In Vitro and In Vivo Imaging-Based Evaluation of Doxorubicin Anticancer Treatment in Combination with the Herbal Medicine Black Cohosh.

As a substitution for hormone replacement therapy, many breast cancer patients use black cohosh (BC) extracts in combination with doxorubicin (DOX)-based chemotherapy. In this study, we evaluated the viability and survival of BC- and DOX-treated MCF-7 cells. A preclinical model of MCF-7 xenografts was used to determine the influence of BC and DOX administration on tumor growth and metabolism. The number of apoptotic cells after incubation with both DOX and BC was significantly increased (~100%) compared to the control. Treatment with DOX altered the potential of MCF-7 cells to form colonies; however, coincubation with BC did not affect this process. In vivo, PET-CT imaging showed that combined treatment of DOX and BC induced a significant reduction in both metabolic activity (29%) and angiogenesis (32%). Both DOX and BC treatments inhibited tumor growth by 20% and 12%, respectively, and combined by 57%, vs. control. We successfully demonstrated that BC increases cytotoxic effects of DOX, resulting in a significant reduction in tumor size. Further studies regarding drug transport and tumor growth biomarkers are necessary to establish the underlying mechanism and potential clinical use of BC in breast cancer patients.

Molecularly targeted nanoparticles: an emerging tool for evaluation of expression of the receptor for advanced glycation end products in a murine model of peripheral artery disease.

BACKGROUND: Molecular imaging with molecularly targeted probes is a powerful tool for studying the spatio-temporal interactions between complex biological processes. The pivotal role of the receptor for advanced glycation end products (RAGE), and its involvement in numerous pathological processes, aroused the demand for RAGE-targeted imaging in various diseases. In the present study, we evaluated the use of a diagnostic imaging agent for RAGE quantification in an animal model of peripheral artery disease, a multimodal dual-labeled probe targeted at RAGE (MMIA-CML). METHODS: PAMAM dendrimer was conjugated with Nε-carboxymethyl-lysine (CML) modified albumin to synthesize the RAGE-targeted probe. A control untargeted agent carried native non-modified human albumin (HSA). Bifunctional p-SCN-Bn-NOTA was used to conjugate the 64Cu radioisotope. Surgical right femoral artery ligation was performed on C57BL/6 male mice. One week after femoral artery ligation, mice were injected with MMIA-CML or MMIA-HSA labeled with 64Cu radioisotope and 60 min later in vivo microPET-CT imaging was performed. Immediately after PET imaging studies, the murine hindlimb muscle tissues were excised and prepared for gene and protein expression analysis. RAGE gene and protein expression was assessed using real-time qPCR and Western blot technique respectively. To visualize RAGE expression in excised tissues, microscopic fluorescence imaging was performed using RAGE-specific antibodies and RAGE-targeted and -control MMIA. RESULTS: Animals subjected to PET imaging exhibited greater MMIA-CML uptake in ischemic hindlimbs than non-ischemic hindlimbs. We observed a high correlation between fluorescent signal detection and radioactivity measurement. Significant RAGE gene and protein overexpression were observed in ischemic hindlimbs compared to non-ischemic hindlimbs at one week after surgical ligation. Fluorescence microscopic staining revealed significantly increased uptake of RAGE-targeted nanoparticles in both ischemic and non-ischemic muscle tissues compared to the control probe but at a higher level in ischemic hindlimbs. Ischemic tissue exhibited explicit RAGE dyeing following anti-RAGE antibody and high colocalization with the MMIA-CML targeted at RAGE. CONCLUSIONS: The present results indicate increased expression of RAGE in the ischemic hindlimb and enable the use of multimodal nanoparticles in both in vitro and in vivo experimental models, creating the possibility for imaging structural and functional changes with a RAGE-targeted tracer.

Quantitative imaging of the receptor for advanced glycation end-products in prostate cancer.

PURPOSE: Current screening and monitoring of prostate cancer (PCa) is insufficient, producing inaccurate diagnoses. Presence of the receptor for advanced glycation end-products (RAGE) is associated with signature characteristics of PCa development such as cell proliferation, anchorage-independent growth, angiogenesis, migration, invasion, and poor patient survival. Therefore, we developed a preclinical multimodal imaging strategy targeted at RAGE to diagnose and monitor PCa. METHODS: In this work, RAGE-targeted multimodal nanoparticles (64Cu-Cy5-G4-CML) were synthesized and rendered functional for nuclear and optical imaging using previously established methods. The probe's binding affinity and targeting specificity was assessed in androgen-dependent (LNCaP) and androgen-independent (DU145) prostate cancer cells using flow cytometry and confocal microscopy. In vivo PET-CT imaging was used to evaluate RAGE levels in DU145 and LNCaP xenograft models in mice. Then, tumors were excised post-imaging for histological staining and autoradiography to further assess RAGE levels and targeting efficiency of the tracer. Finally, RAGE levels from human PCa samples of varying Gleason Scores were evaluated using Western blot and immunohistochemical staining. RESULTS: PCa cell culture studies confirmed adequate RAGE-targeting with 64Cu-Cy5-G4-CML with KD between 360 and 540 nM as measured by flow cytometry. In vivo PET-CT images of PCa xenografts revealed favorable kinetics, rapid blood clearance, and a non-homogenous, enhanced uptake in tumors, which varied based on cell type and tumor size with mean uptake between 0.5 and 1.4%ID/g. RAGE quantification of human samples confirmed increased RAGE uptake corresponding to increased Gleason scoring. CONCLUSIONS: Our study has shown that RAGE-targeted cancer imaging is feasible and could significantly impact PCa management.

Impact of administration route on nanocarrier biodistribution in a murine colitis model.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide. Although current diagnostic and disease monitoring tests for IBD sensitively detect gut inflammation, they lack the molecular and cellular specificity of positron emission tomography (PET). In this proof-of-concept study, we use a radiolabeled macrophage-targeted nanocarrier probe (64Cu-NOTA-D500) administered by oral, enema, and intraperitoneal routes to evaluate the delivery route dependence of biodistribution across healthy and diseased tissues in a murine model of dextran sodium sulfate (DSS)-induced colitis. High inter-subject variability of probe uptake in intestinal tissue was reduced by normalization to uptake in liver or total intestines. Differences in normalized uptake between healthy and DSS colitis animal intestines were highest for oral and IP routes. Differences in absolute liver uptake reflected a possible secondary diagnostic metric of IBD pathology. These results should inform the preclinical development of inflammation-targeted contrast agents for IBD and related gut disorders to improve diagnostic accuracy.

Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging In Vivo, Ex Vivo, and In Situ.

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

Objective Assessments in Diagnoses and Treatment: A Proposed Change in Paradigm.

For patients with psychiatric disorders, current diagnostic and treatment approaches are far from optimal. The clinical interview drives the standard approach-matching symptoms to diagnostic criteria-and results in standardized pharmacological and behavioral treatments, often, with inadequate outcome; but now, recent imaging advances can correlate behavioral assessments with brain function and measure them against normative databases to provide data critical for the reevaluation of patient diagnosis and treatment. This article addresses the data that support a redefinition of our current paradigm. We believe a neurobehavioral approach provides for more personalized treatment approaches unbound from classically defined diagnostic biases.

Nanocarriers targeting adipose macrophages increase glucocorticoid anti-inflammatory potency to ameliorate metabolic dysfunction.

Obesity is associated with systemic inflammation due to macrophage accumulation in adipose tissue (AT). AT macrophages are, therefore, a target for therapeutics to modulate inflammation and prevent comorbidities. Because inflammatory processes have pleiotropic effects throughout the body and are intertwined with metabolic axes, systemic anti-inflammatory therapies are often harmful. We report that targeting AT macrophages using dextran nanocarriers radically alters the pharmacology of anti-inflammatory glucocorticoids, uncoupling the metabolic axis in obese mice. Following a single treatment, expression of inflammatory mediators and markers of inflammatory macrophages decreased with a nearly 20-fold higher potency compared with free drug. As a result, long-term treatment resulted in potent fat mobilization, AT reduction, weight loss, improved glucose tolerance, and altered AT gene expression profiles that led to elevated liver stress. Two weeks after treatment ceased, gene expression of inflammatory mediators in AT remained lower than obese controls, while gene expression related to metabolic function improved. These data demonstrate that nanocarriers show potential for amelioration of obesity-related AT inflammation and metabolic dysfunction, highlighting an important opportunity for nanomedicine to impact chronic metabolic disorders with complex and poorly understood etiology.

Multimodal Nanocarrier Probes Reveal Superior Biodistribution Quantification by Isotopic Analysis over Fluorescence.

Absolute measurements of biodistribution are essential for understanding and optimizing the function of nanomaterials for in vivo diagnostic and therapeutic applications. Biodistribution analysis by optical imaging is desirable due to its low cost, wide accessibility, and high-throughput nature, but it is substantially less accurate than isotopic and chemical techniques. In this work, we developed multimodal probes for optical and nuclear imaging to analyze the quantitative limits of optical contrast in the red and near-infrared spectra for polysaccharide nanocarriers targeting macrophage cells. Probes incorporating three zwitterionic fluorophores together with radioactive copper distributed diffusely to optically dissimilar tissues that were either white (visceral adipose tissue) or dark red (liver and spleen) in obese rodents. We used in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo hyperspectral tomographic fluorescence imaging, and ex vivo optical and isotopic analyses to determine correlations between optical and nuclear signals. PET imaging strongly correlated with standardized ex vivo methods for all tissue types, whereas no fluorescence signals exhibited substantial accuracy in quantification or localization in vivo. Optical imaging of resected tissues was most accurate in the 700 nm wavelength window, but only in white tissues. This work suggests that fluorescence can be used to measure diffuse probe distribution in white tissues over time or across animals, but not red tissues and not deep in the body. This work also highlights the importance of choosing validated experimental protocols and describes how optical measurements are impacted by fluorophore class and spectral properties, tissue properties, and imaging workflow.

Intratumoral generation of photothermal gold nanoparticles through a vectorized biomineralization of ionic gold.

Various cancer cells have been demonstrated to have the capacity to form plasmonic gold nanoparticles when chloroauric acid is introduced to their cellular microenvironment. But their biomedical applications are limited, particularly considering the millimolar concentrations and longer incubation period of ionic gold. Here, we describe a simplistic method of intracellular biomineralization to produce plasmonic gold nanoparticles at micromolar concentrations within 30 min of application utilizing polyethylene glycol as delivery vector for ionic gold. We have characterized this process for intracellular gold nanoparticle formation, which progressively accumulates proteins as the ionic gold clusters migrate to the nucleus. This nano-vectorized application of ionic gold emphasizes its potential biomedical opportunities while reducing the quantity of ionic gold and required incubation time. To demonstrate its biomedical potential, we further induce in-situ biosynthesis of gold nanoparticles within MCF7 tumor mouse xenografts which is followed by its photothermal remediation.

Multimodal imaging of the receptor for advanced glycation end-products with molecularly targeted nanoparticles.

The receptor for advanced glycation end-products (RAGE) is central to multiple disease states, including diabetes-related conditions such as peripheral arterial disease (PAD). Despite RAGE's importance in these pathologies, there remains a need for a molecular imaging agent that can accurately assess RAGE levels in vivo. Therefore, we have developed a multimodal nanoparticle-based imaging agent targeted at RAGE with the well-characterized RAGE ligand, carboxymethyllysine (CML)-modified human serum albumin (HSA). Methods: A multimodal tracer (64Cu-Rho-G4-CML) was developed using a generation-4 (G4) polyamidoamine (PAMAM) dendrimer, conjugated with both rhodamine and copper-64 (64Cu) chelator (NOTA) for optical and PET imaging, respectively. First, 64Cu-Rho-G4-CML and its non-targeted analogue (64Cu-Rho-G4-HSA) were evaluated chemically using techniques such as dynamic light scattering (DLS), electron microscopy and nuclear magnetic resonance (NMR). The tracers' binding capabilities were examined at the cellular level and optimized using live and fixed HUVEC cells grown in 5.5-30 mM glucose, followed by in vivo PET-CT imaging, where the probes' kinetics, biodistribution, and RAGE targeting properties were examined in a murine model of hindlimb ischemia. Finally, histological assessment of RAGE levels in both ischemic and non-ischemic tissues was performed. Conclusions: Our RAGE-targeted probe demonstrated an average size of 450 nm, a Kd of 340-390 nM, rapid blood clearance, and a 3.4 times greater PET uptake in ischemic RAGE-expressing hindlimbs than their non-ischemic counterpart. We successfully demonstrated increased RAGE expression in a murine model of hindlimb ischemia and the feasibility for non-invasive examination of cellular, tissue, and whole-body RAGE levels with a molecularly targeted tracer.

Evaluation of a dimeric-cRGD peptide for targeted PET-CT imaging of peripheral angiogenesis in diabetic mice.

The α V ß3 integrin plays an important role in many physiological functions and pathological disorders. α V ß3 is minimally expressed in normal quiescent endothelial cells, but significantly upregulated during neovascularization. In this study, we evaluated a 64Cu-labeled dimeric cRGD tracer targeted at α V ß3 integrin and report its applicability to assess peripheral angiogenesis in diabetes mellitus (DM). We established a murine model of type-1 DM characterized by elevated glucose, glycated serum protein (GSP), and glycated hemoglobin A1c (HbA1c). We demonstrated that our imaging probe is specific to α V ß3 integrin under both normo- and hyperglycemic conditions. We found that the analysis of in vivo PET-CT images correlated well with gamma well counting (GWC). Both GWC and PET-CT imaging demonstrated increased uptake of 64Cu-NOTA-PEG4-cRGD2 in the ischemic hindlimb in contrast to non-ischemic control. GWC of the distal ischemic tissue from DM mice showed significantly lower probe accumulation than in non-DM mice. The immunofluorescence staining of the ischemic tissues showed a 3-fold reduction in CD31 and 4-fold reduction in the α V ß3 expression in DM vs. non-DM animals. In conclusion, we successfully demonstrated that diabetes-associated reductions in peripheral angiogenesis can be non-invasively detected with PET-CT imaging using targeted dimeric-cRGD probe.

Multimodal Assessment of Mesenchymal Stem Cell Therapy for Diabetic Vascular Complications.

Peripheral arterial disease (PAD) is a debilitating complication of diabetes mellitus (DM) that leads to thousands of injuries, amputations, and deaths each year. The use of mesenchymal stem cells (MSCs) as a regenerative therapy holds the promise of regrowing injured vasculature, helping DM patients live healthier and longer lives. We report the use of muscle-derived MSCs to treat surgically-induced hindlimb ischemia in a mouse model of type 1 diabetes (DM-1). We serially evaluate several facets of the recovery process, including αVß3 -integrin expression (a marker of angiogenesis), blood perfusion, and muscle function. We also perform microarray transcriptomics experiments to characterize the gene expression states of the MSC-treated is- chemic tissues, and compare the results with those of non-ischemic tissues, as well as ischemic tissues from a saline-treated control group. The results show a multifaceted impact of mMSCs on hindlimb ischemia. We determined that the angiogenic activity one week after mMSC treatment was enhanced by approximately 80% relative to the saline group, which resulted in relative increases in blood perfusion and muscle strength of approximately 42% and 1.7-fold, respectively. At the transcriptomics level, we found that several classes of genes were affected by mMSC treatment. The mMSCs appeared to enhance both pro-angiogenic and metabolic genes, while suppressing anti-angiogenic genes and certain genes involved in the inflammatory response. All told, mMSC treatment appears to exert far-reaching effects on the microenvironment of ischemic tissue, enabling faster and more complete recovery from vascular occlusion.