Publications by authors named "Stephan Stern"

68 Publications

Nanomedicine Reformulation of Chloroquine and Hydroxychloroquine.

Molecules 2020 Dec 31;26(1). Epub 2020 Dec 31.

Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD 21702, USA.

The chloroquine family of antimalarials has a long history of use, spanning many decades. Despite this extensive clinical experience, novel applications, including use in autoimmune disorders, infectious disease, and cancer, have only recently been identified. While short term use of chloroquine or hydroxychloroquine is safe at traditional therapeutic doses in patients without predisposing conditions, administration of higher doses and for longer durations are associated with toxicity, including retinotoxicity. Additional liabilities of these medications include pharmacokinetic profiles that require extended dosing to achieve therapeutic tissue concentrations. To improve chloroquine therapy, researchers have turned toward nanomedicine reformulation of chloroquine and hydroxychloroquine to increase exposure of target tissues relative to off-target tissues, thereby improving the therapeutic index. This review highlights these reformulation efforts to date, identifying issues in experimental designs leading to ambiguity regarding the nanoformulation improvements and lack of thorough pharmacokinetics and safety evaluation. Gaps in our current understanding of these formulations, as well as recommendations for future formulation efforts, are presented.
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http://dx.doi.org/10.3390/molecules26010175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794963PMC
December 2020

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Nat Commun 2020 09 9;11(1):4511. Epub 2020 Sep 9.

School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287-1604, USA.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
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http://dx.doi.org/10.1038/s41467-020-18156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481229PMC
September 2020

Application of a Scavenger Receptor A1-Targeted Polymeric Prodrug Platform for Lymphatic Drug Delivery in HIV.

Mol Pharm 2020 10 9;17(10):3794-3812. Epub 2020 Sep 9.

Nanotechnology Characterization Lab, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland 21702-1201, United States.

We have developed a macromolecular prodrug platform based on poly(l-lysine succinylated) (PLS) that targets scavenger receptor A1 (SR-A1), a receptor expressed by myeloid and endothelial cells. We demonstrate the selective uptake of PLS by murine macrophage, RAW 264.7 cells, which was eliminated upon cotreatment with the SR-A inhibitor polyinosinic acid (poly I). Further, we observed no uptake of PLS in an SR-A1-deficient RAW 264.7 cell line, even after 24 h incubation. In mice, PLS distributed to lymphatic organs following i.v. injection, as observed by fluorescent imaging, and accumulated in lymph nodes following both i.v. and i.d. administrations, based on immunohistochemical analysis with high-resolution microscopy. As a proof-of-concept, the HIV antiviral emtricitabine (FTC) was conjugated to the polymer's succinyl groups via ester bonds, with a drug loading of 14.2% (wt/wt). The prodrug (PLS-FTC) demonstrated controlled release properties with a release half-life of 15 h in human plasma and 29 h in esterase-inhibited plasma, indicating that drug release occurs through both enzymatic and nonenzymatic mechanisms. Upon incubation of PLS-FTC with human peripheral blood mononuclear cells (PBMCs), the released drug was converted to the active metabolite FTC triphosphate. In a pharmacokinetic study in rats, the prodrug achieved ∼7-19-fold higher concentrations in lymphatic tissues compared to those in FTC control, supporting lymphatic-targeted drug delivery. We believe that the SR-A1-targeted macromolecular PLS prodrug platform has extraordinary potential for the treatment of infectious diseases.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861197PMC
October 2020

A reanalysis of nanoparticle tumor delivery using classical pharmacokinetic metrics.

Sci Adv 2020 Jul 15;6(29):eaay9249. Epub 2020 Jul 15.

Carolina Center of Cancer Nanotechnology Excellence (C-CCNE), University of North Carolina, Chapel Hill, NC, USA.

Nanoparticle (NP) delivery to solid tumors has recently been questioned. To better understand the magnitude of NP tumor delivery, we reanalyzed published murine NP tumor pharmacokinetic (PK) data used in the Wilhelm . study. Studies included in their analysis reporting matched tumor and blood concentration versus time data were evaluated using classical PK endpoints and compared to the unestablished percent injected dose (%ID) in tumor metric from the Wilhelm . study. The %ID in tumor was poorly correlated with standard PK metrics that describe NP tumor delivery (AUC/AUC ratio) and only moderately associated with maximal tumor concentration. The relative tumor delivery of NPs was ~100-fold greater as assessed by the standard AUC/AUC ratio than by %ID in tumor. These results strongly suggest that PK metrics and calculations can influence the interpretation of NP tumor delivery and stress the need to properly validate novel PK metrics against traditional approaches.
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http://dx.doi.org/10.1126/sciadv.aay9249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439617PMC
July 2020

Challenges in the development of nanoparticle-based imaging agents: Characterization and biology.

Wiley Interdiscip Rev Nanomed Nanobiotechnol 2021 Jan 23;13(1):e1665. Epub 2020 Aug 23.

Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland, USA.

Despite imaging agents being some of the earliest nanomedicines in clinical use, the vast majority of current research and translational activities in the nanomedicine field involves therapeutics, while imaging agents are severely underrepresented. The reasons for this lack of representation are several fold, including difficulties in synthesis and scale-up, biocompatibility issues, lack of suitable tissue/disease selective targeting ligands and receptors, and a high bar for regulatory approval. The recent focus on immunotherapies and personalized medicine, and development of nanoparticle constructs with better tissue distribution and selectivity, provide new opportunities for nanomedicine imaging agent development. This manuscript will provide an overview of trends in imaging nanomedicine characterization and biocompatibility, and new horizons for future development. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.
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http://dx.doi.org/10.1002/wnan.1665DOI Listing
January 2021

Distinguishing Pharmacokinetics of Marketed Nanomedicine Formulations Using a Stable Isotope Tracer Assay.

ACS Pharmacol Transl Sci 2020 Jun 13;3(3):547-558. Epub 2020 Mar 13.

Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, Maryland 21702, United States.

The pharmacokinetics of nanomedicines are complicated by the unique dispositional characteristics of the drug carrier. Most simplistically, the carrier could be a solubilizing platform that allows administration of a hydrophobic drug. Alternatively, the carrier could be stable and release the drug in a controlled manner, allowing for distribution of the carrier to influence distribution of the encapsulated drug. A third potential dispositional mechanism is carriers that are not stably complexed to the drug, but rather bind the drug in a dynamic equilibrium, similar to the binding of unbound drug to protein; since the nanocarrier has distributional and binding characteristics unlike plasma proteins, the equilibrium binding of drug to a nanocarrier can affect pharmacokinetics in unexpected ways, diverging from classical protein binding paradigms. The recently developed stable isotope tracer ultrafiltration assay (SITUA) for nanomedicine fractionation is uniquely suited for distinguishing and comparing these carrier/drug interactions. Here we present the the encapsulated, unencapsulated, and unbound drug fraction pharmacokinetic profiles in rats for marketed nanomedicines, representing examples of controlled release (doxorubicin liposomes, Doxil; and doxorubicin HCl liposome generic), equilibrium binding (paclitaxel cremophor micelle solution, Taxol generic), and solubilizing (paclitaxel albumin nanoparticle, Abraxane; and paclitaxel polylactic acid micelle, Genexol-PM) nanomedicine formulations. The utility of the SITUA method in differentiating these unique pharmacokinetic profiles and its potential for use in establishing generic nanomedicine bioequivalence are discussed.
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http://dx.doi.org/10.1021/acsptsci.0c00011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296544PMC
June 2020

X-ray diffractive imaging of controlled gas-phase molecules: Toward imaging of dynamics in the molecular frame.

J Chem Phys 2020 Feb;152(8):084307

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.

We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array detector, and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to be within 5%. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data, which should be readily available at upcoming high-repetition-rate facilities.
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http://dx.doi.org/10.1063/1.5133963DOI Listing
February 2020

The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation.

J Synchrotron Radiat 2019 May 12;26(Pt 3):660-676. Epub 2019 Apr 12.

European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.
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http://dx.doi.org/10.1107/S1600577519003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510195PMC
May 2019

Blood Interactions, Pharmacokinetics, and Depth-Dependent Ablation of Rat Mammary Tumors with Photoactivatable, Liposomal Doxorubicin.

Mol Cancer Ther 2019 03 26;18(3):592-601. Epub 2018 Dec 26.

Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, New York.

Photosensitizers can be integrated with drug delivery vehicles to develop chemophototherapy agents with antitumor synergy between chemo- and photocomponents. Long-circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) incorporates a phospholipid-like photosensitizer (2 mole %) in the bilayer of Dox-loaded stealth liposomes. Hematological effects of endotoxin-minimized LC-Dox-PoP were characterized via standardized assays. interaction with erythrocytes, platelets, and plasma coagulation cascade were generally unremarkable, whereas complement activation was found to be similar to that of commercial Doxil. Blood partitioning suggested that both the Dox and PoP components of LC-Dox-PoP were stably entrapped or incorporated in liposomes. This was further confirmed with pharmacokinetic studies in Fischer rats, which showed the PoP and Dox components of the liposomes both had nearly identical, long circulation half-lives (25-26 hours). In a large orthotopic mammary tumor model in Fischer rats, following intravenous dosing (2 mg/kg Dox), the depth of enhanced Dox delivery in response to 665 nm laser irradiation was over 1 cm. LC-Dox-PoP with laser treatment cured or potently suppressed tumor growth, with greater efficacy observed in tumors 0.8 to 1.2 cm, compared with larger ones. The skin at the treatment site healed within approximately 30 days. Taken together, these data provide insight into nanocharacterization and photo-ablation parameters for a chemophototherapy agent.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0549DOI Listing
March 2019

Megahertz serial crystallography.

Authors:
Max O Wiedorn Dominik Oberthür Richard Bean Robin Schubert Nadine Werner Brian Abbey Martin Aepfelbacher Luigi Adriano Aschkan Allahgholi Nasser Al-Qudami Jakob Andreasson Steve Aplin Salah Awel Kartik Ayyer Saša Bajt Imrich Barák Sadia Bari Johan Bielecki Sabine Botha Djelloul Boukhelef Wolfgang Brehm Sandor Brockhauser Igor Cheviakov Matthew A Coleman Francisco Cruz-Mazo Cyril Danilevski Connie Darmanin R Bruce Doak Martin Domaracky Katerina Dörner Yang Du Hans Fangohr Holger Fleckenstein Matthias Frank Petra Fromme Alfonso M Gañán-Calvo Yaroslav Gevorkov Klaus Giewekemeyer Helen Mary Ginn Heinz Graafsma Rita Graceffa Dominic Greiffenberg Lars Gumprecht Peter Göttlicher Janos Hajdu Steffen Hauf Michael Heymann Susannah Holmes Daniel A Horke Mark S Hunter Siegfried Imlau Alexander Kaukher Yoonhee Kim Alexander Klyuev Juraj Knoška Bostjan Kobe Manuela Kuhn Christopher Kupitz Jochen Küpper Janine Mia Lahey-Rudolph Torsten Laurus Karoline Le Cong Romain Letrun P Lourdu Xavier Luis Maia Filipe R N C Maia Valerio Mariani Marc Messerschmidt Markus Metz Davide Mezza Thomas Michelat Grant Mills Diana C F Monteiro Andrew Morgan Kerstin Mühlig Anna Munke Astrid Münnich Julia Nette Keith A Nugent Theresa Nuguid Allen M Orville Suraj Pandey Gisel Pena Pablo Villanueva-Perez Jennifer Poehlsen Gianpietro Previtali Lars Redecke Winnie Maria Riekehr Holger Rohde Adam Round Tatiana Safenreiter Iosifina Sarrou Tokushi Sato Marius Schmidt Bernd Schmitt Robert Schönherr Joachim Schulz Jonas A Sellberg M Marvin Seibert Carolin Seuring Megan L Shelby Robert L Shoeman Marcin Sikorski Alessandro Silenzi Claudiu A Stan Xintian Shi Stephan Stern Jola Sztuk-Dambietz Janusz Szuba Aleksandra Tolstikova Martin Trebbin Ulrich Trunk Patrik Vagovic Thomas Ve Britta Weinhausen Thomas A White Krzysztof Wrona Chen Xu Oleksandr Yefanov Nadia Zatsepin Jiaguo Zhang Markus Perbandt Adrian P Mancuso Christian Betzel Henry Chapman Anton Barty

Nat Commun 2018 10 2;9(1):4025. Epub 2018 Oct 2.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607, Hamburg, Germany.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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http://dx.doi.org/10.1038/s41467-018-06156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168542PMC
October 2018

Cholecystokinin Receptor-Targeted Polyplex Nanoparticle Inhibits Growth and Metastasis of Pancreatic Cancer.

Cell Mol Gastroenterol Hepatol 2018 7;6(1):17-32. Epub 2018 Mar 7.

Department of Medicine, Georgetown University, Washington, District of Columbia.

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the cholecystokinin receptor that is overexpressed in this malignancy.

Methods: We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the cholecystokinin receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP.

Results: The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes.

Conclusions: Our polyplex nanoparticle platform establishes both a strong foundation for the development of receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.
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http://dx.doi.org/10.1016/j.jcmgh.2018.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008260PMC
March 2018

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BMC Biol 2018 05 31;16(1):59. Epub 2018 May 31.

Physics Department, University of Wisconsin-Milwaukee, 3135 N. Maryland Ave, Milwaukee, WI, 53211, USA.

Background: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.

Results: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s.

Conclusions: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.
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http://dx.doi.org/10.1186/s12915-018-0524-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977757PMC
May 2018

Mix-and-diffuse serial synchrotron crystallography.

IUCrJ 2017 Nov 9;4(Pt 6):769-777. Epub 2017 Oct 9.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.
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http://dx.doi.org/10.1107/S2052252517013124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668862PMC
November 2017

Designing an In Vivo Efficacy Study of Nanomedicines for Preclinical Tumor Growth Inhibition.

Methods Mol Biol 2018 ;1682:241-253

Cancer Research Technology Program, Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, P.O. Box B, Frederick, MD, 21702, USA.

Novel nanoformulated chemotherapeutics and diagnostics require demonstration of efficacy and safety in appropriate animal models prior to conducting early-phase clinical trials. In vivo efficacy experiments are tailored to the tumor model type and route of administration as well as several parameters related to the nanoformulation, like drug loading to determine dosing volume. When designing in vivo efficacy studies for nanomedicines, understanding the relationship between tumor biology and the nanoformulation characteristics is critical to achieving meaningful results, along with applying appropriate drug and nanoformulation controls. In particular, nanoparticles can have multifunctional roles such as targeting and imaging capabilities that require additional considerations when designing in vivo efficacy studies and choosing tumor models. In this chapter, we outline a general study design for a subcutaneously implanted tumor model along with an example of tumor growth inhibition and survival analysis.
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http://dx.doi.org/10.1007/978-1-4939-7352-1_20DOI Listing
May 2018

Improved Ultrafiltration Method to Measure Drug Release from Nanomedicines Utilizing a Stable Isotope Tracer.

Methods Mol Biol 2018 ;1682:223-239

Cancer Research Technology Program, Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, P.O. Box B, Frederick, MD, 21702, USA.

An important step in the early development of a nanomedicine formulation is the evaluation of stability and drug release in biological matrices. Additionally, the measurement of encapsulated and unencapsulated nanomedicine drug fractions is important for the determination of bioequivalence (pharmacokinetic equivalence) of generic nanomedicines. Unfortunately, current methods to measure drug release in plasma are limited, and all have fundamental disadvantages including non-equilibrium conditions and process-induced artifacts. The primary limitation of current ultrafiltration (and equilibrium dialysis) methods for separation of encapsulated and unencapsulated drug and determination of drug release is the difficulty in accurately differentiating protein bound and encapsulated drug. Since the protein binding of most drugs is high (>70%) and can change in a concentration- and time-dependent manner, it is very difficult to accurately account for the fraction of non-filterable drug that is encapsulated within the nanomedicine and how much is bound to protein. The method in this chapter is an improvement of existing ultrafiltration protocols for nanomedicine fractionation in plasma, in which a stable isotope tracer is spiked into a nanomedicine containing plasma sample in order to precisely measure the degree of plasma protein binding. Determination of protein binding then allows for accurate calculation of encapsulated and unencapsulated nanomedicine drug fractions, as well as free and protein-bound fractions.
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http://dx.doi.org/10.1007/978-1-4939-7352-1_19DOI Listing
May 2018

Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct.

Methods Mol Biol 2018 ;1682:211-219

Cancer Research Technology Program, Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, P.O. Box B, Frederick, MD, 21702, USA.

Autophagy is a catabolic process involved in the degradation and recycling of long-lived proteins and damaged organelles for maintenance of cellular homeostasis, and it has also been proposed as a type II cell death pathway. The cytoplasmic components targeted for catabolism are enclosed in a double-membrane autophagosome that merges with lysosomes, to form autophagosomes, and are finally degraded by lysosomal enzymes. There is substantial evidence that several nanomaterials can cause autophagy and lysosomal dysfunction, either by prevention of autophagolysosome formation, biopersistence or inhibition of lysosomal enzymes. Such effects have emerged as a potential mechanism of cellular toxicity, which is also associated with various pathological conditions. In this chapter, we describe a method to monitor autophagy by fusion of the modifier protein MAP LC3 with green fluorescent protein (GFP; GFP-LC3). This method enables imaging of autophagosome formation in real time by fluorescence microscopy without perturbing the MAP LC3 protein function and the process of autophagy. With the GFP-LC3 protein fusion construct, a longitudinal study of autophagy can be performed in cells after treatment with nanomaterials.
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http://dx.doi.org/10.1007/978-1-4939-7352-1_18DOI Listing
May 2018

Assessing NLRP3 Inflammasome Activation by Nanoparticles.

Methods Mol Biol 2018 ;1682:135-147

Cancer Research Technology Program, Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, P.O. Box B, Frederick, MD, 21702, USA.

NLRP3 inflammasome activation is one of the initial steps in an inflammatory cascade against pathogen/danger-associated molecular patterns (PAMPs/DAMPs), such as those arising from environmental toxins or nanoparticles, and is essential for innate immune response. NLRP3 inflammasome activation in cells can lead to the release of IL-1β cytokine via caspase-1, which is required for inflammatory-induced programmed cell death (pyroptosis). Nanoparticles are commonly used as vaccine adjuvants and drug delivery vehicles to improve the efficacy and reduce the toxicity of chemotherapeutic agents. Several studies indicate that different nanoparticles (e.g., liposomes, polymer-based nanoparticles) can induce NLRP3 inflammasome activation. Generation of a pro-inflammatory response is beneficial for vaccine delivery to provide adaptive immunity, a necessary step for successful vaccination. However, similar immune responses for intravenously injected, drug-containing nanoparticles can result in immunotoxicity (e.g., silica nanoparticles). Evaluation of NLRP3-mediated inflammasome activation by nanoparticles may predict pro-inflammatory responses in order to determine if these effects may be mitigated for drug delivery or optimized for vaccine development. In this protocol, we outline steps to monitor the release of IL-1β using PMA-primed THP-1 cells, a human monocytic leukemia cell line, as a model system. IL-1β release is used as a marker of NLRP3 inflammasome activation.
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http://dx.doi.org/10.1007/978-1-4939-7352-1_12DOI Listing
May 2018

Single-shot determination of focused FEL wave fields using iterative phase retrieval.

Opt Express 2017 Jul;25(15):17892-17903

Determining fluctuations in focus properties is essential for many experiments at Self-Amplified-Spontaneous-Emission (SASE) based Free-Electron-Lasers (FELs), in particular for imaging single non-crystalline biological particles. We report on a diffractive imaging technique to fully characterize highly focused, single-shot pulses using an iterative phase retrieval algorithm, and benchmark it against an existing Hartmann wavefront sensor. The results, both theoretical and experimental, demonstrate the effectiveness of this technique to provide a comprehensive and convenient shot-to-shot measurement of focused-pulse wave fields and source-point positional variations without the need for manipulative optics between the focus and the detector.
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http://dx.doi.org/10.1364/OE.25.017892DOI Listing
July 2017

When Is It Important to Measure Unbound Drug in Evaluating Nanomedicine Pharmacokinetics?

Drug Metab Dispos 2016 12 26;44(12):1934-1939. Epub 2016 Sep 26.

Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland (S.T.S., D.M.S.); and Food and Drug Administration, Center for Veterinary Medicine, Office of New Animal Drug Evaluation, Rockville, Maryland (M.N.M.).

Nanoformulations have become important tools for modifying drug disposition, be it from the perspective of enabling prolonged drug release, protecting the drug molecule from metabolism, or achieving targeted delivery. When examining the in vivo pharmacokinetic properties of these formulations, most investigations either focus on systemic concentrations of total (encapsulated plus unencapsulated) drug, or concentrations of encapsulated and unencapsulated drug. However, it is rare to find studies that differentiate between protein-bound and unbound (free) forms of the unencapsulated drug. In light of the unique attributes of these formulations, we cannot simply assume it appropriate to rely upon the protein-binding properties of the traditionally formulated or legacy drug when trying to define the pharmacokinetic or pharmacokinetic/pharmacodynamic characteristics of these nanoformulations. Therefore, this commentary explores reasons why it is important to consider not only unencapsulated drug, but also the portion of unencapsulated drug that is not bound to plasma proteins. Specifically, we highlight those situations when it may be necessary to include measurement of unencapsulated, unbound drug concentrations as part of the nanoformulation pharmacokinetic evaluation.
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http://dx.doi.org/10.1124/dmd.116.073148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118636PMC
December 2016

A high capacity polymeric micelle of paclitaxel: Implication of high dose drug therapy to safety and in vivo anti-cancer activity.

Biomaterials 2016 09 4;101:296-309. Epub 2016 Jun 4.

Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, 27599, USA; Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119992, Russia. Electronic address:

The poor solubility of paclitaxel (PTX), the commercially most successful anticancer drug, has long been hampering the development of suitable formulations. Here, we present translational evaluation of a nanoformulation of PTX, which is characterized by a facile preparation, extraordinary high drug loading of 50% wt. and PTX solubility of up to 45 g/L, excellent shelf stability and controllable, sub-100 nm size. We observe favorable in vitro and in vivo safety profiles and a higher maximum tolerated dose compared to clinically approved formulations. Pharmacokinetic analysis reveals that the higher dose administered leads to a higher exposure of the tumor to PTX. As a result, we observed improved therapeutic outcome in orthotopic tumor models including particularly faithful and aggressive "T11" mouse claudin-low breast cancer orthotopic, syngeneic transplants. The promising preclinical data on the presented PTX nanoformulation showcase the need to investigate new excipients and is a robust basis to translate into clinical trials.
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http://dx.doi.org/10.1016/j.biomaterials.2016.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035646PMC
September 2016

Coencapsulation of alendronate and doxorubicin in pegylated liposomes: a novel formulation for chemoimmunotherapy of cancer.

J Drug Target 2016 11 6;24(9):878-889. Epub 2016 Jun 6.

a Shaare Zedek Medical Center , Jerusalem , Israel.

We developed a pegylated liposome formulation of a dissociable salt of a nitrogen-containing bisphosphonate, alendronate (Ald), coencapsulated with the anthracycline, doxorubicin (Dox), a commonly used chemotherapeutic agent. Liposome-encapsulated ammonium Ald generates a gradient driving Dox into liposomes, forming a salt that holds both drugs in the liposome water phase. The resulting formulation (PLAD) allows for a high-loading efficiency of Dox, comparable to that of clinically approved pegylated liposomal doxorubicin sulfate (PLD) and is very stable in plasma stability assays. Cytotoxicity tests indicate greater potency for PLAD compared to PLD. This appears to be related to a synergistic effect of the coencapsulated Ald and Dox. PLAD and PLD differed in in vitro monocyte-induced IL-1β release (greater for PLAD) and complement activation (greater for PLD). A molar ratio Ald/Dox of ∼1:1 seems to provide an optimal compromise between loading efficiency of Dox, circulation time and in vivo toxicity of PLAD. In mice, the circulation half-life and tumor uptake of PLAD were comparable to PLD. In the M109R and 4T1 tumor models in immunocompetent mice, PLAD was superior to PLD in the growth inhibition of subcutaneous tumor implants. This new formulation appears to be a promising tool to exploit the antitumor effects of aminobisphosphonates in synergy with chemotherapy.
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http://dx.doi.org/10.1080/1061186X.2016.1191081DOI Listing
November 2016

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

Authors:
Daniel J Klionsky Kotb Abdelmohsen Akihisa Abe Md Joynal Abedin Hagai Abeliovich Abraham Acevedo Arozena Hiroaki Adachi Christopher M Adams Peter D Adams Khosrow Adeli Peter J Adhihetty Sharon G Adler Galila Agam Rajesh Agarwal Manish K Aghi Maria Agnello Patrizia Agostinis Patricia V Aguilar Julio Aguirre-Ghiso Edoardo M Airoldi Slimane Ait-Si-Ali Takahiko Akematsu Emmanuel T Akporiaye Mohamed Al-Rubeai Guillermo M Albaiceta Chris Albanese Diego Albani Matthew L Albert Jesus Aldudo Hana Algül Mehrdad Alirezaei Iraide Alloza Alexandru Almasan Maylin Almonte-Beceril Emad S Alnemri Covadonga Alonso Nihal Altan-Bonnet Dario C Altieri Silvia Alvarez Lydia Alvarez-Erviti Sandro Alves Giuseppina Amadoro Atsuo Amano Consuelo Amantini Santiago Ambrosio Ivano Amelio Amal O Amer Mohamed Amessou Angelika Amon Zhenyi An Frank A Anania Stig U Andersen Usha P Andley Catherine K Andreadi Nathalie Andrieu-Abadie Alberto Anel David K Ann Shailendra Anoopkumar-Dukie Manuela Antonioli Hiroshi Aoki Nadezda Apostolova Saveria Aquila Katia Aquilano Koichi Araki Eli Arama Agustin Aranda Jun Araya Alexandre Arcaro Esperanza Arias Hirokazu Arimoto Aileen R Ariosa Jane L Armstrong Thierry Arnould Ivica Arsov Katsuhiko Asanuma Valerie Askanas Eric Asselin Ryuichiro Atarashi Sally S Atherton Julie D Atkin Laura D Attardi Patrick Auberger Georg Auburger Laure Aurelian Riccardo Autelli Laura Avagliano Maria Laura Avantaggiati Limor Avrahami Suresh Awale Neelam Azad Tiziana Bachetti Jonathan M Backer Dong-Hun Bae Jae-Sung Bae Ok-Nam Bae Soo Han Bae Eric H Baehrecke Seung-Hoon Baek Stephen Baghdiguian Agnieszka Bagniewska-Zadworna Hua Bai Jie Bai Xue-Yuan Bai Yannick Bailly Kithiganahalli Narayanaswamy Balaji Walter Balduini Andrea Ballabio Rena Balzan Rajkumar Banerjee Gábor Bánhegyi Haijun Bao Benoit Barbeau Maria D Barrachina Esther Barreiro Bonnie Bartel Alberto Bartolomé Diane C Bassham Maria Teresa Bassi Robert C Bast Alakananda Basu Maria Teresa Batista Henri Batoko Maurizio Battino Kyle Bauckman Bradley L Baumgarner K Ulrich Bayer Rupert Beale Jean-François Beaulieu George R Beck Christoph Becker J David Beckham Pierre-André Bédard Patrick J Bednarski Thomas J Begley Christian Behl Christian Behrends Georg Mn Behrens Kevin E Behrns Eloy Bejarano Amine Belaid Francesca Belleudi Giovanni Bénard Guy Berchem Daniele Bergamaschi Matteo Bergami Ben Berkhout Laura Berliocchi Amélie Bernard Monique Bernard Francesca Bernassola Anne Bertolotti Amanda S Bess Sébastien Besteiro Saverio Bettuzzi Savita Bhalla Shalmoli Bhattacharyya Sujit K Bhutia Caroline Biagosch Michele Wolfe Bianchi Martine Biard-Piechaczyk Viktor Billes Claudia Bincoletto Baris Bingol Sara W Bird Marc Bitoun Ivana Bjedov Craig Blackstone Lionel Blanc Guillermo A Blanco Heidi Kiil Blomhoff Emilio Boada-Romero Stefan Böckler Marianne Boes Kathleen Boesze-Battaglia Lawrence H Boise Alessandra Bolino Andrea Boman Paolo Bonaldo Matteo Bordi Jürgen Bosch Luis M Botana Joelle Botti German Bou Marina Bouché Marion Bouchecareilh Marie-Josée Boucher Michael E Boulton Sebastien G Bouret Patricia Boya Michaël Boyer-Guittaut Peter V Bozhkov Nathan Brady Vania Mm Braga Claudio Brancolini Gerhard H Braus José M Bravo-San Pedro Lisa A Brennan Emery H Bresnick Patrick Brest Dave Bridges Marie-Agnès Bringer Marisa Brini Glauber C Brito Bertha Brodin Paul S Brookes Eric J Brown Karen Brown Hal E Broxmeyer Alain Bruhat Patricia Chakur Brum John H Brumell Nicola Brunetti-Pierri Robert J Bryson-Richardson Shilpa Buch Alastair M Buchan Hikmet Budak Dmitry V Bulavin Scott J Bultman Geert Bultynck Vladimir Bumbasirevic Yan Burelle Robert E Burke Margit Burmeister Peter Bütikofer Laura Caberlotto Ken Cadwell Monika Cahova Dongsheng Cai Jingjing Cai Qian Cai Sara Calatayud Nadine Camougrand Michelangelo Campanella Grant R Campbell Matthew Campbell Silvia Campello Robin Candau Isabella Caniggia Lavinia Cantoni Lizhi Cao Allan B Caplan Michele Caraglia Claudio Cardinali Sandra Morais Cardoso Jennifer S Carew Laura A Carleton Cathleen R Carlin Silvia Carloni Sven R Carlsson Didac Carmona-Gutierrez Leticia Am Carneiro Oliana Carnevali Serena Carra Alice Carrier Bernadette Carroll Caty Casas Josefina Casas Giuliana Cassinelli Perrine Castets Susana Castro-Obregon Gabriella Cavallini Isabella Ceccherini Francesco Cecconi Arthur I Cederbaum Valentín Ceña Simone Cenci Claudia Cerella Davide Cervia Silvia Cetrullo Hassan Chaachouay Han-Jung Chae Andrei S Chagin Chee-Yin Chai Gopal Chakrabarti Georgios Chamilos Edmond Yw Chan Matthew Tv Chan Dhyan Chandra Pallavi Chandra Chih-Peng Chang Raymond Chuen-Chung Chang Ta Yuan Chang John C Chatham Saurabh Chatterjee Santosh Chauhan Yongsheng Che Michael E Cheetham Rajkumar Cheluvappa Chun-Jung Chen Gang Chen Guang-Chao Chen Guoqiang Chen Hongzhuan Chen Jeff W Chen Jian-Kang Chen Min Chen Mingzhou Chen Peiwen Chen Qi Chen Quan Chen Shang-Der Chen Si Chen Steve S-L Chen Wei Chen Wei-Jung Chen Wen Qiang Chen Wenli Chen Xiangmei Chen Yau-Hung Chen Ye-Guang Chen Yin Chen Yingyu Chen Yongshun Chen Yu-Jen Chen Yue-Qin Chen Yujie Chen Zhen Chen Zhong Chen Alan Cheng Christopher Hk Cheng Hua Cheng Heesun Cheong Sara Cherry Jason Chesney Chun Hei Antonio Cheung Eric Chevet Hsiang Cheng Chi Sung-Gil Chi Fulvio Chiacchiera Hui-Ling Chiang Roberto Chiarelli Mario Chiariello Marcello Chieppa Lih-Shen Chin Mario Chiong Gigi Nc Chiu Dong-Hyung Cho Ssang-Goo Cho William C Cho Yong-Yeon Cho Young-Seok Cho Augustine Mk Choi Eui-Ju Choi Eun-Kyoung Choi Jayoung Choi Mary E Choi Seung-Il Choi Tsui-Fen Chou Salem Chouaib Divaker Choubey Vinay Choubey Kuan-Chih Chow Kamal Chowdhury Charleen T Chu Tsung-Hsien Chuang Taehoon Chun Hyewon Chung Taijoon Chung Yuen-Li Chung Yong-Joon Chwae Valentina Cianfanelli Roberto Ciarcia Iwona A Ciechomska Maria Rosa Ciriolo Mara Cirone Sofie Claerhout Michael J Clague Joan Clària Peter Gh Clarke Robert Clarke Emilio Clementi Cédric Cleyrat Miriam Cnop Eliana M Coccia Tiziana Cocco Patrice Codogno Jörn Coers Ezra Ew Cohen David Colecchia Luisa Coletto Núria S Coll Emma Colucci-Guyon Sergio Comincini Maria Condello Katherine L Cook Graham H Coombs Cynthia D Cooper J Mark Cooper Isabelle Coppens Maria Tiziana Corasaniti Marco Corazzari Ramon Corbalan Elisabeth Corcelle-Termeau Mario D Cordero Cristina Corral-Ramos Olga Corti Andrea Cossarizza Paola Costelli Safia Costes Susan L Cotman Ana Coto-Montes Sandra Cottet Eduardo Couve Lori R Covey L Ashley Cowart Jeffery S Cox Fraser P Coxon Carolyn B Coyne Mark S Cragg Rolf J Craven Tiziana Crepaldi Jose L Crespo Alfredo Criollo Valeria Crippa Maria Teresa Cruz Ana Maria Cuervo Jose M Cuezva Taixing Cui Pedro R Cutillas Mark J Czaja Maria F Czyzyk-Krzeska Ruben K Dagda Uta Dahmen Chunsun Dai Wenjie Dai Yun Dai Kevin N Dalby Luisa Dalla Valle Guillaume Dalmasso Marcello D'Amelio Markus Damme Arlette Darfeuille-Michaud Catherine Dargemont Victor M Darley-Usmar Srinivasan Dasarathy Biplab Dasgupta Srikanta Dash Crispin R Dass Hazel Marie Davey Lester M Davids David Dávila Roger J Davis Ted M Dawson Valina L Dawson Paula Daza Jackie de Belleroche Paul de Figueiredo Regina Celia Bressan Queiroz de Figueiredo José de la Fuente Luisa De Martino Antonella De Matteis Guido Ry De Meyer Angelo De Milito Mauro De Santi Wanderley de Souza Vincenzo De Tata Daniela De Zio Jayanta Debnath Reinhard Dechant Jean-Paul Decuypere Shane Deegan Benjamin Dehay Barbara Del Bello Dominic P Del Re Régis Delage-Mourroux Lea Md Delbridge Louise Deldicque Elizabeth Delorme-Axford Yizhen Deng Joern Dengjel Melanie Denizot Paul Dent Channing J Der Vojo Deretic Benoît Derrien Eric Deutsch Timothy P Devarenne Rodney J Devenish Sabrina Di Bartolomeo Nicola Di Daniele Fabio Di Domenico Alessia Di Nardo Simone Di Paola Antonio Di Pietro Livia Di Renzo Aaron DiAntonio Guillermo Díaz-Araya Ines Díaz-Laviada Maria T Diaz-Meco Javier Diaz-Nido Chad A Dickey Robert C Dickson Marc Diederich Paul Digard Ivan Dikic Savithrama P Dinesh-Kumar Chan Ding Wen-Xing Ding Zufeng Ding Luciana Dini Jörg Hw Distler Abhinav Diwan Mojgan Djavaheri-Mergny Kostyantyn Dmytruk Renwick Cj Dobson Volker Doetsch Karol Dokladny Svetlana Dokudovskaya Massimo Donadelli X Charlie Dong Xiaonan Dong Zheng Dong Terrence M Donohue Kelly S Doran Gabriella D'Orazi Gerald W Dorn Victor Dosenko Sami Dridi Liat Drucker Jie Du Li-Lin Du Lihuan Du André du Toit Priyamvada Dua Lei Duan Pu Duann Vikash Kumar Dubey Michael R Duchen Michel A Duchosal Helene Duez Isabelle Dugail Verónica I Dumit Mara C Duncan Elaine A Dunlop William A Dunn Nicolas Dupont Luc Dupuis Raúl V Durán Thomas M Durcan Stéphane Duvezin-Caubet Umamaheswar Duvvuri Vinay Eapen Darius Ebrahimi-Fakhari Arnaud Echard Leopold Eckhart Charles L Edelstein Aimee L Edinger Ludwig Eichinger Tobias Eisenberg Avital Eisenberg-Lerner N Tony Eissa Wafik S El-Deiry Victoria El-Khoury Zvulun Elazar Hagit Eldar-Finkelman Chris Jh Elliott Enzo Emanuele Urban Emmenegger Nikolai Engedal Anna-Mart Engelbrecht Simone Engelender Jorrit M Enserink Ralf Erdmann Jekaterina Erenpreisa Rajaraman Eri Jason L Eriksen Andreja Erman Ricardo Escalante Eeva-Liisa Eskelinen Lucile Espert Lorena Esteban-Martínez Thomas J Evans Mario Fabri Gemma Fabrias Cinzia Fabrizi Antonio Facchiano Nils J Færgeman Alberto Faggioni W Douglas Fairlie Chunhai Fan Daping Fan Jie Fan Shengyun Fang Manolis Fanto Alessandro Fanzani Thomas Farkas Mathias Faure Francois B Favier Howard Fearnhead Massimo Federici Erkang Fei Tania C Felizardo Hua Feng Yibin Feng Yuchen Feng Thomas A Ferguson Álvaro F Fernández Maite G Fernandez-Barrena Jose C Fernandez-Checa Arsenio Fernández-López Martin E Fernandez-Zapico Olivier Feron Elisabetta Ferraro Carmen Veríssima Ferreira-Halder Laszlo Fesus Ralph Feuer Fabienne C Fiesel Eduardo C Filippi-Chiela Giuseppe Filomeni Gian Maria Fimia John H Fingert Steven Finkbeiner Toren Finkel Filomena Fiorito Paul B Fisher Marc Flajolet Flavio Flamigni Oliver Florey Salvatore Florio R Andres Floto Marco Folini Carlo Follo Edward A Fon Francesco Fornai Franco Fortunato Alessandro Fraldi Rodrigo Franco Arnaud Francois Aurélie François Lisa B Frankel Iain Dc Fraser Norbert Frey Damien G Freyssenet Christian Frezza Scott L Friedman Daniel E Frigo Dongxu Fu José M Fuentes Juan Fueyo Yoshio Fujitani Yuuki Fujiwara Mikihiro Fujiya Mitsunori Fukuda Simone Fulda Carmela Fusco Bozena Gabryel Matthias Gaestel Philippe Gailly Malgorzata Gajewska Sehamuddin Galadari Gad Galili Inmaculada Galindo Maria F Galindo Giovanna Galliciotti Lorenzo Galluzzi Luca Galluzzi Vincent Galy Noor Gammoh Sam Gandy Anand K Ganesan Swamynathan Ganesan Ian G Ganley Monique Gannagé Fen-Biao Gao Feng Gao Jian-Xin Gao Lorena García Nannig Eleonora García Véscovi Marina Garcia-Macía Carmen Garcia-Ruiz Abhishek D Garg Pramod Kumar Garg Ricardo Gargini Nils Christian Gassen Damián Gatica Evelina Gatti Julie Gavard Evripidis Gavathiotis Liang Ge Pengfei Ge Shengfang Ge Po-Wu Gean Vania Gelmetti Armando A Genazzani Jiefei Geng Pascal Genschik Lisa Gerner Jason E Gestwicki David A Gewirtz Saeid Ghavami Eric Ghigo Debabrata Ghosh Anna Maria Giammarioli Francesca Giampieri Claudia Giampietri Alexandra Giatromanolaki Derrick J Gibbings Lara Gibellini Spencer B Gibson Vanessa Ginet Antonio Giordano Flaviano Giorgini Elisa Giovannetti Stephen E Girardin Suzana Gispert Sandy Giuliano Candece L Gladson Alvaro Glavic Martin Gleave Nelly Godefroy Robert M Gogal Kuppan Gokulan Gustavo H Goldman Delia Goletti Michael S Goligorsky Aldrin V Gomes Ligia C Gomes Hernando Gomez Candelaria Gomez-Manzano Rubén Gómez-Sánchez Dawit Ap Gonçalves Ebru Goncu Qingqiu Gong Céline Gongora Carlos B Gonzalez Pedro Gonzalez-Alegre Pilar Gonzalez-Cabo Rosa Ana González-Polo Ing Swie Goping Carlos Gorbea Nikolai V Gorbunov Daphne R Goring Adrienne M Gorman Sharon M Gorski Sandro Goruppi Shino Goto-Yamada Cecilia Gotor Roberta A Gottlieb Illana Gozes Devrim Gozuacik Yacine Graba Martin Graef Giovanna E Granato Gary Dean Grant Steven Grant Giovanni Luca Gravina Douglas R Green Alexander Greenhough Michael T Greenwood Benedetto Grimaldi Frédéric Gros Charles Grose Jean-Francois Groulx Florian Gruber Paolo Grumati Tilman Grune Jun-Lin Guan Kun-Liang Guan Barbara Guerra Carlos Guillen Kailash Gulshan Jan Gunst Chuanyong Guo Lei Guo Ming Guo Wenjie Guo Xu-Guang Guo Andrea A Gust Åsa B Gustafsson Elaine Gutierrez Maximiliano G Gutierrez Ho-Shin Gwak Albert Haas James E Haber Shinji Hadano Monica Hagedorn David R Hahn Andrew J Halayko Anne Hamacher-Brady Kozo Hamada Ahmed Hamai Andrea Hamann Maho Hamasaki Isabelle Hamer Qutayba Hamid Ester M Hammond Feng Han Weidong Han James T Handa John A Hanover Malene Hansen Masaru Harada Ljubica Harhaji-Trajkovic J Wade Harper Abdel Halim Harrath Adrian L Harris James Harris Udo Hasler Peter Hasselblatt Kazuhisa Hasui Robert G Hawley Teresa S Hawley Congcong He Cynthia Y He Fengtian He Gu He Rong-Rong He Xian-Hui He You-Wen He Yu-Ying He Joan K Heath Marie-Josée Hébert Robert A Heinzen Gudmundur Vignir Helgason Michael Hensel Elizabeth P Henske Chengtao Her Paul K Herman Agustín Hernández Carlos Hernandez Sonia Hernández-Tiedra Claudio Hetz P Robin Hiesinger Katsumi Higaki Sabine Hilfiker Bradford G Hill Joseph A Hill William D Hill Keisuke Hino Daniel Hofius Paul Hofman Günter U Höglinger Jörg Höhfeld Marina K Holz Yonggeun Hong David A Hood Jeroen Jm Hoozemans Thorsten Hoppe Chin Hsu Chin-Yuan Hsu Li-Chung Hsu Dong Hu Guochang Hu Hong-Ming Hu Hongbo Hu Ming Chang Hu Yu-Chen Hu Zhuo-Wei Hu Fang Hua Ya Hua Canhua Huang Huey-Lan Huang Kuo-How Huang Kuo-Yang Huang Shile Huang Shiqian Huang Wei-Pang Huang Yi-Ran Huang Yong Huang Yunfei Huang Tobias B Huber Patricia Huebbe Won-Ki Huh Juha J Hulmi Gang Min Hur James H Hurley Zvenyslava Husak Sabah Na Hussain Salik Hussain Jung Jin Hwang Seungmin Hwang Thomas Is Hwang Atsuhiro Ichihara Yuzuru Imai Carol Imbriano Megumi Inomata Takeshi Into Valentina Iovane Juan L Iovanna Renato V Iozzo Nancy Y Ip Javier E Irazoqui Pablo Iribarren Yoshitaka Isaka Aleksandra J Isakovic Harry Ischiropoulos Jeffrey S Isenberg Mohammad Ishaq Hiroyuki Ishida Isao Ishii Jane E Ishmael Ciro Isidoro Ken-Ichi Isobe Erika Isono Shohreh Issazadeh-Navikas Koji Itahana Eisuke Itakura Andrei I Ivanov Anand Krishnan V Iyer José M Izquierdo Yotaro Izumi Valentina Izzo Marja Jäättelä Nadia Jaber Daniel John Jackson William T Jackson Tony George Jacob Thomas S Jacques Chinnaswamy Jagannath Ashish Jain Nihar Ranjan Jana Byoung Kuk Jang Alkesh Jani Bassam Janji Paulo Roberto Jannig Patric J Jansson Steve Jean Marina Jendrach Ju-Hong Jeon Niels Jessen Eui-Bae Jeung Kailiang Jia Lijun Jia Hong Jiang Hongchi Jiang Liwen Jiang Teng Jiang Xiaoyan Jiang Xuejun Jiang Xuejun Jiang Ying Jiang Yongjun Jiang Alberto Jiménez Cheng Jin Hongchuan Jin Lei Jin Meiyan Jin Shengkan Jin Umesh Kumar Jinwal Eun-Kyeong Jo Terje Johansen Daniel E Johnson Gail Vw Johnson James D Johnson Eric Jonasch Chris Jones Leo Ab Joosten Joaquin Jordan Anna-Maria Joseph Bertrand Joseph Annie M Joubert Dianwen Ju Jingfang Ju Hsueh-Fen Juan Katrin Juenemann Gábor Juhász Hye Seung Jung Jae U Jung Yong-Keun Jung Heinz Jungbluth Matthew J Justice Barry Jutten Nadeem O Kaakoush Kai Kaarniranta Allen Kaasik Tomohiro Kabuta Bertrand Kaeffer Katarina Kågedal Alon Kahana Shingo Kajimura Or Kakhlon Manjula Kalia Dhan V Kalvakolanu Yoshiaki Kamada Konstantinos Kambas Vitaliy O Kaminskyy Harm H Kampinga Mustapha Kandouz Chanhee Kang Rui Kang Tae-Cheon Kang Tomotake Kanki Thirumala-Devi Kanneganti Haruo Kanno Anumantha G Kanthasamy Marc Kantorow Maria Kaparakis-Liaskos Orsolya Kapuy Vassiliki Karantza Md Razaul Karim Parimal Karmakar Arthur Kaser Susmita Kaushik Thomas Kawula A Murat Kaynar Po-Yuan Ke Zun-Ji Ke John H Kehrl Kate E Keller Jongsook Kim Kemper Anne K Kenworthy Oliver Kepp Andreas Kern Santosh Kesari David Kessel Robin Ketteler Isis do Carmo Kettelhut Bilon Khambu Muzamil Majid Khan Vinoth Km Khandelwal Sangeeta Khare Juliann G Kiang Amy A Kiger Akio Kihara Arianna L Kim Cheol Hyeon Kim Deok Ryong Kim Do-Hyung Kim Eung Kweon Kim Hye Young Kim Hyung-Ryong Kim Jae-Sung Kim Jeong Hun Kim Jin Cheon Kim Jin Hyoung Kim Kwang Woon Kim Michael D Kim Moon-Moo Kim Peter K Kim Seong Who Kim Soo-Youl Kim Yong-Sun Kim Yonghyun Kim Adi Kimchi Alec C Kimmelman Tomonori Kimura Jason S King Karla Kirkegaard Vladimir Kirkin Lorrie A Kirshenbaum Shuji Kishi Yasuo Kitajima Katsuhiko Kitamoto Yasushi Kitaoka Kaio Kitazato Rudolf A Kley Walter T Klimecki Michael Klinkenberg Jochen Klucken Helene Knævelsrud Erwin Knecht Laura Knuppertz Jiunn-Liang Ko Satoru Kobayashi Jan C Koch Christelle Koechlin-Ramonatxo Ulrich Koenig Young Ho Koh Katja Köhler Sepp D Kohlwein Masato Koike Masaaki Komatsu Eiki Kominami Dexin Kong Hee Jeong Kong Eumorphia G Konstantakou Benjamin T Kopp Tamas Korcsmaros Laura Korhonen Viktor I Korolchuk Nadya V Koshkina Yanjun Kou Michael I Koukourakis Constantinos Koumenis Attila L Kovács Tibor Kovács Werner J Kovacs Daisuke Koya Claudine Kraft Dimitri Krainc Helmut Kramer Tamara Kravic-Stevovic Wilhelm Krek Carole Kretz-Remy Roswitha Krick Malathi Krishnamurthy Janos Kriston-Vizi Guido Kroemer Michael C Kruer Rejko Kruger Nicholas T Ktistakis Kazuyuki Kuchitsu Christian Kuhn Addanki Pratap Kumar Anuj Kumar Ashok Kumar Deepak Kumar Dhiraj Kumar Rakesh Kumar Sharad Kumar Mondira Kundu Hsing-Jien Kung Atsushi Kuno Sheng-Han Kuo Jeff Kuret Tino Kurz Terry Kwok Taeg Kyu Kwon Yong Tae Kwon Irene Kyrmizi Albert R La Spada Frank Lafont Tim Lahm Aparna Lakkaraju Truong Lam Trond Lamark Steve Lancel Terry H Landowski Darius J R Lane Jon D Lane Cinzia Lanzi Pierre Lapaquette Louis R Lapierre Jocelyn Laporte Johanna Laukkarinen Gordon W Laurie Sergio Lavandero Lena Lavie Matthew J LaVoie Betty Yuen Kwan Law Helen Ka-Wai Law Kelsey B Law Robert Layfield Pedro A Lazo Laurent Le Cam Karine G Le Roch Hervé Le Stunff Vijittra Leardkamolkarn Marc Lecuit Byung-Hoon Lee Che-Hsin Lee Erinna F Lee Gyun Min Lee He-Jin Lee Hsinyu Lee Jae Keun Lee Jongdae Lee Ju-Hyun Lee Jun Hee Lee Michael Lee Myung-Shik Lee Patty J Lee Sam W Lee Seung-Jae Lee Shiow-Ju Lee Stella Y Lee Sug Hyung Lee Sung Sik Lee Sung-Joon Lee Sunhee Lee Ying-Ray Lee Yong J Lee Young H Lee Christiaan Leeuwenburgh Sylvain Lefort Renaud Legouis Jinzhi Lei Qun-Ying Lei David A Leib Gil Leibowitz Istvan Lekli Stéphane D Lemaire John J Lemasters Marius K Lemberg Antoinette Lemoine Shuilong Leng Guido Lenz Paola Lenzi Lilach O Lerman Daniele Lettieri Barbato Julia I-Ju Leu Hing Y Leung Beth Levine Patrick A Lewis Frank Lezoualc'h Chi Li Faqiang Li Feng-Jun Li Jun Li Ke Li Lian Li Min Li Min Li Qiang Li Rui Li Sheng Li Wei Li Wei Li Xiaotao Li Yumin Li Jiqin Lian Chengyu Liang Qiangrong Liang Yulin Liao Joana Liberal Pawel P Liberski Pearl Lie Andrew P Lieberman Hyunjung Jade Lim Kah-Leong Lim Kyu Lim Raquel T Lima Chang-Shen Lin Chiou-Feng Lin Fang Lin Fangming Lin Fu-Cheng Lin Kui Lin Kwang-Huei Lin Pei-Hui Lin Tianwei Lin Wan-Wan Lin Yee-Shin Lin Yong Lin Rafael Linden Dan Lindholm Lisa M Lindqvist Paul Lingor Andreas Linkermann Lance A Liotta Marta M Lipinski Vitor A Lira Michael P Lisanti Paloma B Liton Bo Liu Chong Liu Chun-Feng Liu Fei Liu Hung-Jen Liu Jianxun Liu Jing-Jing Liu Jing-Lan Liu Ke Liu Leyuan Liu Liang Liu Quentin Liu Rong-Yu Liu Shiming Liu Shuwen Liu Wei Liu Xian-De Liu Xiangguo Liu Xiao-Hong Liu Xinfeng Liu Xu Liu Xueqin Liu Yang Liu Yule Liu Zexian Liu Zhe Liu Juan P Liuzzi Gérard Lizard Mila Ljujic Irfan J Lodhi Susan E Logue Bal L Lokeshwar Yun Chau Long Sagar Lonial Benjamin Loos Carlos López-Otín Cristina López-Vicario Mar Lorente Philip L Lorenzi Péter Lõrincz Marek Los Michael T Lotze Penny E Lovat Binfeng Lu Bo Lu Jiahong Lu Qing Lu She-Min Lu Shuyan Lu Yingying Lu Frédéric Luciano Shirley Luckhart John Milton Lucocq Paula Ludovico Aurelia Lugea Nicholas W Lukacs Julian J Lum Anders H Lund Honglin Luo Jia Luo Shouqing Luo Claudio Luparello Timothy Lyons Jianjie Ma Yi Ma Yong Ma Zhenyi Ma Juliano Machado Glaucia M Machado-Santelli Fernando Macian Gustavo C MacIntosh Jeffrey P MacKeigan Kay F Macleod John D MacMicking Lee Ann MacMillan-Crow Frank Madeo Muniswamy Madesh Julio Madrigal-Matute Akiko Maeda Tatsuya Maeda Gustavo Maegawa Emilia Maellaro Hannelore Maes Marta Magariños Kenneth Maiese Tapas K Maiti Luigi Maiuri Maria Chiara Maiuri Carl G Maki Roland Malli Walter Malorni Alina Maloyan Fathia Mami-Chouaib Na Man Joseph D Mancias Eva-Maria Mandelkow Michael A Mandell Angelo A Manfredi Serge N Manié Claudia Manzoni Kai Mao Zixu Mao Zong-Wan Mao Philippe Marambaud Anna Maria Marconi Zvonimir Marelja Gabriella Marfe Marta Margeta Eva Margittai Muriel Mari Francesca V Mariani Concepcio Marin Sara Marinelli Guillermo Mariño Ivanka Markovic Rebecca Marquez Alberto M Martelli Sascha Martens Katie R Martin Seamus J Martin Shaun Martin Miguel A Martin-Acebes Paloma Martín-Sanz Camille Martinand-Mari Wim Martinet Jennifer Martinez Nuria Martinez-Lopez Ubaldo Martinez-Outschoorn Moisés Martínez-Velázquez Marta Martinez-Vicente Waleska Kerllen Martins Hirosato Mashima James A Mastrianni Giuseppe Matarese Paola Matarrese Roberto Mateo Satoaki Matoba Naomichi Matsumoto Takehiko Matsushita Akira Matsuura Takeshi Matsuzawa Mark P Mattson Soledad Matus Norma Maugeri Caroline Mauvezin Andreas Mayer Dusica Maysinger Guillermo D Mazzolini Mary Kate McBrayer Kimberly McCall Craig McCormick Gerald M McInerney Skye C McIver Sharon McKenna John J McMahon Iain A McNeish Fatima Mechta-Grigoriou Jan Paul Medema Diego L Medina Klara Megyeri Maryam Mehrpour Jawahar L Mehta Yide Mei Ute-Christiane Meier Alfred J Meijer Alicia Meléndez Gerry Melino Sonia Melino Edesio Jose Tenorio de Melo Maria A Mena Marc D Meneghini Javier A Menendez Regina Menezes Liesu Meng Ling-Hua Meng Songshu Meng Rossella Menghini A Sue Menko Rubem Fs Menna-Barreto Manoj B Menon Marco A Meraz-Ríos Giuseppe Merla Luciano Merlini Angelica M Merlot Andreas Meryk Stefania Meschini Joel N Meyer Man-Tian Mi Chao-Yu Miao Lucia Micale Simon Michaeli Carine Michiels Anna Rita Migliaccio Anastasia Susie Mihailidou Dalibor Mijaljica Katsuhiko Mikoshiba Enrico Milan Leonor Miller-Fleming Gordon B Mills Ian G Mills Georgia Minakaki Berge A Minassian Xiu-Fen Ming Farida Minibayeva Elena A Minina Justine D Mintern Saverio Minucci Antonio Miranda-Vizuete Claire H Mitchell Shigeki Miyamoto Keisuke Miyazawa Noboru Mizushima Katarzyna Mnich Baharia Mograbi Simin Mohseni Luis Ferreira Moita Marco Molinari Maurizio Molinari Andreas Buch Møller Bertrand Mollereau Faustino Mollinedo Marco Mongillo Martha M Monick Serena Montagnaro Craig Montell Darren J Moore Michael N Moore Rodrigo Mora-Rodriguez Paula I Moreira Etienne Morel Maria Beatrice Morelli Sandra Moreno Michael J Morgan Arnaud Moris Yuji Moriyasu Janna L Morrison Lynda A Morrison Eugenia Morselli Jorge Moscat Pope L Moseley Serge Mostowy Elisa Motori Denis Mottet Jeremy C Mottram Charbel E-H Moussa Vassiliki E Mpakou Hasan Mukhtar Jean M Mulcahy Levy Sylviane Muller Raquel Muñoz-Moreno Cristina Muñoz-Pinedo Christian Münz Maureen E Murphy James T Murray Aditya Murthy Indira U Mysorekar Ivan R Nabi Massimo Nabissi Gustavo A Nader Yukitoshi Nagahara Yoshitaka Nagai Kazuhiro Nagata Anika Nagelkerke Péter Nagy Samisubbu R Naidu Sreejayan Nair Hiroyasu Nakano Hitoshi Nakatogawa Meera Nanjundan Gennaro Napolitano Naweed I Naqvi Roberta Nardacci Derek P Narendra Masashi Narita Anna Chiara Nascimbeni Ramesh Natarajan Luiz C Navegantes Steffan T Nawrocki Taras Y Nazarko Volodymyr Y Nazarko Thomas Neill Luca M Neri Mihai G Netea Romana T Netea-Maier Bruno M Neves Paul A Ney Ioannis P Nezis Hang Tt Nguyen Huu Phuc Nguyen Anne-Sophie Nicot Hilde Nilsen Per Nilsson Mikio Nishimura Ichizo Nishino Mireia Niso-Santano Hua Niu Ralph A Nixon Vincent Co Njar Takeshi Noda Angelika A Noegel Elsie Magdalena Nolte Erik Norberg Koenraad K Norga Sakineh Kazemi Noureini Shoji Notomi Lucia Notterpek Karin Nowikovsky Nobuyuki Nukina Thorsten Nürnberger Valerie B O'Donnell Tracey O'Donovan Peter J O'Dwyer Ina Oehme Clara L Oeste Michinaga Ogawa Besim Ogretmen Yuji Ogura Young J Oh Masaki Ohmuraya Takayuki Ohshima Rani Ojha Koji Okamoto Toshiro Okazaki F Javier Oliver Karin Ollinger Stefan Olsson Daniel P Orban Paulina Ordonez Idil Orhon Laszlo Orosz Eyleen J O'Rourke Helena Orozco Angel L Ortega Elena Ortona Laura D Osellame Junko Oshima Shigeru Oshima Heinz D Osiewacz Takanobu Otomo Kinya Otsu Jing-Hsiung James Ou Tiago F Outeiro Dong-Yun Ouyang Hongjiao Ouyang Michael Overholtzer Michelle A Ozbun P Hande Ozdinler Bulent Ozpolat Consiglia Pacelli Paolo Paganetti Guylène Page Gilles Pages Ugo Pagnini Beata Pajak Stephen C Pak Karolina Pakos-Zebrucka Nazzy Pakpour Zdena Palková Francesca Palladino Kathrin Pallauf Nicolas Pallet Marta Palmieri Søren R Paludan Camilla Palumbo Silvia Palumbo Olatz Pampliega Hongming Pan Wei Pan Theocharis Panaretakis Aseem Pandey Areti Pantazopoulou Zuzana Papackova Daniela L Papademetrio Issidora Papassideri Alessio Papini Nirmala Parajuli Julian Pardo Vrajesh V Parekh Giancarlo Parenti Jong-In Park Junsoo Park Ohkmae K Park Roy Parker Rosanna Parlato Jan B Parys Katherine R Parzych Jean-Max Pasquet Benoit Pasquier Kishore Bs Pasumarthi Daniel Patschan Cam Patterson Sophie Pattingre Scott Pattison Arnim Pause Hermann Pavenstädt Flaminia Pavone Zully Pedrozo Fernando J Peña Miguel A Peñalva Mario Pende Jianxin Peng Fabio Penna Josef M Penninger Anna Pensalfini Salvatore Pepe Gustavo Js Pereira Paulo C Pereira Verónica Pérez-de la Cruz María Esther Pérez-Pérez Diego Pérez-Rodríguez Dolores Pérez-Sala Celine Perier Andras Perl David H Perlmutter Ida Perrotta Shazib Pervaiz Maija Pesonen Jeffrey E Pessin Godefridus J Peters Morten Petersen Irina Petrache Basil J Petrof Goran Petrovski James M Phang Mauro Piacentini Marina Pierdominici Philippe Pierre Valérie Pierrefite-Carle Federico Pietrocola Felipe X Pimentel-Muiños Mario Pinar Benjamin Pineda Ronit Pinkas-Kramarski Marcello Pinti Paolo Pinton Bilal Piperdi James M Piret Leonidas C Platanias Harald W Platta Edward D Plowey Stefanie Pöggeler Marc Poirot Peter Polčic Angelo Poletti Audrey H Poon Hana Popelka Blagovesta Popova Izabela Poprawa Shibu M Poulose Joanna Poulton Scott K Powers Ted Powers Mercedes Pozuelo-Rubio Krisna Prak Reinhild Prange Mark Prescott Muriel Priault Sharon Prince Richard L Proia Tassula Proikas-Cezanne Holger Prokisch Vasilis J Promponas Karin Przyklenk Rosa Puertollano Subbiah Pugazhenthi Luigi Puglielli Aurora Pujol Julien Puyal Dohun Pyeon Xin Qi Wen-Bin Qian Zheng-Hong Qin Yu Qiu Ziwei Qu Joe Quadrilatero Frederick Quinn Nina Raben Hannah Rabinowich Flavia Radogna Michael J Ragusa Mohamed Rahmani Komal Raina Sasanka Ramanadham Rajagopal Ramesh Abdelhaq Rami Sarron Randall-Demllo Felix Randow Hai Rao V Ashutosh Rao Blake B Rasmussen Tobias M Rasse Edward A Ratovitski Pierre-Emmanuel Rautou Swapan K Ray Babak Razani Bruce H Reed Fulvio Reggiori Markus Rehm Andreas S Reichert Theo Rein David J Reiner Eric Reits Jun Ren Xingcong Ren Maurizio Renna Jane Eb Reusch Jose L Revuelta Leticia Reyes Alireza R Rezaie Robert I Richards Des R Richardson Clémence Richetta Michael A Riehle Bertrand H Rihn Yasuko Rikihisa Brigit E Riley Gerald Rimbach Maria Rita Rippo Konstantinos Ritis Federica Rizzi Elizete Rizzo Peter J Roach Jeffrey Robbins Michel Roberge Gabriela Roca Maria Carmela Roccheri Sonia Rocha Cecilia Mp Rodrigues Clara I Rodríguez Santiago Rodriguez de Cordoba Natalia Rodriguez-Muela Jeroen Roelofs Vladimir V Rogov Troy T Rohn Bärbel Rohrer Davide Romanelli Luigina Romani Patricia Silvia Romano M Isabel G Roncero Jose Luis Rosa Alicia Rosello Kirill V Rosen Philip Rosenstiel Magdalena Rost-Roszkowska Kevin A Roth Gael Roué Mustapha Rouis Kasper M Rouschop Daniel T Ruan Diego Ruano David C Rubinsztein Edmund B Rucker Assaf Rudich Emil Rudolf Ruediger Rudolf Markus A Ruegg Carmen Ruiz-Roldan Avnika Ashok Ruparelia Paola Rusmini David W Russ Gian Luigi Russo Giuseppe Russo Rossella Russo Tor Erik Rusten Victoria Ryabovol Kevin M Ryan Stefan W Ryter David M Sabatini Michael Sacher Carsten Sachse Michael N Sack Junichi Sadoshima Paul Saftig Ronit Sagi-Eisenberg Sumit Sahni Pothana Saikumar Tsunenori Saito Tatsuya Saitoh Koichi Sakakura Machiko Sakoh-Nakatogawa Yasuhito Sakuraba María Salazar-Roa Paolo Salomoni Ashok K Saluja Paul M Salvaterra Rosa Salvioli Afshin Samali Anthony Mj Sanchez José A Sánchez-Alcázar Ricardo Sanchez-Prieto Marco Sandri Miguel A Sanjuan Stefano Santaguida Laura Santambrogio Giorgio Santoni Claudia Nunes Dos Santos Shweta Saran Marco Sardiello Graeme Sargent Pallabi Sarkar Sovan Sarkar Maria Rosa Sarrias Minnie M Sarwal Chihiro Sasakawa Motoko Sasaki Miklos Sass Ken Sato Miyuki Sato Joseph Satriano Niramol Savaraj Svetlana Saveljeva Liliana Schaefer Ulrich E Schaible Michael Scharl Hermann M Schatzl Randy Schekman Wiep Scheper Alfonso Schiavi Hyman M Schipper Hana Schmeisser Jens Schmidt Ingo Schmitz Bianca E Schneider E Marion Schneider Jaime L Schneider Eric A Schon Miriam J Schönenberger Axel H Schönthal Daniel F Schorderet Bernd Schröder Sebastian Schuck Ryan J Schulze Melanie Schwarten Thomas L Schwarz Sebastiano Sciarretta Kathleen Scotto A Ivana Scovassi Robert A Screaton Mark Screen Hugo Seca Simon Sedej Laura Segatori Nava Segev Per O Seglen Jose M Seguí-Simarro Juan Segura-Aguilar Ekihiro Seki Christian Sell Iban Seiliez Clay F Semenkovich Gregg L Semenza Utpal Sen Andreas L Serra Ana Serrano-Puebla Hiromi Sesaki Takao Setoguchi Carmine Settembre John J Shacka Ayesha N Shajahan-Haq Irving M Shapiro Shweta Sharma Hua She C-K James Shen Chiung-Chyi Shen Han-Ming Shen Sanbing Shen Weili Shen Rui Sheng Xianyong Sheng Zu-Hang Sheng Trevor G Shepherd Junyan Shi Qiang Shi Qinghua Shi Yuguang Shi Shusaku Shibutani Kenichi Shibuya Yoshihiro Shidoji Jeng-Jer Shieh Chwen-Ming Shih Yohta Shimada Shigeomi Shimizu Dong Wook Shin Mari L Shinohara Michiko Shintani Takahiro Shintani Tetsuo Shioi Ken Shirabe Ronit Shiri-Sverdlov Orian Shirihai Gordon C Shore Chih-Wen Shu Deepak Shukla Andriy A Sibirny Valentina Sica Christina J Sigurdson Einar M Sigurdsson Puran Singh Sijwali Beata Sikorska Wilian A Silveira Sandrine Silvente-Poirot Gary A Silverman Jan Simak Thomas Simmet Anna Katharina Simon Hans-Uwe Simon Cristiano Simone Matias Simons Anne Simonsen Rajat Singh Shivendra V Singh Shrawan K Singh Debasish Sinha Sangita Sinha Frank A Sinicrope Agnieszka Sirko Kapil Sirohi Balindiwe Jn Sishi Annie Sittler Parco M Siu Efthimios Sivridis Anna Skwarska Ruth Slack Iva Slaninová Nikolai Slavov Soraya S Smaili Keiran Sm Smalley Duncan R Smith Stefaan J Soenen Scott A Soleimanpour Anita Solhaug Kumaravel Somasundaram Jin H Son Avinash Sonawane Chunjuan Song Fuyong Song Hyun Kyu Song Ju-Xian Song Wei Song Kai Y Soo Anil K Sood Tuck Wah Soong Virawudh Soontornniyomkij Maurizio Sorice Federica Sotgia David R Soto-Pantoja Areechun Sotthibundhu Maria João Sousa Herman P Spaink Paul N Span Anne Spang Janet D Sparks Peter G Speck Stephen A Spector Claudia D Spies Wolfdieter Springer Daret St Clair Alessandra Stacchiotti Bart Staels Michael T Stang Daniel T Starczynowski Petro Starokadomskyy Clemens Steegborn John W Steele Leonidas Stefanis Joan Steffan Christine M Stellrecht Harald Stenmark Tomasz M Stepkowski Stęphan T Stern Craig Stevens Brent R Stockwell Veronika Stoka Zuzana Storchova Björn Stork Vassilis Stratoulias Dimitrios J Stravopodis Pavel Strnad Anne Marie Strohecker Anna-Lena Ström Per Stromhaug Jiri Stulik Yu-Xiong Su Zhaoliang Su Carlos S Subauste Srinivasa Subramaniam Carolyn M Sue Sang Won Suh Xinbing Sui Supawadee Sukseree David Sulzer Fang-Lin Sun Jiaren Sun Jun Sun Shi-Yong Sun Yang Sun Yi Sun Yingjie Sun Vinod Sundaramoorthy Joseph Sung Hidekazu Suzuki Kuninori Suzuki Naoki Suzuki Tadashi Suzuki Yuichiro J Suzuki Michele S Swanson Charles Swanton Karl Swärd Ghanshyam Swarup Sean T Sweeney Paul W Sylvester Zsuzsanna Szatmari Eva Szegezdi Peter W Szlosarek Heinrich Taegtmeyer Marco Tafani Emmanuel Taillebourg Stephen Wg Tait Krisztina Takacs-Vellai Yoshinori Takahashi Szabolcs Takáts Genzou Takemura Nagio Takigawa Nicholas J Talbot Elena Tamagno Jerome Tamburini Cai-Ping Tan Lan Tan Mei Lan Tan Ming Tan Yee-Joo Tan Keiji Tanaka Masaki Tanaka Daolin Tang Dingzhong Tang Guomei Tang Isei Tanida Kunikazu Tanji Bakhos A Tannous Jose A Tapia Inmaculada Tasset-Cuevas Marc Tatar Iman Tavassoly Nektarios Tavernarakis Allen Taylor Graham S Taylor Gregory A Taylor J Paul Taylor Mark J Taylor Elena V Tchetina Andrew R Tee Fatima Teixeira-Clerc Sucheta Telang Tewin Tencomnao Ba-Bie Teng Ru-Jeng Teng Faraj Terro Gianluca Tettamanti Arianne L Theiss Anne E Theron Kelly Jean Thomas Marcos P Thomé Paul G Thomes Andrew Thorburn Jeremy Thorner Thomas Thum Michael Thumm Teresa Lm Thurston Ling Tian Andreas Till Jenny Pan-Yun Ting Vladimir I Titorenko Lilach Toker Stefano Toldo Sharon A Tooze Ivan Topisirovic Maria Lyngaas Torgersen Liliana Torosantucci Alicia Torriglia Maria Rosaria Torrisi Cathy Tournier Roberto Towns Vladimir Trajkovic Leonardo H Travassos Gemma Triola Durga Nand Tripathi Daniela Trisciuoglio Rodrigo Troncoso Ioannis P Trougakos Anita C Truttmann Kuen-Jer Tsai Mario P Tschan Yi-Hsin Tseng Takayuki Tsukuba Allan Tsung Andrey S Tsvetkov Shuiping Tu Hsing-Yu Tuan Marco Tucci David A Tumbarello Boris Turk Vito Turk Robin Fb Turner Anders A Tveita Suresh C Tyagi Makoto Ubukata Yasuo Uchiyama Andrej Udelnow Takashi Ueno Midori Umekawa Rika Umemiya-Shirafuji Benjamin R Underwood Christian Ungermann Rodrigo P Ureshino Ryo Ushioda Vladimir N Uversky Néstor L Uzcátegui Thomas Vaccari Maria I Vaccaro Libuše Váchová Helin Vakifahmetoglu-Norberg Rut Valdor Enza Maria Valente Francois Vallette Angela M Valverde Greet Van den Berghe Ludo Van Den Bosch Gijs R van den Brink F Gisou van der Goot Ida J van der Klei Luc Jw van der Laan Wouter G van Doorn Marjolein van Egmond Kenneth L van Golen Luc Van Kaer Menno van Lookeren Campagne Peter Vandenabeele Wim Vandenberghe Ilse Vanhorebeek Isabel Varela-Nieto M Helena Vasconcelos Radovan Vasko Demetrios G Vavvas Ignacio Vega-Naredo Guillermo Velasco Athanassios D Velentzas Panagiotis D Velentzas Tibor Vellai Edo Vellenga Mikkel Holm Vendelbo Kartik Venkatachalam Natascia Ventura Salvador Ventura Patrícia St Veras Mireille Verdier Beata G Vertessy Andrea Viale Michel Vidal Helena L A Vieira Richard D Vierstra Nadarajah Vigneswaran Neeraj Vij Miquel Vila Margarita Villar Victor H Villar Joan Villarroya Cécile Vindis Giampietro Viola Maria Teresa Viscomi Giovanni Vitale Dan T Vogl Olga V Voitsekhovskaja Clarissa von Haefen Karin von Schwarzenberg Daniel E Voth Valérie Vouret-Craviari Kristina Vuori Jatin M Vyas Christian Waeber Cheryl Lyn Walker Mark J Walker Jochen Walter Lei Wan Xiangbo Wan Bo Wang Caihong Wang Chao-Yung Wang Chengshu Wang Chenran Wang Chuangui Wang Dong Wang Fen Wang Fuxin Wang Guanghui Wang Hai-Jie Wang Haichao Wang Hong-Gang Wang Hongmin Wang Horng-Dar Wang Jing Wang Junjun Wang Mei Wang Mei-Qing Wang Pei-Yu Wang Peng Wang Richard C Wang Shuo Wang Ting-Fang Wang Xian Wang Xiao-Jia Wang Xiao-Wei Wang Xin Wang Xuejun Wang Yan Wang Yanming Wang Ying Wang Ying-Jan Wang Yipeng Wang Yu Wang Yu Tian Wang Yuqing Wang Zhi-Nong Wang Pablo Wappner Carl Ward Diane McVey Ward Gary Warnes Hirotaka Watada Yoshihisa Watanabe Kei Watase Timothy E Weaver Colin D Weekes Jiwu Wei Thomas Weide Conrad C Weihl Günther Weindl Simone Nardin Weis Longping Wen Xin Wen Yunfei Wen Benedikt Westermann Cornelia M Weyand Anthony R White Eileen White J Lindsay Whitton Alexander J Whitworth Joëlle Wiels Franziska Wild Manon E Wildenberg Tom Wileman Deepti Srinivas Wilkinson Simon Wilkinson Dieter Willbold Chris Williams Katherine Williams Peter R Williamson Konstanze F Winklhofer Steven S Witkin Stephanie E Wohlgemuth Thomas Wollert Ernst J Wolvetang Esther Wong G William Wong Richard W Wong Vincent Kam Wai Wong Elizabeth A Woodcock Karen L Wright Chunlai Wu Defeng Wu Gen Sheng Wu Jian Wu Junfang Wu Mian Wu Min Wu Shengzhou Wu William Kk Wu Yaohua Wu Zhenlong Wu Cristina Pr Xavier Ramnik J Xavier Gui-Xian Xia Tian Xia Weiliang Xia Yong Xia Hengyi Xiao Jian Xiao Shi Xiao Wuhan Xiao Chuan-Ming Xie Zhiping Xie Zhonglin Xie Maria Xilouri Yuyan Xiong Chuanshan Xu Congfeng Xu Feng Xu Haoxing Xu Hongwei Xu Jian Xu Jianzhen Xu Jinxian Xu Liang Xu Xiaolei Xu Yangqing Xu Ye Xu Zhi-Xiang Xu Ziheng Xu Yu Xue Takahiro Yamada Ai Yamamoto Koji Yamanaka Shunhei Yamashina Shigeko Yamashiro Bing Yan Bo Yan Xianghua Yan Zhen Yan Yasuo Yanagi Dun-Sheng Yang Jin-Ming Yang Liu Yang Minghua Yang Pei-Ming Yang Peixin Yang Qian Yang Wannian Yang Wei Yuan Yang Xuesong Yang Yi Yang Ying Yang Zhifen Yang Zhihong Yang Meng-Chao Yao Pamela J Yao Xiaofeng Yao Zhenyu Yao Zhiyuan Yao Linda S Yasui Mingxiang Ye Barry Yedvobnick Behzad Yeganeh Elizabeth S Yeh Patricia L Yeyati Fan Yi Long Yi Xiao-Ming Yin Calvin K Yip Yeong-Min Yoo Young Hyun Yoo Seung-Yong Yoon Ken-Ichi Yoshida Tamotsu Yoshimori Ken H Young Huixin Yu Jane J Yu Jin-Tai Yu Jun Yu Li Yu W Haung Yu Xiao-Fang Yu Zhengping Yu Junying Yuan Zhi-Min Yuan Beatrice Yjt Yue Jianbo Yue Zhenyu Yue David N Zacks Eldad Zacksenhaus Nadia Zaffaroni Tania Zaglia Zahra Zakeri Vincent Zecchini Jinsheng Zeng Min Zeng Qi Zeng Antonis S Zervos Donna D Zhang Fan Zhang Guo Zhang Guo-Chang Zhang Hao Zhang Hong Zhang Hong Zhang Hongbing Zhang Jian Zhang Jian Zhang Jiangwei Zhang Jianhua Zhang Jing-Pu Zhang Li Zhang Lin Zhang Lin Zhang Long Zhang Ming-Yong Zhang Xiangnan Zhang Xu Dong Zhang Yan Zhang Yang Zhang Yanjin Zhang Yingmei Zhang Yunjiao Zhang Mei Zhao Wei-Li Zhao Xiaonan Zhao Yan G Zhao Ying Zhao Yongchao Zhao Yu-Xia Zhao Zhendong Zhao Zhizhuang J Zhao Dexian Zheng Xi-Long Zheng Xiaoxiang Zheng Boris Zhivotovsky Qing Zhong Guang-Zhou Zhou Guofei Zhou Huiping Zhou Shu-Feng Zhou Xu-Jie Zhou Hongxin Zhu Hua Zhu Wei-Guo Zhu Wenhua Zhu Xiao-Feng Zhu Yuhua Zhu Shi-Mei Zhuang Xiaohong Zhuang Elio Ziparo Christos E Zois Teresa Zoladek Wei-Xing Zong Antonio Zorzano Susu M Zughaier

Autophagy 2016 ;12(1):1-222

kb Emory University, School of Medicine , Department of Microbiology and Immunology , Atlanta , GA , USA.

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http://dx.doi.org/10.1080/15548627.2015.1100356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835977PMC
October 2016

Stable isotope method to measure drug release from nanomedicines.

J Control Release 2015 Dec 24;220(Pt A):169-174. Epub 2015 Oct 24.

Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, United States. Electronic address:

Existing methods to measure nanomedicine drug release in biological matrices are inadequate. A novel drug release method utilizing a stable isotope tracer has been developed. Stable isotope-labeled drug is spiked into plasma containing nanomedicine. The labeled drug equilibrates with plasma components identical to the normoisotopic drug released from the nanomedicine formulation. Therefore, the ultrafilterable fraction of the isotope-labeled drug represents a reliable measure of free normoisotopic drug fraction in plasma, and can be used to calculate nanomedicine encapsulated and unencapsulated drug fractions. To demonstrate the utility of this method, we performed a plasma drug release study with both a fast releasing commercial docetaxel formulation, Taxotere®, and a delayed releasing nanomicellar formulation of a docetaxel prodrug, Procet 8. The instability of the unencapsulated prodrug in plasma allowed us to compare our calculated prodrug release and docetaxel conversion with the actual docetaxel concentration measured directly without fractionation. Drug release estimates for the fast releasing Taxotere formulation demonstrated accuracy deviation and precision (%CV) of <15%. For the controlled release Procet 8 formulation, we calculated a slow release and conversion of the prodrug in rat plasma that was highly correlated with the direct docetaxel measurement (R(2)=0.98). We believe that this method will have tremendous utility in the development and regulatory evaluation of nanomedicines, and aid in determination of generic bioequivalence.
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http://dx.doi.org/10.1016/j.jconrel.2015.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688069PMC
December 2015

Nanoparticles for cancer imaging: The good, the bad, and the promise.

Nano Today 2013 Oct;8(5):454-460

Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322, United States.

Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.
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http://dx.doi.org/10.1016/j.nantod.2013.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240321PMC
October 2013

Imaging molecular structure through femtosecond photoelectron diffraction on aligned and oriented gas-phase molecules.

Faraday Discuss 2014 14;171:57-80. Epub 2014 Aug 14.

Deutsches Elektronen-Synchrotron (DESY), 22607 Hamburg, Germany.

This paper gives an account of our progress towards performing femtosecond time-resolved photoelectron diffraction on gas-phase molecules in a pump-probe setup combining optical lasers and an X-ray free-electron laser. We present results of two experiments aimed at measuring photoelectron angular distributions of laser-aligned 1-ethynyl-4-fluorobenzene (C(8)H(5)F) and dissociating, laser-aligned 1,4-dibromobenzene (C(6)H(4)Br(2)) molecules and discuss them in the larger context of photoelectron diffraction on gas-phase molecules. We also show how the strong nanosecond laser pulse used for adiabatically laser-aligning the molecules influences the measured electron and ion spectra and angular distributions, and discuss how this may affect the outcome of future time-resolved photoelectron diffraction experiments.
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http://dx.doi.org/10.1039/c4fd00037dDOI Listing
June 2015

Spatial separation of molecular conformers and clusters.

J Vis Exp 2014 Jan 9(83):e51137. Epub 2014 Jan 9.

Center for Free-Electron Laser Science, CFEL, DESY.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.
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http://dx.doi.org/10.3791/51137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089472PMC
January 2014

PLGA/liposome hybrid nanoparticles for short-chain ceramide delivery.

Pharm Res 2014 Mar 25;31(3):684-93. Epub 2013 Sep 25.

Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, 48108, USA.

Purpose: Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA).

Methods: BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor).

Results: FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h.

Conclusions: The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.
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http://dx.doi.org/10.1007/s11095-013-1190-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164208PMC
March 2014

A novel gadolinium-based trimetasphere metallofullerene for application as a magnetic resonance imaging contrast agent.

Invest Radiol 2013 Nov;48(11):745-54

From the *Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick, Inc, Frederick National Laboratory for Cancer Research, Frederick, MD; †Luna nanoWorks, Luna Innovations, Inc, Danville, VA; ‡Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, Greensboro, NC; §Small Animal Imaging Program, Laboratory Animal Sciences Program, SAIC-Frederick, Inc, Frederick National Laboratory for Cancer Research, Frederick; and ∥Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD.

Objective: Macromolecular contrast agents for magnetic resonance imaging (MRI) are useful blood-pool agents because of their long systemic half-life and have found applications in monitoring tumor vasculature and angiogenesis. Macromolecular contrast agents have been able to overcome some of the disadvantages of the conventional small-molecule contrast agent Magnevist (gadolinium-diethylenetriaminepentaacetic acid), such as rapid extravasation and quick renal clearance, which limits the viable MRI time. There is an urgent need for new MRI contrast agents that increase the sensitivity of detection with a higher relaxivity, longer blood half-life, and reduced toxicity from free Gd3+ ions. Here, we report on the characterization of a novel water-soluble, derivatized, gadolinium-enclosed metallofullerene nanoparticle (Hydrochalarone-1) in development as an MRI contrast agent.

Materials And Methods: The physicochemical properties of Hydrochalarone-1 were characterized by dynamic light scattering (hydrodynamic diameter), atomic force microscopy (particle height), ζ potential analysis (surface charge), and inductively coupled plasma-mass spectrometry (gadolinium concentration). The blood compatibility of Hydrochalarone-1 was also assessed in vitro through analysis of hemolysis, platelet aggregation, and complement activation of human blood. In vitro relaxivities, in vivo pharmacokinetics, and a pilot in vivo acute toxicity study were also performed.

Results: An extensive in vitro and in vivo characterization of Hydrochalarone-1 is described here. The hydrodynamic size of Hydrochalarone-1 was 5 to 7 nm depending on the dispersing media, and it was negatively charged at physiological pH. Hydrochalarone-1 showed compatibility with blood cells in vitro, and no significant hemolysis, platelet aggregation, or complement activation was observed in vitro. In addition, Hydrochalarone-1 had significantly higher r1 and r2 in vitro relaxivities in human plasma in comparison with Magnevist and was not toxic at the doses administered in an in vivo pilot acute-dose toxicity study in mice.In vivo MRI pharmacokinetic analysis after a single intravenous injection of Hydrochalarone-1 (0.2 mmol Gd/kg) showed that the volume of distribution at steady state was approximately 100 mL/kg, suggesting prolonged systemic circulation. Hydrochalarone-1 also had a long blood half-life (88 minutes) and increased relaxivity, suggesting application as a promising blood-pool MRI contrast agent.

Conclusions: The evidence suggests that Hydrochalarone-1, with its long systemic half-life, may have significant utility as a blood-pool MRI contrast agent.
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http://dx.doi.org/10.1097/RLI.0b013e318294de5dDOI Listing
November 2013

Prediction of nanoparticle prodrug metabolism by pharmacokinetic modeling of biliary excretion.

J Control Release 2013 Dec 9;172(2):558-67. Epub 2013 May 9.

Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick Inc., Frederick National Lab for Cancer Research, Frederick, 21702, USA. Electronic address:

Pharmacokinetic modeling and simulation is a powerful tool for the prediction of drug concentrations in the absence of analytical techniques that allow for direct quantification. The present study applied this modeling approach to determine active drug release from a nanoparticle prodrug formulation. A comparative pharmacokinetic study of a nanoscale micellar docetaxel (DTX) prodrug, Procet 8, and commercial DTX formulation, Taxotere, was conducted in bile duct cannulated rats. The nanoscale (~40nm) size of the Procet 8 formulation resulted in confinement within the plasma space and high prodrug plasma concentrations. Ex vivo prodrug hydrolysis during plasma sample preparation resulted in unacceptable error that precluded direct measurement of DTX concentrations. Pharmacokinetic modeling of Taxotere and Procet 8 plasma concentrations, and their associated biliary metabolites, allowed for prediction of the DTX concentration profile and DTX bioavailability, and thereby evaluation of Procet 8 metabolism. Procet 8 plasma decay and in vitro plasma hydrolytic rates were identical, suggesting that systemic clearance of the prodrug was primarily metabolic. The Procet 8 and Taxotere plasma profiles, and associated docetaxel hydroxy-tert-butyl carbamate (HDTX) metabolite biliary excretion, were best fit by a two compartment model, with both linear and non-linear DTX clearance, and first order Procet 8 hydrolysis. The model estimated HDTX clearance rate agreed with in vitro literature values, supporting the predictability of the proposed model. Model simulation at the 10mg DTX equivalent/kg dose level predicted DTX formation rate-limited kinetics and a peak plasma DTX concentration of 39ng/mL at 4h for Procet 8, in comparison to 2826ng/mL for Taxotere. As a result of nonlinear DTX clearance, the DTX AUCinf for the Procet 8 formulation was predicted to be 2.6 times lower than Taxotere (775 vs. 2017h×ng/mL, respectively), resulting in an absolute bioavailability estimate of 38%. As DTX clearance in man is considered linear, this low bioavailability is likely species-dependent. These data support the use of pharmacokinetic modeling and simulation in cases of complex formulations, where analytical methods for direct measurement of free (released) drug concentrations are unavailable. Uses of such models may include interpretation of preclinical toxicology studies, selection of first in man dosing regimens, and PK/PD model development.
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http://dx.doi.org/10.1016/j.jconrel.2013.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788091PMC
December 2013