a method for synthesis of multimodal imaging gold-fluorescein isothiocyanate (FITC) nanoparticles with preclinical application via induction of nanoparticle-labeled endometriosis-like lesions in mice. Labeling donor endometrial tissue fragments with gold-FITC nanoparticles prior to induction of endometriosis in recipients enables in vivo detection of the gold-labeled lesions with photoacoustic imaging. The same imaging method can be used to visualize embryos noninvasively in pregnant mice. Furthermore, the conjugated FITC dye on the gold nanoparticles allows easy isolation of labeled lesion tissue under a fluorescence dissection microscope. After dissection, the presence of gold-FITC nanoparticles and endometrium-like histology of lesions can be verified through fluorescence imaging, gold enhancement, and immunostaining. This method for in vivo imaging of endometriosis-like lesions and fluorescence-guided dissection will permit new experimental possibilities for the longitudinal study of endometriosis development and progression as well as endometriosis-related infertility.

Ceria-based nanotheranostic agent for rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that affects 1-2% of the human population worldwide, and effective therapies with targeted delivery for local immune suppression have not been described. We address this problem by developing a novel theranostic nanoparticle for RA and assessed its therapeutic and targeting effects under image-guidance. Methods: Albumin-cerium oxide nanoparticles were synthesized by the biomineralization process and further conjugated with near-infrared, indocyanine green (ICG) dye. Enzymatic-like properties and reactive oxygen species (ROS) scavenging activities, as well as the ability to reprogram macrophages, were determined on a monocyte cell line in culture. The therapeutic effect and systemic targeting potential were evaluated in collagen-induced arthritis (CIA) mouse model using optical/optoacoustic tomographic imaging. Results: Small nanotheranostics with narrow size distribution and high colloidal stability were fabricated and displayed high ROS scavenging and enzymatic-like activity, as well as advanced efficacy in a converting pro-inflammatory macrophage phenotype into anti-inflammatory phenotype. When administrated into affected animals, these nanoparticles accumulated in inflamed joints and revealed a therapeutic effect similar to the gold-standard therapy for RA, methotrexate. Conclusions: The inflammation-targeting, inherent contrast and therapeutic activity of this new albumin-cerium oxide nanoparticle may make it a relevant agent for assessing severity in RA, and other inflammatory diseases, and controlling inflammation with image-guidance. The design of these nanotheranostics will enable potential clinical translation as systemic therapy for RA.

Biodegradable Hollow Manganese Silicate Nanocomposites to Alleviate Tumor Hypoxia toward Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT) has been extensively explored as a minimally invasive treatment strategy for malignant cancers. It works with the help of a photosensitizer located within cancer cells that is irradiated by near-infrared light to produce potent toxins and singlet oxygen (1O2) and induce cell death. However, reactive oxygen species can be overexpressed in tumor tissue because of the rapid metabolic activity in cancer cells, and the insufficient oxygenation (hypoxia) can lead to low production of singlet oxygen (1O2) during PDT. In this study, we developed nanocomposites composed of a hollow manganese silicate (HMnOSi) nanoparticle and a photosensitizer (Ce6) that can generate significant amounts of O2 to relieve tumor hypoxia and enhance the therapeutic efficacy of PDT. Our nanocomposites were characterized by UV-vis, fluorescence spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray, and dynamic light scattering. Our particles' hollow mesoporous structures were shown to retain large amounts of Ce6 on the particle surface with high loading capacity (33%). TEM imaging showed that the nanoparticles could be biodegradable over time in simulated body fluid, which can imply clinical potentials. Significant H2O2 quenching capabilities to alleviate hypoxic conditions in a solid tumor were also presented. For breast cancer cells, the nanocomposite-treated group revealed that 91% of cells were dead under laser activation compared to 51% for the control group (free Ce6). In an animal study, our nanocomposites showed almost fourfold tumor growth inhibition versus the control and more than twofold over free Ce6 in orthotopic tumor xenografts. In addition, the oxygen saturation contrast inside tumors was evaluated by photoacoustic imaging to demonstrate the alleviated hypoxia in vivo. Our works provide a smart nanosystem to ameliorate the hypoxic tumor microenvironment and augment the efficacy of PDT in a targeted cancer treatment.

Tumor-Associated Macrophages Enhance Tumor Hypoxia and Aerobic Glycolysis.

Tumor hypoxia and aerobic glycolysis are well-known resistance factors for anticancer therapies. Here, we demonstrate that tumor-associated macrophages (TAM) enhance tumor hypoxia and aerobic glycolysis in mice subcutaneous tumors and in patients with non-small cell lung cancer (NSCLC). We found a strong correlation between CD68 TAM immunostaining and PET 18fluoro-deoxyglucose (FDG) uptake in 98 matched tumors of patients with NSCLC. We also observed a significant correlation between CD68 and glycolytic gene signatures in 513 patients with NSCLC from The Cancer Genome Atlas database. TAM secreted TNFα to promote tumor cell glycolysis, whereas increased AMP-activated protein kinase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in TAM facilitated tumor hypoxia. Depletion of TAM by clodronate was sufficient to abrogate aerobic glycolysis and tumor hypoxia, thereby improving tumor response to anticancer therapies. TAM depletion led to a significant increase in programmed death-ligand 1 (PD-L1) expression in aerobic cancer cells as well as T-cell infiltration in tumors, resulting in antitumor efficacy by PD-L1 antibodies, which were otherwise completely ineffective. These data suggest that TAM can significantly alter tumor metabolism, further complicating tumor response to anticancer therapies, including immunotherapy. SIGNIFICANCE: These findings show that tumor-associated macrophages can significantly modulate tumor metabolism, hindering the efficacy of anticancer therapies, including anti-PD-L1 immunotherapy.

18F-FEDAC as a Targeting Agent for Activated Macrophages in DBA/1 Mice with Collagen-Induced Arthritis: Comparison with 18F-FDG.

Activated macrophages have been known to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). 18F-FEDAC (N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-18F-fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide) is a radiolabeled ligand for the 18-kDa translocator protein (TSPO), which is abundant in activated macrophages. We evaluated the feasibility of using 18F-FEDAC in a murine RA model. Methods: RAW 264.7 mouse macrophages were activated by lipopolysaccharide. TSPO expression levels in activated and inactivated macrophages were measured by quantitative polymerase chain reaction and Western blotting. The cellular uptake and specific binding of 18F-FEDAC were measured using a γ-counter. For the in vivo study, collagen-induced arthritis (CIA) was developed in DBA/1 mice, and the clinical score for arthritis was measured regularly. 18F-FEDAC and 18F-FDG PET images were acquired on days 23 and 37 after the first immunization. Histologic examinations were performed to evaluate macrophages and TSPO expression. Results: We found increased TSPO messenger RNA and protein expression in activated macrophages. Uptake of 18F-FEDAC in activated macrophages was higher than that in nonactivated cells and was successfully blocked by the competitor, PK11195. In CIA mice, joint swelling was apparent on day 26 after the first immunization, and the condition worsened by day 37. 18F-FEDAC uptake by arthritic joints increased early on (day 23), whereas 18F-FDG uptake did not. However, 18F-FDG uptake by arthritic joints markedly increased at later stages (day 37) to a higher level than 18F-FEDAC uptake. The 18F-FEDAC uptake correlated weakly with summed severity score (P = 0.019, r = 0.313), whereas the 18F-FDG uptake correlated strongly with summed severity score (P < 0.001, r = 0.897). Histologic sections of arthritic joints demonstrated an influx of macrophages compared with that in normal joints. Conclusion:18F-FEDAC enabled the visualization of active inflammation sites in arthritic joints in a CIA model by targeting TSPO expression in activated macrophages. The results suggest the potential usefulness of 18F-FEDAC imaging in the early phase of RA.

Noninvasive Imaging of Myocardial Inflammation in Myocarditis using 68Ga-tagged Mannosylated Human Serum Albumin Positron Emission Tomography.

The diagnosis of myocarditis traditionally relies on invasive endomyocardial biopsy but none of the imaging studies so far are specific for infiltration of the inflammatory cells itself. We synthesized 68Ga-2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) mannosylated human serum albumin (MSA) by conjugating human serum albumin with mannose, followed by conjugation with NOTA and labeling it with 68Ga. The efficacy of 68Ga-NOTA-MSA positron emission tomography (PET) for imaging myocardial inflammation was tested in a rat myocarditis model. A significant number of mannose receptor-positive inflammatory cells infiltrated the myocardium in both human and rat myocarditis tissue. 68Ga-NOTA-MSA uptake was upregulated in organs of macrophage accumulation, such as liver, spleen, bone marrow and myocardium (0.32 (0.31~0.33) for normal versus 1.02 (0.86~1.06) for myocarditis (median (range), SUV); n=4~6 per group, p-value=0.01). 68Ga-NOTA-MSA uptake in the left ventricle was upregulated in myocarditis compared with normal rats (2.29 (1.42~3.40) for normal versus 4.18 (3.43~6.15) for myocarditis (median (range), average standard uptake value ratio against paraspinal muscle); n=6 per group, p-value<0.01), which was downregulated in rats with cyclosporine-A treated myocarditis (3.69 (2.59~3.86) for myocarditis versus 2.28 (1.76~2.60) for cyclosporine-A treated myocarditis; n=6 per group, p-value<0.01). The specificity of the tracer was verified by administration of excess non-labeled MSA. 68Ga-NOTA-MSA uptake was significantly enhanced earlier in the evolution of myocarditis before any signs of inflammation could be seen on echocardiography. These results demonstrate the potential utility of visualizing infiltration of mannose receptor-positive macrophages with 68Ga-NOTA-MSA PET in the early diagnosis of as well as in the monitoring of treatment response of myocarditis.

Simultaneous Detection of EGFR and VEGF in Colorectal Cancer using Fluorescence-Raman Endoscopy.

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy.