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Chang Y.-C.,National Changhua University of Education | Chang Y.-C.,University of Michigan | Ye J.Y.,University of Michigan | Ye J.Y.,Michigan Nanotechnology Institute for Medicine and Biological science | And 6 more authors.
Journal of Biomedical Optics | Year: 2010

Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using freespace optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1(-dioctadecyl-3,3,3',3'- tetramethylindoldicarbocyanine, 4- chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiDlabeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood. © 2010 Society of Photo Optical Instrumentation Engineers.


Erickson B.,University of Michigan | Erickson B.,Michigan Nanotechnology Institute for Medicine and Biological science | Fang M.,Michigan Nanotechnology Institute for Medicine and Biological science | Fang M.,University of Michigan | And 6 more authors.
Biotechnology Journal | Year: 2013

This article details a quantitative method to measure the D-periodic spacing of type I collagen fibrils using atomic force microscopy coupled with analysis using a two-dimensional fast fourier transform approach. Instrument calibration, data sampling and data analysis are discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of type I collagen morphology in disease models of estrogen depletion and osteogenesis imperfecta (OI) are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage osteoporosis and OI. The ability to quantitatively characterize nanoscale morphological features of type I collagen fibrils will provide important structural information regarding type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knockout studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments. © 2013.


Waddell J.N.,University of Michigan | Mullen D.G.,Program in Macromolecular Science and Engineering | Mullen D.G.,Michigan Nanotechnology Institute for Medicine and Biological science | Orr B.G.,University of Michigan | And 4 more authors.
Physical Review E - Statistical, Nonlinear, and Soft Matter Physics | Year: 2010

Nanoparticles with multiple ligands have been proposed for use in nanomedicine. The multiple targeting ligands on each nanoparticle can bind to several locations on a cell surface facilitating both drug targeting and uptake. Experiments show that the distribution of conjugated ligands is unexpectedly broad, and the desorption rate appears to depend exponentially upon the mean number of attached ligands. These two findings are explained with a model in which ligands conjugate to the nanoparticle with a positive cooperativity of 4kT, and that nanoparticles bound to a surface by multiple bonds are permanently affixed. This drives new analysis of the data, which confirms that there is only one time constant for desorption, that of a nanoparticle bound to the surface by a single bond. © 2010 The American Physical Society.


Choi S.K.,Michigan Nanotechnology Institute for Medicine and Biological science | Thomas T.P.,Michigan Nanotechnology Institute for Medicine and Biological science | Leroueil P.,Michigan Nanotechnology Institute for Medicine and Biological science | Kotlyar A.,Michigan Nanotechnology Institute for Medicine and Biological science | And 4 more authors.
Journal of Physical Chemistry B | Year: 2012

Oximes are important in the treatment of organophosphate (OP) poisoning, but have limited biological half-lives. Complexing these drugs with a macromolecule, such as a dendrimer, could improve their pharmacokinetics. The present study investigates the intermolecular interactions that drive the complexation of oxime-based drug molecules with fifth generation poly(amidoamine) (PAMAM) dendrimers. We performed steady-state binding studies of two molecules, pralidoxime and obidoxime, employing multiple NMR methods, including 1D titration, 1H-1H 2D spectroscopy (COSY, NOESY), and 1H diffusion-ordered spectroscopy (DOSY). Several important insights were gained in understanding the host-guest interactions occurring between the drug molecules and the polymer. First, the guest molecules bind to the dendrimer macromolecule through a specific interaction rather than through random, hydrophobic encapsulation. Second, this specificity is driven primarily by the electrostatic or H-bond interaction of the oxime at a dendrimer amine site. Also, the average strength for each drug and dendrimer interaction is affected by the surface modification of the polymer. Third, individual binding events between oximes and a dendrimer have a negative cooperative effect on subsequent oxime binding. In summary, this report provides a novel perspective important for designing host systems for drug delivery. © 2012 American Chemical Society.


Goonewardena S.N.,Michigan Nanotechnology Institute for Medicine and Biological science | Goonewardena S.N.,University of Michigan | Kratz J.D.,Michigan Nanotechnology Institute for Medicine and Biological science | Zong H.,Michigan Nanotechnology Institute for Medicine and Biological science | And 6 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2013

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer-ester MTX conjugates were much more cytotoxic than the dendrimer-amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics. © 2013 Elsevier Ltd. All rights reserved.


Leistra A.N.,Calvin College | Han J.H.,Calvin College | Tang S.,Michigan Nanotechnology Institute for Medicine and Biological science | Orr B.G.,Michigan Nanotechnology Institute for Medicine and Biological science | And 6 more authors.
Journal of Physical Chemistry B | Year: 2015

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates. © 2015 American Chemical Society.


PubMed | University of Michigan and Michigan Nanotechnology Institute for Medicine and Biological science
Type: Journal Article | Journal: Bioorganic & medicinal chemistry | Year: 2014

Bioorthogonal click reactions have recently emerged as promising tools for chemistry and biological applications. By using a combination of two different click reactions, double-click strategies have been developed to attach multiple labels onto biomacromolecules. These strategies require multi-step modifications of the biomacromolecules that can lead to heterogeneity in the final conjugates. Herein, we report the synthesis and characterization of a set of three trifunctional linkers. The linkers having alkyne and cyclooctyne moieties that are capable of participating in sequential copper(I)-catalyzed and copper-free cycloaddition reactions with azides. We have also prepared a linker comprised of an alkyne and a 1,2,4,5-terazine moiety that allows for simultaneous cycloaddition reactions with azides and trans-cyclooctenes, respectively. These linkers can be attached to synthetic or biological macromolecules to create a platform capable of sequential or parallel double-click labeling in biological systems. We show this potential using a generation 5 (G5) polyamidoamine (PAMAM) dendrimer in combination with the clickable linkers. The dendrimers were successfully modified with these linkers and we demonstrate both sequential and parallel double-click labeling with fluorescent reporters. We anticipate that these linkers will have a variety of application including molecular imaging and monitoring of macromolecule interactions in biological systems.


PubMed | Michigan Nanotechnology Institute for Medicine and Biological science
Type: | Journal: Journal of translational medicine | Year: 2014

BID functions as a bridge molecule between death-receptor and mitochondrial related apoptotic pathways to amplify apoptotic signaling. Our previous studies have demonstrated a substantial increase in BID expression in primary normal thyroid epithelia cells treated with inflammatory cytokines, including the combination of IFN and IL-1 or IFN and TNF. The aim of this study was to determine whether an increase in BID expression in thyroid can induce autoimmune thyroiditis.A transgenic mouse line that expresses human BID in thyroid cells was established by fusing a mouse thyroglobulin (Tg) promoter upstream of human BID (Tg-BID). We tested whether the increased expression of pro-apoptotic BID in thyroid would induce autoimmune thyroiditis, both in the presence and absence of 0.3% iodine water.Our data show that Tg-BID mice in a CBA/J (H-2k) background do not spontaneously develop autoimmune thyroiditis for over a year. However, upon ingestion of iodine in the drinking water, autoimmune thyroiditis does develop in Tg-BID transgenic mice, as shown by a significant increase in anti-Tg antibody and mononuclear cell infiltration in the thyroid glands in 30% of mice tested. Serum T4 levels, however, were similar between iodine-treated Tg-BID transgenic mice and the wild type mice.Our data demonstrate that increased thyroid expression of BID facilitates the development of autoimmune thyroiditis induced by iodine uptake. However, the overexpression of BID itself is not sufficient to initiate thyroiditis in CBA/J (H-2k) mice.


PubMed | Michigan Nanotechnology Institute for Medicine and Biological science and University of Wisconsin - Madison
Type: Journal Article | Journal: Cardiovascular drugs and therapy | Year: 2016

Over the past several decades, tremendous advances have been made in the understanding, diagnosis, and treatment of coronary artery disease (CAD). However, with shifting demographics and evolving risk factors we now face new challenges that must be met in order to further advance are management of patients with CAD. In parallel with advances in our mechanistic appreciation of CAD and atherosclerosis, nanotechnology approaches have greatly expanded, offering the potential for significant improvements in our diagnostic and therapeutic management of CAD. To realize this potential we must go beyond to recognize new frontiers including knowledge gaps between understanding atherosclerosis to the translation of targeted molecular tools. This review highlights nanotechnology applications for imaging and therapeutic advancements in CAD.


PubMed | Michigan Nanotechnology Institute for Medicine and Biological science
Type: Journal Article | Journal: The journal of physical chemistry. B | Year: 2015

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

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