Name: an optimized protocol for the efficient radiolabeling of gold nanoparticles by using a 125i-labeled azide prosthetic group
Uploaded: 2016-10-10T15:00:00.000+00:00
Duration: 7 min 23 s
Description: Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

注意:放射性碘的氧化形式是相当挥发性的，且必须有足够的铅屏蔽和铅的小瓶处理。所有放射化学步骤应在通风良好的木炭过滤通风橱中进行，实验程序需要通过放射性检测装置进行监控。 1.化学品和反向相萃取柱的制备125 I标记的叠氮合成<ol><li> 在溶液试剂的制备 <ol><li>溶解1毫克150微升叠氮化物前体（2）无水乙醇（ 图1）。 注:叠氮化物前体的详细合成步骤（2）据报道，在以前的论文22。 </li><li>在20μl1×磷酸盐缓冲盐水（pH = 7.4）的溶解1毫克的氯胺T。 </li><li>溶解2毫克焦亚硫酸钠在20μlH 2 O </li></ol></li><li> Prepara墨盒化<ol><li>用10ml无水乙醇，然后用10毫升水2 O洗tC18柱不干燥与空气筒的矩阵。 </li></ol></li></ol> 2. 125 I标记的叠氮化辅基的放射合成<ol><li> 前体的放射性碘化反应 <ol><li>添加叠氮化物前体溶液（1毫克150微升绝对乙醇）和乙酸（10微升）到1.5ml微量离心管中。 </li><li>在0.1M的NaOH（50微升）添加的[125 I]碘化钠150活度到反应混合物中。 </li><li>添加氯胺T溶液（1mg在20μl1×磷酸盐缓冲盐水），并关闭含有微量离心管反应混合物。 </li><li>在室温下孵育该反应混合物15分钟，直到放射性碘化反应完成。 </li><li>一个焦亚硫酸钠溶液（2毫克在20μlH 2 O）加入到该反应混合物以猝灭放射性碘化反应。 </li><li>撤回0.2微升粗产物，然后用100微升的溶液稀释（H 2 O / CH 3 CN，1:1）进行HPLC分析。 注:对于所有的HPLC实验中，使用含有H 2 O（溶剂A）0.1％甲酸和0.1％甲酸含酸乙腈（溶剂B）作为洗脱剂。 </li><li>通过使用反相分析放射性HPLC（分析稀释粗产物C18反相柱;流速:1ml /分钟;洗脱剂梯度:0-2分钟，20％溶剂B，20-80％溶剂B为2-22分钟，为22-23分钟80-100％溶剂B，和100％溶剂B为23-28分钟;保留时间:16.4分钟）（ 图2）。 </li></ol></li><li> 用制备HPLC纯化粗产物 注意:提供围绕型HPLC份足够铅屏蔽诸如喷射器，柱，检测器，收集瓶，并在其中流出物被收集在容器。 <ol><li>第撤Ë整个反应混合物倒入HPLC小瓶。冲洗，用乙腈（0.5毫升）的反应管中，加入漂洗到相同的注射小瓶中。稀释用H 2 O（1毫升）中收集的溶液。 </li><li>注入粗产物到制备放射性HPLC（C18反相柱;流速:10ml /分钟;洗脱剂梯度:20％溶剂B为0-2分钟，20-80％溶剂B为2-22分钟，为22-23分钟80-100％溶剂B，以及用于23-28分钟100％溶剂B）。 </li><li>收集表示125 I标记的叠氮化物（1）的放射性峰（ 叔 R这些HPLC条件下是17.8-18.8分钟）中的玻璃试管（ 图2）。 </li><li>测量使用根据制造商的协议的放射性剂量校准馏分的放射化学产率。 </li><li>注入纯化产品上使用，用于确定产物的放射化学纯度的相同HPLC条件分析放射性HPLC。 </li></ol></li><li> 固相萃取的产物 <ol><li>稀释含用40ml纯H 2 O所需的产物（1）的级分</li><li>添加稀释的溶液成预处理tC18柱。 </li><li>用另外的15毫升水2 O洗盒</li><li>洗脱被困在盒用2毫升丙酮到由一铅屏蔽保护的10-ml玻璃管制瓶内的产品（1）。测量使用根据制造商的协议的放射性剂量校准洗脱产物的放射性。 注:二甲基亚砜（DMSO）或无水乙醇也可用于从盒中的产品的溶出。放射性的约5-10％，通常粘附在盒中，而剩余的放射性标记的产物不能通过使用过量的有机溶剂完全洗脱。 </li><li>蒸发用氮气或氩气的气流的丙酮。 </li><li>化解重用DMSO（100-200微升）为下放射性标记步骤sidue。 </li></ol></li></ol> 3. DBCO群结合的金纳米粒子的合成<ol><li> 13纳米大小的金表面改性与DBCO含基的聚乙二醇纳米粒 <ol><li>制备钠-柠檬酸盐稳定的金纳米粒子（3）（平均粒度= 13纳米）根据先前的报告24。 </li><li>添加吐温20（1毫米，1.5毫升）的水溶液的柠檬酸盐稳定的金纳米粒子（10纳米，15毫升）。摇上轨道摇床进行20分钟的溶液中。 </li><li>添加DBCO含有基团的聚乙二醇硫醇（平均分子量= 5,000，100μM，1.5毫升）的水溶液。摇上轨道摇床2小时的溶液中。 </li></ol></li><li> 所述DBCO团改性金纳米颗粒的纯化 <ol><li>净化DBCO团改性的金纳米粒子（4）&lt;/ STRONG&gt;是通过连续离心（11400 XG，15分钟×3）。 </li><li>倒出上清液并加入纯水金纳米颗粒粒料再悬浮。 </li></ol></li></ol> 4.通过无铜点击反应DBCO团改性金纳米粒子的放射性标记<ol><li> 使用125 I标记的叠氮化物125 I标记的金纳米颗粒的合成（1） <ol><li>通过使用离心分离（11400 xg离心，15分钟）制备DBCO团改性金纳米颗粒的浓溶液，并调整金纳米颗粒的浓度为2μM。 </li><li>添加在DMSO（5微升）的125 I标记的叠氮化物（1）的4.1活度的悬浮液金纳米粒子（4）（2μM，50微升）。 </li><li>孵育在40℃下将所得反应混合物60分钟。 </li><li>从粗产物撤回等分试样（0.2微升），并将其应用到二氧化硅 - 共ated薄层色谱（TLC）板。 </li><li>开发使用乙酸乙酯作为流动相的TLC板上。 </li><li>放置在无线电型TLC扫描器TLC板上并运行扫描器监视根据制造商的方案的放射性标记反应（ 图3）。 </li></ol></li><li> 粗产物的纯化 <ol><li>净化含有125 I标记的金纳米颗粒（4）通过离心（11400 xg离心，15分钟），将反应混合物。 </li><li>倒出上清液并加入纯水金纳米颗粒粒料再悬浮。 </li><li>从纯化的产物撤回等分试样（0.2微升），并将其应用到一个二氧化硅包覆TLC板上。 </li><li>开发使用乙酸乙酯作为流动相的TLC板上。 </li><li>放置在无线电型TLC扫描器TLC板上并运行扫描器，以确定125 I标记勒的放射化学产率和放射化学纯度ð纳米颗粒（4）（ 图3）根据制造商的协议。 </li></ol></li></ol>

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The authors have nothing to disclose.

在一般情况下，纯化的125 I标记的叠氮化物（1）所观察到的放射化学产率为75±10％（N = 8）。放射性标记用放射性50-150活度完成，放射化学的结果是相当一致的。如果[125 I]碘化钠（T 1/2 = 59.4 D）将经历放射性衰变了一个多月的放射性碘化反应时，观察到1放化收益率将略有下降（53-65％）。因此，我们建议，因为它产生的[125 I]碘化钠?...

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

<table><tbody><tr><td>Chloramine T trihydrate</td><td>Sigma</td><td>402869</td><td></td></tr><tr><td>[&lt;sup&gt;125&lt;/sup&gt;I]NaI in aq. NaOH</td><td>Perkin-Elmer</td><td>NEZ033A010MC</td><td></td></tr><tr><td>Sodium metabisulfite&amp;nbsp;</td><td>Sigma</td><td>S9000</td><td></td></tr><tr><td>Formic acid</td><td>Sigma</td><td>251364</td><td></td></tr><tr><td>Sep-Pak tC18 plus cartridge</td><td>Waters</td><td>WAT036800</td><td></td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma</td><td>520918</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>DBCO PEG SH (MW 5,000)</td><td>NANOCS</td><td>PG2-DBTH-5k</td><td></td></tr><tr><td>TLC silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck</td><td></td><td></td></tr><tr><td>Analytical HPLC</td><td>Agilent</td><td>1290 Infinity</td><td>Model number</td></tr><tr><td>Preparative HPLC</td><td>Agilent</td><td>1260 Infinity</td><td>Model number</td></tr><tr><td>Analytical C18 reverse-phase column</td><td>Agilent</td><td>Zorbax Eclipse XDB-C18</td><td></td></tr><tr><td>Preparative C18 reverse-phase column</td><td>Agilent</td><td>PrepHT XDB-C18</td><td></td></tr><tr><td>Radio TLC scanner</td><td>Bioscan</td><td>AR-2000</td><td>Model number</td></tr><tr><td>Radioisotope dose calibrator</td><td>Capintec, Inc</td><td>CRC -25R dose calibrator</td><td>Model number</td></tr></tbody></table>

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.

所述stannylated前体（2）的放射性碘化反应进行了使用[125 I]碘化钠，乙酸，和氯胺T 150活度在室温下15分钟，以提供放射性标记的产物（1）。粗混合物的制备HPLC纯化后，用放射化学产率的75±10％（N = 8）中得到所需产物。分析性HPLC显示，125 I标记产物的放射化学纯度是99％以上（ 图2），和产品1所观察到的?...

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

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

对于一个125 I标记的叠氮化物的合成和dibenzocyclooctyne（DBCO） -基共轭，13纳米大小的金使用无铜点击反应的纳米粒子的放射性标记的详细过程进行说明。

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

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

an-optimized-protocol-for-efficient-radiolabeling-gold-nanoparticles

Universidad San Sebastian

Research

JoVE Journal

Chemistry

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

8.1K 次观看.  Korea Atomic Energy Research Institute. 对于一个125 I标记的叠氮化物的合成和dibenzocyclooctyne（DBCO） -基共轭，13纳米大小的金使用无铜点击反应的纳米粒子的放射性标记的详细过程进行说明。 

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

科研

生物化学

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.

该生物耦合和重新布置<sup&gt; 89</sup&gt;锆DFO标记的抗体

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

化学

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.

利用生物正交反电子需求的Diels-Alder环的预靶向PET成像

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

生物工程

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.

基于逆电子需求函数-alder 反应的隐形放射免疫治疗

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

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

开发用于对程序性死亡配体 1 表达进行成像的 68镓标记的 D 肽 PET 示踪剂

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

癌症研究

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.

细胞运输的正电子发射断层扫描成像:一种细胞放射性标记方法

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

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

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).

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

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

免疫与感染

<meta charset="utf8"></head><body>使用 Loop 方法甲基化高效合成碳 11 放射性示踪剂

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

<meta charset="utf8"></head><body>使用 Loop 方法提高用于临床研究的碳 11 放射性配体的效率和放射性标记产量

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

使用 Loop 方法提高用于临床研究的碳 11 放射性配体的效率和放射性标记产量

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

一个优化的协议通过使用金纳米粒子的放射性标记效率

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Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

小鼠淋巴细胞通过标签

使用 Loop 方法提高用于临床研究的碳 11 放射性配体的效率和放射性标记产量

使用 Loop 方法提高用于临床研究的碳 11 放射性配体的效率和放射性标记产量