[89Zr]Oxinate4 for long-term in vivo cell tracking by positron emission tomography

Purpose 111In (typically as [111In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an 89Zr PET tracer for cell labelling and compare it with [111In]oxinate3 single photon emission computed tomography (SPECT). Methods [89Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [89Zr]oxinate4 or [111In]oxinate3 was monitored for up to 14 days. 89Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting. Results Zr labelling was effective in all cell types with yields comparable with 111In labelling. Retention of 89Zr in cells in vitro after 24 h was significantly better (range 71 to >90 %) than 111In (43–52 %). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with 111In or 89Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for 111In. In liver, spleen and bone marrow at least 92 % of 89Zr remained associated with eGFP-positive cells after 7 days in vivo. Conclusion [89Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types

Nanocolloidal albumin-IRDye 800CW: a near-infrared fluorescent tracer with optimal retention in the sentinel lymph node

Purpose: At present, the only approved fluorescent tracer for clinical near-infrared fluorescence-guided sentinel node (SN) detection is indocyanine green (ICG), but the use of this tracer is limited due to its poor retention in the SN resulting in the detection of higher tier nodes. We describe the development and characterization of a next-generation fluorescent tracer, nanocolloidal albumin-IRDye 800CW that has optimal properties for clinical SN detection Methods: The fluorescent dye IRDye 800CW was covalently coupled to colloidal human serum albumin (HSA) particles present in the labelling kit Nanocoll in a manner compliant with current Good Manufacturing Practice. Characterization of nanocolloidal albumin-IRDye 800CW included determination of conjugation efficiency, purity, stability and particle size. Quantum yield was determined in serum and compared to that of ICG. For in vivo evaluation a lymphogenic metastatic tumour model in rabbits was used. Fluorescence imaging was performed directly after peritumoral injection of nanocolloidal albumin-IRDye 800CW or the reference ICG/HSA (i.e. ICG mixed with HSA), and was repeated after 24 h, after which fluorescent lymph nodes were excised. Results: Conjugation of IRDye 800CW to nanocolloidal albumin was always about 50% efficient and resulted in a stable and pure product without affecting the particle size of the nanocolloidal albumin. The quantum yield of nanocolloidal albumin-IRDye 800CW was similar to that of ICG. In vivo evaluation revealed noninvasive detection of the SN within 5 min of injection of either nanocolloidal albumin-IRDye 800CW or ICG/HSA. No decrease in the fluorescence signal from SN was observed 24 h after injection of the nanocolloidal albumin-IRDye 800CW, while a strong decrease or complete disappearance of the fluorescence signal was seen 24 h after injection of ICG/HSA. Fluorescence-guided SN biopsy was very easy. Conclusion: Nanocolloidal albumin-IRDye 800CW is a promising fluorescent tracer with optimal kinetic features for SN detection. © The Author(s) 2012

Injektionsglottoplastik mit Fazia lata - Beschreibung einer modifizierten Injektionstechnik mit verbesserter Anwendungsgenauigkeit

Hintergrund: Die glottale Insuffizienz führt oft zu einer eingeschränkten Stimmfunktion mit eingeschränkter stimmlicher Belastbarkeit und/oder Ausbildung von muskulären Verspannungen im Hals- und Nackenbereich. Bei ausgeprägten Fällen bspw. mit begleitenden Sensibilitätsdefiziten können zudem Schluckbeschwerden vorliegen. Klinische Konstellationen, in denen die Stimmfunktion den individuellen Anforderungen des Patienten nicht gewachsen ist, sind typische Indikationen zur Injektionsglottoplastik. Hiermit kann durch Augmentierung des Volumens einer oder beider Stimmlippen eine effizientere und leistungsfähigere Stimmfunktion erreicht werden. Zur Injektion in die Stimmlippen kann u.a. autologe Faszia lata verwendet werden. Vorteile liegen in der vollständigen Bio-Kompatibilität sowie in der nicht notwendigen Überkorrektur. Beschrieben ist die Anwendung mittels Hoch-Druck-Injektionssystemen unter Verwendung von starren Injektionskanülen mit relativ großen Außendurchmessern bis zu 1,3 mm. Wenngleich das Faszien Material bspw. vom äußeren Oberschenkel relativ unkompliziert offen chirurgisch zu gewinnen ist, stellt die Injektion den Anwender vor einige technische Probleme: Die Platzierung der starren Kanüle in der individuell variablem glottischen Zielposition ist stark abhängig von der Einstellbarkeit; die Injektion mittels Hoch-Druck-Kanüle erlaubt nur die Abgabe definierter Einzelportionen von 0,1 ml und liefert kein haptisches Feedback.Material und Methoden: Das Poster beschreibt die Injektionsglottoplastik mit Fazia lata unter Verwendung einer 19 G Butterfly Nadel.Diskussion: Vorteile gegenüber der etablierten Methode bestehen in einer höheren Flexibilität der Nadelpositionierung, einem kleinen Nadelaußendurchmesser mit Vermeidung oder Reduktion eines Wiederaustritts des Materials aus dem Stichkanal und dem zusätzlich vorhanden haptischen Feedback des Gewebewiderstands beim Injizieren. Die Methode ist gleichermaßen geeignet für die Injektion von Eigenfett. Die zusätzliche Anschaffung wiederaufbereitbarer Komponenten entfällt.Fazit: Die Technik erlaubt eine Optimierung der plastischen Rekonstruktion und Modellierung der Stimmlippe mit verlässlicher Injektion und verbesserter lokaler Portionierung

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.We thank FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B., and 2013/25504-1 to A.C.B.B.), Xunta de Galicia (M.J.M.), FCT Portugal (FCT Investigator to G.J.L.B., IF/00624/2015), the EU (Marie Sklodowska-Curie ITN Protein Conjugates, GA 675007, including a PhD Studentship to X.F.), the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R and UNLR13-4E-1931 to F.C. and CTQ2015-70524-R and RYC-2013-14706 to G.J.O.) and the Universidad de La Rioja (FPI Studentship to I.C.). We also thank S. Massa and N. Devoogdt (Vrije Universiteit Brussel (VUM), Brussels) for the generous gift of the Her2-targeting nanobody 2Rb17c, Genentech for providing Thiomab LC-V205C and trastuzumab antibodies, and D. Neri’s laboratory (Swiss Federal Institute of Technology (ETH Zürich), Zurich) for the generous gift of the F16 antibody. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF/R/180019) and the recipient of a European Research Council Starting Grant (TagIt, GA 676832)