Name: preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry
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Description: Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

Los estudios con animales fueron aprobados por el Consejo de Ética de Experimentación Animal de la Universidad McMaster, de acuerdo con el Consejo Canadiense de Protección de los Animales (CCPA) directrices. 1. El radiomarcado de ligandos Tz-tridentado con 99m Tc PRECAUCIÓN: Los siguientes procedimientos requieren el uso de compuestos radiactivos. El trabajo sólo debe hacerse en un laboratorio autorizado con el cumplimiento de las normas de seguridad y eliminación. reacciones de microondas se deben realizar en un horno de microondas diseñado específicamente para la síntesis química. <ol><li> Síntesis de [99m Tc (CO) 3 (H2O) 3] + 19, 20 <ol><li> En un vial para microondas, combinar 8 mg K 2 [BH 3 CO 2], 15 mg Na 2 CO 3, 20 mg Na 2 B 4 O 7 · 10H 2 O, y 25 mg KOCO [CH (OH)]2 COONa · 4H 2 O. Purgar el vial durante 10 minutos con gas argón. </li><li> Añadir 4 ml de 99m TcO 4 - (~ 1.100 MBq, ~ 30 mCi) en 0,9% de solución salina al vial. </li><li> La reacción se calienta en un microondas durante 3,5 min a 110 ° C después de 10 s de agitación para asegurar la mezcla completa de los reactivos. </li><li> Ajustar el pH de la solución a 3,5 a 4 utilizando ~ 400 l de HCl 1 M. Comprobar el uso de papel de pH. </li></ol></li><li> El radiomarcado de tetrazina ligandos 1-5 <ol><li> Disolver 2 mg de cada ligando (compuestos 1-5) en 250 ml de MeOH 21. </li><li> Añadir 250 l de [99m Tc (CO) 3 (H2O) 3] + (~ 74 MBq, ~ 2 mCi) a cada solución. </li><li> Se calienta la mezcla de reacción usando un horno de microondas durante 20 min a 60 ° C. NOTA: Este paso fue idéntica para los 5 tetrazinas. </li><li> Para los compuestos 2 a 5, se evapora el disolvente y se re-DISSOLve los productos resultantes en 1 ml de 1: 1 v / v DCM: TFA. </li><li> Calentar los productos de reacción disueltos (2-5) a 60 ° C en un microondas durante 6 min (2-4) o 10 min (5). </li><li> Después de enfriar a temperatura ambiente, se evapora el disolvente usando un evaporador (36 ° C, 8 mbar, 3 min, 6000 rpm) y se disuelve el compuesto se seca en 1: 1 ACN: H 2 O o 1: 1 MeOH: H 2 O, antes de la purificación por HPLC. </li><li> Purificar los compuestos marcados con Tc 99m (1-5), que incluyen la separación del producto etiquetado de ligando no marcado tetrazina, usando HPLC (C18 de fase inversa). Típicamente, usar un gradiente de elución de 30:70 ACN: H 2 O (ambos con 0,1% de TFA) a 40:60 ACN: H 2 O durante 20 min (18 min) y una columna de 4,6 x 100 mm analítica C 18. Usar tanto UV (254 nm) y detección gamma. <ol><li> Tomar una pequeña muestra de cada producto marcado y comparar su tiempo de retención HPLC a la de un co-injeja, no radiactivo, estándar marcado con Re (0,125 mg en 20% de metanol-H 2 O). El estándar Re-marcado se identifica en la traza de HPLC UV, y eluir en el mismo tiempo que el compuesto marcado con Tc 99m en la traza de γ-HPLC. Esta co-inyección de muestra picos a tiempos de retención comparables, lo que confirma la identidad del compuesto 99m Tc-etiquetados. </li></ol></li><li> Se evapora el disolvente de las fracciones de HPLC utilizando un evaporador (36 ° C, 8 mbar, 3 min, 6000 rpm). </li><li> Formular el compuesto purificado a una concentración de 7,4 kBq / l en PBS, que contenía 0,5% de BSA y 0,01% de Tween-80. </li><li> Para garantizar los compuestos marcados son estables, realice un estudio in vitro de la estabilidad en. Incubar el compuesto formulado a 37 ° C durante 1, 4 y 6 h, la inyección de una pequeña cantidad (3,7 MBq) de la mezcla en la HPLC en cada punto de tiempo para evaluar la estabilidad. </li></ol></li></ol> 2. Pre-Estudios dirigidos biodistribución <ol><li&gt; Preparación de los animales <ol><li> Utilizando, ratones Balb / c hembras de 7-9 semanas de edad (n = 3), administrar TCO-BP formulado en solución salina (20 mg / kg) (5 mg / l), a través de la cola de inyección venosa. </li><li> Coloque el ratón en el dispositivo de restricción física, e identificar las venas situadas en las superficies laterales de la cola y limpie con un algodón con alcohol. En aproximadamente 2 cm desde el extremo de la cola, insertar una aguja de calibre 30 con un ángulo pequeño, en paralelo a la vena. Presione lentamente el émbolo para inyectar, retire la aguja y aplicar esponja de gasa limpia en el sitio de la inyección con una ligera presión hasta que el sangrado se detenga. </li><li> A 1 h después de la inyección de TCO-BP, administrar ~ 0,74 MBq (20 Ci) de Tc-99m tetrazina formulados en 100 l de BSA al 0,5%, 0,01% de Tween-80 en PBS, a través de la cola de inyección venosa. </li></ol></li><li> Los estudios de biodistribución <ol><li> En el punto de tiempo deseado (t = 6 h), anestesiar los ratones utilizando 3% de isoflurano y mezcla de gases de oxígeno 2%. Demostrar una dee pizca de retirada en el ratón anestesiado para asegurarse de que están bajo plano quirúrgico de anestesia. </li><li> Recoger la sangre (1 ml) mediante punción cardíaca usando una jeringa pre-tratado con heparina. Coloque el ratón sobre su espalda con la nariz en el cono de la nariz para la anestesia continua y localizar la apófisis xifoides en el animal. <ol><li> Insertar una aguja de 25 G, ligeramente a la izquierda de la línea media del animal en el marco del proceso xifoides, en un ángulo de 20 °. Inserte completamente la aguja y tire lentamente el émbolo hacia atrás para ver la sangre en el cubo de la aguja si se perfora el corazón. Ligeramente reajustar la aguja mientras mantiene el émbolo en caso necesario, para perforar el corazón. extraer lentamente sangre en la jeringa. </li></ol></li><li> La eutanasia a los animales por dislocación cervical, mientras que bajo anestesia. </li><li> Coloque cada animal en una bolsa de plástico y utilizar un calibrador de dosis (99m Tc configuración) para medir el nivel de actividad todo el cuerpo. </li><li> Recoge los siguientes tejidos y fluidos pre-pesajeed contando tubos: sangre, médula (rodilla y hombro), la vesícula biliar, los riñones, el hígado, el estómago (con su contenido), los intestinos pequeños (con su contenido), intestino grueso y ciego (con su contenido), la tiroides y la tráquea, vejiga urinaria con orina , y la cola. </li><li> Enjuague tejidos apropiados (con exclusión de la sangre, la vesícula biliar y la vejiga urinaria) en PBS para eliminar la sangre y secar antes de colocar los tejidos en tubos de recuento apropiados. </li><li> Coloque cadáver animal en una bolsa de plástico y medir la actividad de todo el cuerpo residual utilizando un calibrador de dosis. </li><li> Pesar cada tubo que contiene una muestra de tejido. Restar el peso inicial del tubo para obtener la masa del tejido. </li><li> Utilice un calibrador de dosis (99m Tc ajuste) para medir la cantidad de actividad en una muestra de ensayo (100 l) en el momento de la inyección para cada ratón. NOTA: Este muestra de ensayo es igual al volumen de inyección, dando así el recuento de actividad en el momento de la inyección. </li><li> En el momento de la medición de tejido, unaliquot 5 l de la muestra de ensayo utilizado anteriormente. Use un contador gamma multi-detector (99m Tc configuración) y contar hasta obtener el recuento por minuto (CPM) para la muestra 5 mL. </li><li> Usa los dos valores obtenidos en 2.2.9 y 2.2.10 para calcular la actividad y la relación CPM utilizando la ecuación 1 para obtener un factor de conversión (CPM Ci -1). (1) <img alt="Ecuación 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li> Utilice el contador gamma para medir la cantidad de radiactividad en cada muestra de tejido o fluido. </li><li> Usar la ecuación 1 para calcular la cantidad de actividad en cada tejido o fluido en el momento de la medición en relación con la dosis total inyectada. Este valor se normalizó por el peso de órganos y se expresa como porcentaje de dosis inyectada por gramo (es decir,% ID / g) de tejido. </li><li> Siga los pasos 2.1.2 a 2.2.13 para llevar a cabo un experimento de control negativo utilizando las marcado con 99mTc ligandos tetrazina en el Absencina de TCO-BP. ratones Sacrificio (n = 3) a las 0,5, 1, 4 y 6 h después de la inyección y obtener tejido o fluido como se describe anteriormente. </li></ol></li></ol>

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The authors declare they have no competing financial interests.

Se preparó una colección de quelatos tridentado tetrazina ligados de distintas polaridades, y se evaluó la utilidad de sus complejos 99m Tc en la reacción con un derivado IEDDA TCO in vivo. Un método eficaz y reproducible 99m Tc fue desarrollado para cinco tetrazina quelatos, donde la concentración de ligando se 10 -3 M. La etapa de marcaje fue seguido por la desprotección de t- butilo (para los compuestos 2-5). La alta concentración de ligando ...

99m Tc sigue siendo el radioisótopo más utilizado en medicina nuclear de diagnóstico, con más de 50 millones de procedimientos de imágenes realizadas por año en todo el mundo 1, 2, 3. La mayoría de los agentes de Tc 99m usados clínicamente son de tipo perfusión radiofármacos. Hay un número limitado de compuestos dirigidos activamente en la que 99m Tc se dirige para unir un marcador biológico específico a través de la ligación a un constructo de reconocimiento. La creación de 99m dirigida radiofármacos Tc es a menudo dificultada por la influencia de 99m complejos de Tc-ligando en la capacidad de la molécula de direccionamiento de obligar a la biomarcador de interés, o los isótopos de vida media no es lo suficientemente largo para su uso con biomoléculas de mayor peso molecular tales como anticuerpos. Este último requiere típicamente varios días antes de las imágenes se adquieren a fin de que la biomolécula para borrar de no diana tiss<html> UES. Pre-focalización ofrece un enfoque alternativo para superar estos desafíos. Pre-orientación combinada con la química bioorthogonal ha demostrado ser una forma eficaz para desarrollar nuevas sondas de imagen molecular tanto para fluorescencia y radio-formación de imágenes 4, 5, 6, 7, 8. La reacción de la demanda de electrones inversa Diels-Alder (IEDDA) entre 1,2,4,5-tetrazina (Tz) y derivados -cyclooctene trans (TCO), tal como se muestra en la Figura 1, ha demostrado ser particularmente eficaz 6. La reacción IEDDA con estos componentes puede exhibir cinética rápida en PBS (2 k ≈ 6.000 M -1 s -1) y una alta selectividad, por lo que es ideal para in vivo de orientación previa aplicaciones 9, 10. e_content &quot;&gt; El enfoque más común utilizado implica la administración de un vector de orientación TCO-derivado y después de un período de retardo suficiente, se administra una tetrazina radiomarcado. tetrazinas radiomarcados basado en 11 C, 18 F, 64 Cu, 89 Zr, y 111 En haber sido reportado 11, 12, 13, 14, 15. por el contrario, sólo hay un informe de un marcado con 99mTc Tz, que se preparó usando un ligando de tipo HYNIC requerir el uso de co-ligandos para prevenir la unión a proteínas y la degradación in vivo 16. Como alternativa, se presenta aquí la síntesis de 99m Tc (I) marcado tetrazinas usando una familia de ligandos que forman complejos estables con un tridentados [99m Tc (CO) 3] + núcleo. Figura 1: La reacción entre IEDDA bioorthogonal tetrazina y -cyclooctene trans. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> La familia de ligandos preparadas contienen quelatos tridentados que varían en la polaridad y la naturaleza del grupo de enlace entre la región de unión de metal y el Tz (Figura 2). El objetivo fue identificar una Tc-99m tetrazina constructo que podría localizar y reaccionar con los sitios marcados con TCO en vivo y rápidamente claro cuando no estén unidas de manera efectiva, a fin de producir alta objetivo a no objetivo proporciones. Para probar los ligandos, se utilizó un TCO-derivado de un bisfosfonato (TCO-BP) 17. Hemos demostrado previamente que el TCO-BP se localiza en áreas de metabolismo óseo activo y puede reaccionar contetrazinas radiomarcados en vivo 18. Es un reactivo conveniente para probar nuevos tetrazinas, ya que se puede preparar en un solo paso y los experimentos se puede realizar en los ratones normales, donde la localización se produce principalmente en las articulaciones (rodillas y hombros).

<table><tbody><tr><td>Argon gas</td><td>Alphagaz</td><td>---</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD Millipore</td><td>106395</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#183;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>S9640</td><td>---</td></tr><tr><td>KNaC&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;&amp;#183;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>217255</td><td>---</td></tr><tr><td>Technelite &lt;sup&gt;99m&lt;/sup&gt;Tc generator</td><td>Lantheus medical imaging</td><td>---</td><td>Source of &lt;sup&gt;99m&lt;/sup&gt;TcO&lt;sup&gt;4-&lt;/sup&gt;</td></tr><tr><td>0.9% Saline</td><td>Lantheus medical imaging</td><td>---</td><td>To elute generator</td></tr><tr><td>1 M HCl</td><td>Lab Chem</td><td>---</td><td>---</td></tr><tr><td>MeOH</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>ACN</td><td>Caledon</td><td>---</td><td>HPLC grade</td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>Barnstead Nanopure</td><td>---</td></tr><tr><td>DCM</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>TFA</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH 7.4 1x</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>---</td></tr><tr><td>Tween80</td><td>Sigma Aldrich</td><td>P8047</td><td>---</td></tr><tr><td>Isoflurane</td><td>CDMV</td><td>108737</td><td>Supplier: Fresenius Kabi Animal Health</td></tr><tr><td>HPLC</td><td>Waters</td><td>1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td><td>---</td></tr><tr><td>HPLC column for analysis and purification of compounds 2-4</td><td>Phenomenex</td><td>00G-4435-E0</td><td>Gemini&amp;reg; 5 &amp;micro;m C18 110&amp;nbsp;&amp;Aring;, LC Column 250 x 4.6 mm</td></tr><tr><td>HPLC column for analysis and purification of compounds 1 and 5</td><td>Waters&amp;nbsp;</td><td>186003115</td><td>XBridge BEH C18 Column, 130 &amp;Aring;, 5 &amp;micro;m, 4.6 mm x 100 mm</td></tr><tr><td>Microwave Reactor&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>Initiator 8</td><td>---</td></tr><tr><td>Biotage V10 Evaporator</td><td>Biotage&amp;nbsp;</td><td>Serial # V1041</td><td>---</td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.&amp;nbsp;</td><td>CRC-25R</td><td>---</td></tr><tr><td>Gamma counter</td><td>Perkin Elmer</td><td>Wizard 1470 Automatic Gamma Counter</td><td>---</td></tr><tr><td>Animal room scale&amp;nbsp;</td><td>Mettler Toledo</td><td>XP105 Delta Range</td><td>---</td></tr><tr><td>Microwave vials&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>355629</td><td>0.5-2 mL&amp;nbsp;</td></tr></tbody></table>

preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Los ligandos se sintetizaron utilizando diferentes enlazadores y quelantes a través de una simple estrategia de aminación reductora (Figura 2), seguido de acoplamiento del producto a un tetrazina disponible en el mercado 22, 23. El radiomarcado se realizó utilizando el mismo método para todos los compuestos y era altamente reproducible. El proceso se ha optimizado variando el pH, la cantidad de ligando, tiemp...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Preparación y Evaluación de<sup&gt; 99m</sup&gt; Tc-etiquetados tridentado Los quelatos de orientación previa Usando Bioorthogonal Química

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Universidad San Sebastian

Research

JoVE Journal

Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

8.1K vistas.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

Video: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Microfluidic Reacción en el chip de captura-cicloadición para inmovilizar reversiblemente pequeñas moléculas o estructuras de múltiples componentes para aplicaciones de biosensores

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Investigación

Química

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

El bioconjugación y Radiosíntesis de<sup&gt; 89</sup&gt; Los anticuerpos marcados con Zr-DFO

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Un protocolo optimizado para el radiomarcaje eficaz de nanopartículas de oro mediante el uso de una<sup&gt; 125</sup&gt; Azida grupo prostético marcado con I

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

El aprovechamiento de la demanda Bioorthogonal Inverse Electron Diels-Alder Cicloadición para Pretargeted TEP

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioingeniería

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imagen Molecular del cáncer de próstata con un agente de contraste dirigidos Pequeño Molecular CLT1 Péptido

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicina

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

Radioinmunoterapia pretargeted basada en la reacción de Diels-Alder de demanda electrónica inversa

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Síntesis y Bioconjugation de tioles reactivos reactivos para la creación de sitio selectivamente modificados Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Tomografía por emisión de positrones Imágenes del tráfico celular: un método de radiomarcaje celular

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Investigación sobre el cáncer

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Evaluación inicial de los conjugados de anticuerpos modificados con péptidos virales derivados para aumentar la acumulación celular y mejorar Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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