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.

주의 : 방사성 요오드의 산화 된 형태가 매우 휘발성 적절한 리드 방패와 리드 유리 병 취급해야합니다. 모든 방사 단계는 통풍이 잘되는 숯을 여과 후드에서 수행 한 실험 절차는 방사능 검출 장치에 의해 모니터링해야한다. I 125 - 표지 된 아 지드의 합성 화학 및 역상 카트리지 (1)의 제조 <ol><li> 용액의 시약의 조제 <ol><li> 150 μL의 아 지드 전구체 (2) 무수 에탄올 (그림 1)의 1 mg의 용해. 참고 : 아 지드 전구체에 대한 자세한 합성 절차 (2) 이전의 종이 (22)에보고되었다. </li><li> 1X 인산 완충 생리 식염수 (pH는 7.4) 20 μL를 1 mg을 클로라민 T를 녹인다. </li><li> 20 μL의 H 2 O 2 mg의 나트륨 메타 비설 파이트를 녹여 </li></ol></li><li> Prepara카트리지 기 <ol><li> 10 ㎖의 H 2 O이어서 10 ㎖의 무수 에탄올로 TC18 카트리지 워시 공기와 카트리지의 행렬을 건조하지 마십시오. </li></ol></li></ol> 125 I 표지 아 지드 보철 그룹의 2 Radiosynthesis <ol><li> 전구체의 방사성 요오드화 반응 <ol><li> 1.5 ml의 마이크로 원심 튜브에 지드 전구체 용액 및 아세트산 (10 μL) (무수 에탄올 150 ㎕의 1 mg)을 추가한다. </li><li> 반응 혼합물에 0.1 M의 NaOH (50 μL)에 [125 I]의 NaI 150 MBq의 추가. </li><li> 클로라민 T 용액 (1X 인산 완충 식염수 20 ㎕의 1 mg)을 첨가하고 튜브를 마이크로 원심 함유하는 반응 혼합물을 닫는다. </li><li> 방사성 요오드화 반응이 완료 될 때까지 실온에서 15 분간 반응 혼합물을 인큐베이션. </li><li> 반응 혼합물에 소듐 메타 바이 설 파이트 용액 (2 ㎎을 20 μL의 H 2 O)를 추가방사성 요오드화 반응을 해소합니다. </li><li> 조 생성물을 0.2 μl를 인출 한 후 용액을 100 ㎕로 희석 (H 2 O / CH 3 CN, 1 : 1) HPLC 분석. 참고 : 모든 HPLC 실험의 경우, H 2 O (용매 A)를 포함하는 0.1 % 포름산 및 이동상 0.1 % 포름산 함유 아세토 니트릴 (용매 B)를 사용합니다. </li><li> 칼럼 상 반전 (역상 분석 무선-HPLC를 이용하여 희석하고, 조 생성물을 C18 분석; 유속 : 1 ml / 분 용리액 구배 : 0-2 분 동안 20 % 용매 B, 20 % ~ 80 % 용매 B에 대한 2-22 분, 22 ~ 23 분 80~100% 용매 B, 그리고 23-28 분 동안 100 % 용매 B, 체류 시간 : 16.4 분) (그림 2). </li></ol></li><li> 분취 HPLC로 조 생성물을 정제하여 주 : 이러한 주입기, 컬럼, 검출기 수집 튜브 및 폐수 수집 된 컨테이너 HPLC 부품 주위에 충분한 납 차폐를 제공한다. <ol><li> 일 철수HPLC 바이알에 전체 반응 혼합물을 전자. 아세토 니트릴 (0.5 mL)로 반응 관을 씻어 동일한 주입 병에 린스를 추가합니다. H 2 O (1 mL)로 수집 된 용액을 희석. </li><li> (조제 무선 HPLC 상에 주입하여 조 생성물을 C18 컬럼 상 역방향 유속 : 10 ㎖ / 분 용리액 구배 : 2-22 분 동안 0-2 분, 20 % ~ 80 % 용매 B 20 % 용매 B를 22 ~ 23 분 동안 80~100% 용매 B, 그리고 23-28 분 동안 100 % 용매 B). </li><li> I 125 - 표지 지드 (1)를 나타내는 피크 방사능 수집을 유리 시험관에 (도 2) (이들의 HPLC 조건에서의 t R은 17.8-18.8 분이다). </li><li> 제조사의 프로토콜에 따라 방사선 도즈 교정기를 사용하여 분획의 방사 화학적 수율을 측정한다. </li><li> 제품의 방사 화학적 순도를 결정하기위한 동일한 HPLC 조건을 사용하여 분석 무선 HPLC로 정제 된 생성물을 주입한다. </li></ol></li><li> 제품의 고체상 추출 <ol><li> 40ml의 순수 H 2 O로 원하는 생성물 (1)을 포함하는 분획을 희석 </li><li> 컨디셔닝 TC18 카트리지에 희석액을 추가합니다. </li><li> 추가로 15 ML의 H 2 O와 카트리지를 세척 </li><li> 리드 방패에 의해 보호되는 10 ml의 유리 바이알에 2 ㎖의 아세톤으로 카트리지에 갇혀 제품 (1) 용출. 제조사의 프로토콜에 따라 방사선 도즈 교정을 사용하여 용출 된 생성물의 방사능을 측정한다. 주 : 디메틸 설폭 사이드 (DMSO) 또는 무수 에탄올 또한 카트리지로부터의 생성물의 용출을 위해 사용될 수있다. 방사능의 약 5-10 %는 보통 카트리지 스틱, 남은 방사선 표지 생성물은 완전히 유기 용매의 과잉을 사용하여 용출 될 수 없다. </li><li> 질소 또는 아르곤 가스의 흐름과 함께 아세톤을 증발시켰다. </li><li> 재를 녹여다음 방사성 표지 단계에 대한 DMSO (100-200 μL)와 sidue. </li></ol></li></ol> DBCO 기 - 금 나노 입자 컨쥬 게이트 (3)의 합성 <ol><li> 13 나노 크기의 금 표면 변형 DBCO 기 함유 폴리에틸렌 글리콜 나노 입자 <ol><li> 준비 나트륨 구연산 안정화 된 금 나노 입자 (3) (평균 크기 = 13 ㎚) 이전의 보고서 (24)에 따라. </li><li> (10 nM 내지 15 mL) 중 시트 레이트 - 안정화 된 금 나노 입자에 트윈 20 (1 ㎜, 1.5 ㎖)의 수용액을 추가한다. 궤도 통에 20 분의 솔루션을 흔들어. </li><li> DBCO 기 함유 폴리에틸렌 글리콜 티올 (평균 분자량 = 5,000, 100 μM, 1.5 ㎖)의 수용액을 추가한다. 오비탈 진탕 기에서 2 시간 동안 용액을 흔들어. </li></ol></li><li> DBCO 그룹 - 개질 된 금 나노 입자의 정제 <ol><li> 정제 DBCO 그룹 - 개질 된 금 나노 입자 (4) &lt;/ 연속 원심 분리 (11,400 XG, 15 분 × 3)로&gt; 강한. </li><li> 뜨는을 가만히 따르다 및 금 나노 입자 펠릿의 재 부유 순수을 추가합니다. </li></ol></li></ol> 4. 구리 무료 클릭 반응을 통해 DBCO - 그룹 - 개질 된 금 나노 입자의 방사성 표지 <ol><li> I 125 - 표지 된 아 지드를 사용하여 125 I 표지 된 금 나노 입자의 합성 (1) <ol><li> 원심 분리 (11,400 × g으로 15 분간)을 사용하여 DBCO 그룹 - 개질 된 금 나노 입자의 농축 용액을 제조하고, 2 μM로 금 나노 입자의 농도를 조절한다. </li><li> 금 나노 입자의 현탁액을 DMSO (5 μL)의 125I 표지 지드 (1) 4.1 MBq의 추가 (4) (2 μM, 50 μL). </li><li> 60 분 동안 40 ° C에서 얻어진 반응 혼합물을 인큐베이션. </li><li> 조 생성물로부터 분취 량 (0.2 μL)를 인출하고, 실리카 - 코에 적용ated 박층 크로마토 그래피 (TLC) 판. </li><li> 이동상으로서 에틸 아세테이트를 사용하여 TLC 판을 개발한다. </li><li> 전파 TLC 스캐너의 TLC 플레이트를 배치하고, 제조자의 프로토콜에 따라 방사성 표지 반응 (도 3)을 감시하는 스캐너를 실행. </li></ol></li><li> 조 생성물을 정제하여 <ol><li> 원심 분리하여 125 I 표지 된 금 나노 입자 (4) (11,400 × g으로 15 분간)를 포함하는 반응 혼합물을 정제 하였다. </li><li> 뜨는을 가만히 따르다 및 금 나노 입자 펠릿의 재 부유 순수을 추가합니다. </li><li> 정제 된 제품에서 나누어지는 (0.2 μl를) 철회 및 실리카 코팅 된 TLC 판에 적용합니다. </li><li> 이동상으로서 에틸 아세테이트를 사용하여 TLC 판을 개발한다. </li><li> 무선-TLC 스캐너에 TLC 판을 놓고 125I 표지 GOL의 방사 화학적 수율 및 방사 화학적 순도를 결정하기 위해 스캐너를 실행제조 업체의 프로토콜에 따라 D 나노 입자 (4) (그림 3). </li></ol></li></ol>

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	<li>Jewett, J. C., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cu-free+Click+Cycloaddition+Reactions+in+Chemical+Biology.">Cu-free Click Cycloaddition Reactions in Chemical Biology.</a> Chem. Soc. Rev. 39, 1272-1279 (2010).</li><li>Debets, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioconjugation+with+Strained+Alkenes+and+Alkyne.">Bioconjugation with Strained Alkenes and Alkyne.</a> Acc. Chem. Res. 44, 805-815 (2011).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Mechanism+to+Mouse:+A+Tale+of+Two+Bioorthogonal+Reactions.">From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions.</a> Acc. Chem. Res. 44, 666-676 (2011).</li><li>Koo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+Cu-Free+Click+Chemistry+in+vivo+for+Tumor-Targeted+Delivery+of+Nanoparticles.">Bioorthogonal Cu-Free Click Chemistry in vivo for Tumor-Targeted Delivery of Nanoparticles.</a> Angew. Chem. Int. Ed. 51, 11836-11840 (2012).</li><li>Chang, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Free+Click+Chemistry+in+Living+Animals.">Copper-Free Click Chemistry in Living Animals.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 1821-1826 (2010).</li><li>Bostic, H. E., Smith, M. D., Poloukhtine, A. A., Popik, V. V., Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Labeling+and+Immobilization+via+copper-free+Click+Chemistry.">Membrane Labeling and Immobilization via copper-free Click Chemistry.</a> Chem. Commun. 48, 1431-1433 (2012).</li><li>Someya, T., Ando, A., Kimoto, M., Hirao, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Labeling+of+RNA+by+Combining+Genetic+Alphabet+Expansion+Transcription+and+Copper-Free+Click+Chemistry.">Site-Specific Labeling of RNA by Combining Genetic Alphabet Expansion Transcription and Copper-Free Click Chemistry.</a> Nucl. Acids Res. 43, 6665-6676 (2015).</li><li>Lee, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoporous+Silica+Nanoparticle+Pretargeting+for+PET+Imaging+Based+on+a+Rapid+Bioorthogonal+Reaction+in+a+Living+Body.">Mesoporous Silica Nanoparticle Pretargeting for PET Imaging Based on a Rapid Bioorthogonal Reaction in a Living Body.</a> Angew. Chem. Int. Ed. 52, 10549-10552 (2013).</li><li>Sachin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-18+Labeling+Protocol+of+Peptides+Based+on+Chemically+Orthogonal+Strain-Promoted+Cycloaddition+under+Physiologically+Friendly+Reaction+Conditions.">F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions.</a> Bioconjugate Chem. 23, 1680-1686 (2012).</li><li>Evans, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Free+Click+-+A+Promising+Tool+for+Pre-targeted+PET+Imaging.">Copper-Free Click - A Promising Tool for Pre-targeted PET Imaging.</a> Chem. Commun. 48, 991-993 (2012).</li><li>Campbell-Verduyn, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-Promoted+Copper-Free+&amp;#34;Click&amp;#34;+Chemistry+for+18F+Radiolabeling+of+Bombesin.">Strain-Promoted Copper-Free &amp;#34;Click&amp;#34; Chemistry for 18F Radiolabeling of Bombesin.</a> Angew. Chem. Int. Ed. 50, 11117-11120 (2011).</li><li>Arumugam, S., Chin, J., Schirrmacher, R., Popik, V. V., Kostikov, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F]Azadibenzocyclooctyne+([18F]ADIBO):+A+Biocompatible+Radioactive+Labeling+Synthon+for+Peptides+using+Catalyst+Free+[3+2]+Cycloaddition.">18F]Azadibenzocyclooctyne ([18F]ADIBO): A Biocompatible Radioactive Labeling Synthon for Peptides using Catalyst Free [3+2] Cycloaddition.</a> Bioorg. Med. Chem. Lett. 21, 6987-6991 (2011).</li><li>Bouvet, V., Wuest, M., Wuest, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Free+Click+Chemistry+with+the+Short-Lived+Positron+Emitter+Fluorine-18.">Copper-Free Click Chemistry with the Short-Lived Positron Emitter Fluorine-18.</a> Org. Biomol. Chem. 9, 7393-7399 (2011).</li><li>Satpati, D., Bauer, N., Hausner, S. H., Sutcliffe, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+[64Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2+via+Copper-Free+Click+Chemistry+for+PET+Imaging+of+Integrin+&#945;v&#946;6.">Synthesis of [64Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 via Copper-Free Click Chemistry for PET Imaging of Integrin &#945;v&#946;6.</a> J. Radioanal. Nucl. Chem. 302, 765-771 (2014).</li><li>Lee, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Method+To+Radiolabel+Glycol+Chitosan+Nanoparticles+with+64Cu+via+Copper-Free+Click+Chemistry+for+MicroPET+Imaging.">Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging.</a> Mol. Pharmaceutics. 10, 2190-2198 (2013).</li><li>Zeng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=64Cu+Core-Labeled+Nanoparticles+with+High+Specific+Activity+via+Metal-Free+Click+Chemistry.">64Cu Core-Labeled Nanoparticles with High Specific Activity via Metal-Free Click Chemistry.</a> ACS Nano. 6, 5209-5219 (2012).</li><li>Jeon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosynthesis+and+in+vivo+Evaluation+of+[125I]2-4(iodophenethyl)-2-Methylmalonic+Acid+as+a+Potential+Radiotracer+for+Detection+of+Apoptosis.">Radiosynthesis and in vivo Evaluation of [125I]2-4(iodophenethyl)-2-Methylmalonic Acid as a Potential Radiotracer for Detection of Apoptosis.</a> J. Radioanal. Nucl. Chem. 308, 23-29 (2016).</li><li>Adam, M. J., Wilbur, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiohalogens+for+Imaging+and+Therapy.">Radiohalogens for Imaging and Therapy.</a> Chem. Soc. Rev. 34, 153-163 (2005).</li><li>Jeon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosynthesis+of+123I-Labeld+Hesperetin+for+Biodistribution+Study+of+Orally+Administered+Hesperetin.">Radiosynthesis of 123I-Labeld Hesperetin for Biodistribution Study of Orally Administered Hesperetin.</a> J. Radioanal. Nucl. Chem. 306, 437-443 (2015).</li><li>Kil, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+[123I]IPEB+and+[123I]IMPEB+as+SPECT+Radioligands+for+Metabotropic+Glutamate+Receptor+Subtype.">Development of [123I]IPEB and [123I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype.</a> ACS Med. Chem. Lett. 5, 652-656 (2014).</li><li>Chen, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+I-123+Pretherapy+Scan+in+I-131+Radioiodine+Therapy+for+Thyroid+Cancer.">The Utility of I-123 Pretherapy Scan in I-131 Radioiodine Therapy for Thyroid Cancer.</a> Thyroid. 22, 304-309 (2012).</li><li>Jeon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Method+for+Iodine+Radioisotope+Labeling+of+Cyclooctyne-Containing+Molecules+using+Strain-Promoted+Copper-Free+Click+Reaction.">Efficient Method for Iodine Radioisotope Labeling of Cyclooctyne-Containing Molecules using Strain-Promoted Copper-Free Click Reaction.</a> Bioorg. Med. Chem. 23, 3303-3308 (2015).</li><li>Choi, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Evaluation+of+an+125I-Labeled+Azide+Prosthetic+Group+for+Efficient+and+Bioorthogonal+Radiolabeling+of+Cyclooctyne-Group+Containing+Molecules+using+Copper-Free+Click+Reaction.">Synthesis and Evaluation of an 125I-Labeled Azide Prosthetic Group for Efficient and Bioorthogonal Radiolabeling of Cyclooctyne-Group Containing Molecules using Copper-Free Click Reaction.</a> Bioorg. Med. Chem. Lett. 26, 875-878 (2016).</li><li>Kim, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Targeting+and+Imaging+Using+Cyclic+RGD-PEGylated+Gold+Nanoparticle+Probes+with+Directly+Conjugated+Iodine-125.">Tumor Targeting and Imaging Using Cyclic RGD-PEGylated Gold Nanoparticle Probes with Directly Conjugated Iodine-125.</a> Small. 7, 2052-2060 (2011).</li></ol>

The authors have nothing to disclose.

일반적으로, 정제 된 I-125 표지 지드 (1)의 관찰 방사 화학적 수율은 75 ± 10 %이었다 (N = 8). 방사성 표지는 방사능 MBq의 50-150로 수행하고, 그 결과 방사 상당히 일치한다. [125 I]의 NaI (t 1/2 = 59.4 d) 방사성 요오드화 반응에 사용 하였다 개월 이상 방사성 붕괴를 시행 한의 방사 화학적 수율로 관찰하면 약간 (53~65%) 감소 하였다. 따라서, [125 I]의 NaI 그것이 생성 되 자마자 이용 될 최적의 방사 또는 수율을 획득하기 위해 실험실로 전달하는 것이 좋다. 또한, 새로 제조 클로라민 T 용액은 원하는 방사 화학적 수율을 획득하기 위해 상기 반응에 사용되어야한다. 프리 커서 (2)는 매우 소수성이기 때문에, 150 μL의 절대 에탄올을 1 mg의 용해에 추가되어야 <st방사성 요오드화 반응에서 룽&gt; 2. 그렇지 않으면, 전체 반응 혼합물을 수성 산화제 용액 [125 I]의 NaI를 첨가 한 후 혼탁해질 수있다. 전구체의 감소 용해도는 종종 1의 낮은 방사 화학적 수율을 초래한다. DMSO는 또한 방사성 표지 단계 2에 녹여 사용할 수있다. 또한, 아세트산은 방사성 요오드화 단계 높은 방사 화학적 수율을 획득하기 위해 상기 전구체 용액에 첨가한다. 용리액 구배, 10 ㎖ / 분 : 0~10 분 동안 100 % 용매 B, 100 (1)을 함유하는 조 생성물의 정제를 위해 조제 적 HPLC를 사용하기 전에, 역상 HPLC 컬럼은 용매 A 및 B (유속으로 세척해야 10-25 분 동안 -0 % 용매 B, 0 % 용매 B 25-30 분) 시스템에서의 불순물의 미량을 제거한다. 이어서, 역상 HPLC 컬럼 얻기 적어도 20 분 동안 80 % 용매 A 20 % 용매 B로 평형화1의 일관성 유지 시간. 정제 된 하나를 포함하는 분획은 고상 추출 과정에서 H 2 O 이상 4 배 부피로 희석한다. 그렇지 않으면, 정제 된 생성물의 일부는 TC18 카트리지에 포집 될 수 없다. 아세톤 카트리지 1 정제를 용출하는데 사용되는 경우, 최종 부피는 주위 온도에서 질소 또는 아르곤 가스의 흐름과 함께 아세톤 증발에 의해 감소 될 수있다. 여러 방사성 iodines 중 125 내가 선택하고, 현재의 연구에 사용됩니다. 요오드 방사성 동위 원소의 여러 가지 다른 생물학적, 의학적 연구에서, 본 방법을 사용하여 시험 될 필요 (예를 들어, PET 이미징 124 I, 치료 목적 131 I). 지금까지 우리가 이해, 본 방사성 표지 프로토콜 상세 syntheti을 설명하는 첫 번째 보고서입니다방사성 표지 아 지드 그룹 C 단계. 최근에, 우리는 다른 구조 (23)가 다른 아 지드 보철 그룹을 발표했다. 그러나, 현재의 방법에서 방사성 표지 지드 (1) DBCO 기 함유 분자를 방사성 표지 효율의 관점에서 다른 것보다 약간 더 방사 결과를 제공 하였다. 방사성 요오드의 표시를위한 보철 그룹 (즉, N의 -hydroxysuccinimide 및 말레이 미드를) 존재하는 사이트 특이성을 제공 할 수 있습니다. 그러나, 본 방법은 우수한 bioorthogonality 함께 직접적인 방사성 표지 효율을 보여준다. 아 지드 작용기 생리적 조건 및 생체 내 환경에서의 안정성이 높은 것으로 알려져 있기 때문에, 방사성 표지 된 생성물 (1)은 생체 내 영상화 연구 미리 타겟에 이용 될 수있다. 우리는이 방법이 효율적 라 모두 생체 외 및 생체 요오드 방사성 동위 원소에 적용 할 것으로 예상변형 된 cyclooctyne 구조를 포함하는 생체 분자 및 나노 물질의 beling. . (1) 125 I 표지 된 금 나노 입자 (4)은 나노 물질의 분자 이미징 및 생체 분포 연구에서 사용될 수있다 : 1의 비 방사능에 기초하여, I (125)과 금 나노 입자의 환산 몰비는 ~ 1이다. 현재의 방법은 서로 다른 크기와 모양의 금 나노 물질의 방사성 요오드 라벨에 적용 할 수있다.

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.

stannylated 전구체 (2)의 방사성 요오드화 반응은 방사능 표지 된 생성물을 제공하기 위해 실온에서 15 분 동안 [125 I] NaI를 아세트산 및 클로라민 T 150 MBq의를 사용하여 수행 하였다 (1). 조 혼합물을 예비 HPLC 정제 후, 목적 생성물을 방사 화학적 수율은 75 ± 10 % (N = 8)을 얻었다. HPLC 분석은 125 I 표지 된 생성물의 방사 화학적 순도는 99 % 이상 (도 2)이고, 제품 (1)의 관찰 특정 방사능 40.7 MBq의 / μmol 않은 것을 확인했다. 카트리지를 사용하여 정제 된 생성물을 함유하는 분획을 고체상 추출 (1)의 아세톤 용액을 제공 하였다. 질소 또는 아르곤 가스의 스트림을 사용하여, 유기 용매가 증발 될 수 있고, 그 잔류 물을 다음 인트 대한 DMSO 또는 무수 에탄올에 다시 용해시킬 수있다피. 폴리에틸렌 글리콜 - 개질 된 금 나노 입자 (125) I-라벨링은 DBCO 그룹 - 개질 된 금 나노 입자를도 1에 도시 된 절차에 의해 제조 하였다. 폴리에틸렌 글리콜 티올 과량 (MW 5,000) DBCO 그룹과 반응시켰다 구연산은 13 나노 금 나노 입자를 안정화. 개질 단계 후, 생성물은 DBCO 기능화 된 금 나노 입자를 수득 연속 원심 분리에 의해 정제 하였다 (3). 방사성 표지 단계 1 3.7 MBq 내지 3의 2 μM합니다 (DBCO 그룹 ~ 400 μM)에 첨가하고, 라벨링 반응물을 1 시간 동안 40 ℃에서 수행 하였다. 무선 TLC 분석은 하나의 95 % 이상이 60 분 이내에 DBCO 기 - 작용 화 된 금 나노 입자 (3)와 반응되었음을 보여 주었다. 반응물을 60 분 동안 수행 한 다음, 조 생성물을 끝까지 마음이었다무선 TLC (도 3)에 의해 측정하여 원심. ified하게는 (4) 125 I 표지 된 금 나노 입자가&gt; 99 % (N = 4) 방사성 수율로 수득 하였다. <img alt="그림 1" src="/files/ftp_upload/54759/54759fig1.jpg" /> I 125 - 표지 된 아 지드 Radiosynthesis도 1 (1), 125 I 표지 된 금 나노 입자 (4). 시약 및 조건 : (a) [125 I] NaI를 아세트산, 클로라민 T, RT, 15 분, 75 ± 10 % (N = 8) 방사 화학적 수율; (b) DBCO-PEG-SH (MW 5000), H 2 O, RT, 2 시간; ~ 40 ° C, 60 분,&gt; 99 % 방사 화학적 수율. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54759/54759fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img al<html> t = &quot;그림 2&quot;SRC = &quot;/ 파일 / ftp_upload / 54759 / 54759fig2.jpg&quot;/&gt; 도 125 I 표지 지드 2. 분석 HPLC 크로마토 그램 (1). (a) 미정 제 생성물의 라디오 크로마토 그램. 정제 된 생성물 (b)의 라디오 크로마토 그램. 정제 된 제품의 (c)에 UV 크로마토 그램 (254 ㎚). <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54759/54759fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54759/54759fig3.jpg" /> 125 I 표지 된 금 나노 입자도 3 무선 TLC 결과 (4) (4 = 0.05의 Rf는, R F 1 = 0.45, 용리액 : 에틸 아세테이트) (a) 60 분 반응 후, (b) 정제 후./ecsource.jove.com/files/ftp_upload/54759/54759fig3large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.

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

I 125 - 표지 지드의 합성 및 무도 클릭 반응을 이용 dibenzocyclooctyne (DBCO) - 기 - 컨쥬 게이트 13 나노 크기의 금 나노 입자의 방사성 표지에 대한 세부 절차를 설명한다.

를 사용하여 금 나노 입자의 효율적인 방사성 표지를위한 최적화 된 프로토콜<sup&gt; (125)</sup&gt; I-표시 아 지드 보철 그룹

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

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

8.1K 조회수.  Korea Atomic Energy Research Institute.  I 125 - 표지 지드의 합성 및 무도 클릭 반응을 이용 dibenzocyclooctyne (DBCO) - 기 - 컨쥬 게이트 13 나노 크기의 금 나노 입자의 방사성 표지에 대한 세부 절차를 설명한다. 

비디오: 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).

효율적이고 사이트 별 스트레인 승진 아 지드 - 알킨 사이클로에 의해 항체 라벨링

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

연구

생화학

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.

바이오 콘쥬 게이션과의 Radiosynthesis<sup&gt; 89</sup&gt; ZR-DFO 표지 항체

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

화학

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.

Pretargeted PET 영상에 대한 Bioorthogonal 역 전자 수요 딜스 - 알더 고리 모으고

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

생체공학

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.

Pretargeted Radioimmunotherapy 역 전자 수요 Diels 오리 나무 반응에 따라

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.

프로그래밍된 죽음 리간드 1 발현 이미징을 위한 68갈륨 표지 D-펩타이드 PET 추적자 개발

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

암 연구학

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.

세포 이동의 양전자 방출 단층 촬영 이미징: 세포 방사선 표지 방법

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).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

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

면역학 및 감염병학

<meta charset="utf8"></head><body>Loop Method Methylation을 사용한 Carbon-11 방사성 추적자의 효율적인 합성

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>Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for clinical research with the loop method(루프 방법을 사용한 임상 연구를 위한 탄소-11 방사성 리간드의 효율성 및 방사성 표지

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.

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for clinical research with the loop method(루프 방법을 사용한 임상 연구를 위한 탄소-11 방사성 리간드의 효율성 및 방사성 표지 수율 향상)

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

를 사용하여 금 나노 입자의 효율적인 방사성 표지를위한 최적화 된 프로토콜

요약

더 많은 비디오 탐색

이 비디오의 챕터

효율적이고 사이트 별 스트레인 승진 아 지드 - 알킨 사이클로에 의해 항체 라벨링

바이오 콘쥬 게이션과의 Radiosynthesis

Pretargeted PET 영상에 대한 Bioorthogonal 역 전자 수요 딜스 - 알더 고리 모으고

Pretargeted Radioimmunotherapy 역 전자 수요 Diels 오리 나무 반응에 따라

프로그래밍된 죽음 리간드 1 발현 이미징을 위한 ⁶⁸갈륨 표지 D-펩타이드 PET 추적자 개발

세포 이동의 양전자 방출 단층 촬영 이미징: 세포 방사선 표지 방법

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

에 의해 쥐 림프구 라벨링

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for clinical research with the loop method(루프 방법을 사용한 임상 연구를 위한 탄소-11 방사성 리간드의 효율성 및 방사성 표지 수율 향상)

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for clinical research with the loop method(루프 방법을 사용한 임상 연구를 위한 탄소-11 방사성 리간드의 효율성 및 방사성 표지 수율 향상)