Roberta Napolitano, Alessia Adamo, Luca Biondi, Alessia Cordaro, Margherita Iaboni, Francesco Stummo, Francesca La Cava, Francesca Arena, Daniele Faletto, Federica Chianale, Erika Reitano, Luisa Poggi, Francesco Blasi
{"title":"新型近红外染料靶向碳酸酐酶IX荧光成像应用。","authors":"Roberta Napolitano, Alessia Adamo, Luca Biondi, Alessia Cordaro, Margherita Iaboni, Francesco Stummo, Francesca La Cava, Francesca Arena, Daniele Faletto, Federica Chianale, Erika Reitano, Luisa Poggi, Francesco Blasi","doi":"10.1097/RLI.0000000000001204","DOIUrl":null,"url":null,"abstract":"<p><strong>Objectives: </strong>Fluorescence tumor-targeted imaging is a sensitive technique that may assist surgeons to remove residual cancer tissue during resection. Carbonic anhydrase IX (CAIX) is a tumor-associated cell-surface glycoprotein, upregulated in hypoxic environments, and a suitable biomarker to develop targeted dyes for fluorescence-guided surgery. This study describes design, synthesis, in vitro and in vivo assessment of novel CAIX-targeting fluorescent probes based on the well-known drug acetazolamide, addressing the contribution of both targeting moiety and fluorophore structure on imaging efficacy.</p><p><strong>Materials and methods: </strong>All the CAIX-targeting heptamethine cyanines synthesized and described in the present work were characterized in terms of their optical properties in different media. The affinity to human serum albumin was evaluated by UV-VIS spectrophotometry. The affinity to the CA catalytic site was determined on a recombinant bovine CAII enzyme (bCAII), with a fluorescent-based assay. Human colon adenocarcinoma HT-29 cells, highly expressing CAIX, were used for the in vitro characterization, including cell binding, uptake and competition assays, by flow cytometry. Finally, the in vivo tumor targeting efficacy of a selected group of probes was assessed by Optical Imaging in a mouse subcutaneous tumor from HT-29 cells, characterized by both expression of CAIX and a hypoxic tumor microenvironment.</p><p><strong>Results: </strong>First, a family of CAIX-targeting probes was prepared by functionalizing a novel glucamine-bearing heptamethine cyanine (Dye1) with a modified acetazolamide moiety, whose acetyl group was replaced with i) aminooctanoic acid C8, ii) phenylalanine, iii) amino-PEG2-acid and iv) the longer linker 4a, or 2 commercially available benzenesulfonamides. From the in vitro screening of this first group of compounds, the C8-AZA targeting moiety was selected due to its highest affinity. Indeed, Dye1-C8-AZA exhibited the lowest KD values for both bCAII (6.1 ± 1.6 nM) and CAIX-expressing HT-29 cells (58 ± 9 nM), even lower than HypoxyFluor-1 (HF-1), a CAIX-targeted dye already reported in the literature. Then, other heptamethine cyanines (Dye2-Dye5, linear or cyclic, with different substituents on the indolenines and different conjugation position of the targeting vector) were functionalized with C8-AZA and fully characterized both in vitro and in vivo, to evaluate the combinatory effect of vector and fluorophore on the performance of the resulting probes. The different chemical features of the cyanines influenced the optical properties, solubility, binding with albumin, biodistribution, and imaging efficacy of the probes, while leaving unaffected the high affinity to the target. When tested in vivo for the visualization of CAIX-expressing HT-29 tumors, all C8-AZA probes showed high and specific tumor accumulation, often superior to HF-1.</p><p><strong>Conclusions: </strong>Several CAIX-targeting probes were synthesized to test the combinatory effect of different molecular vectors and dyes on the biological properties. All probes containing the C8-AZA targeting moiety displayed higher affinity and specificity to the target, while imaging efficacy in vivo was strongly influenced also by the structure of the labelling dye. All probes, and among them especially Dye1-C8-AZA, displayed efficient in vivo tumor accumulation. These results support further studies toward clinical testing of CAIX as suitable target for tumor fluorescence imaging and pave the way for future clinical applications.</p>","PeriodicalId":14486,"journal":{"name":"Investigative Radiology","volume":" ","pages":""},"PeriodicalIF":8.0000,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Novel Near Infrared Dyes Targeting Carbonic Anhydrase IX for Fluorescence Imaging Applications.\",\"authors\":\"Roberta Napolitano, Alessia Adamo, Luca Biondi, Alessia Cordaro, Margherita Iaboni, Francesco Stummo, Francesca La Cava, Francesca Arena, Daniele Faletto, Federica Chianale, Erika Reitano, Luisa Poggi, Francesco Blasi\",\"doi\":\"10.1097/RLI.0000000000001204\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objectives: </strong>Fluorescence tumor-targeted imaging is a sensitive technique that may assist surgeons to remove residual cancer tissue during resection. Carbonic anhydrase IX (CAIX) is a tumor-associated cell-surface glycoprotein, upregulated in hypoxic environments, and a suitable biomarker to develop targeted dyes for fluorescence-guided surgery. This study describes design, synthesis, in vitro and in vivo assessment of novel CAIX-targeting fluorescent probes based on the well-known drug acetazolamide, addressing the contribution of both targeting moiety and fluorophore structure on imaging efficacy.</p><p><strong>Materials and methods: </strong>All the CAIX-targeting heptamethine cyanines synthesized and described in the present work were characterized in terms of their optical properties in different media. The affinity to human serum albumin was evaluated by UV-VIS spectrophotometry. The affinity to the CA catalytic site was determined on a recombinant bovine CAII enzyme (bCAII), with a fluorescent-based assay. Human colon adenocarcinoma HT-29 cells, highly expressing CAIX, were used for the in vitro characterization, including cell binding, uptake and competition assays, by flow cytometry. Finally, the in vivo tumor targeting efficacy of a selected group of probes was assessed by Optical Imaging in a mouse subcutaneous tumor from HT-29 cells, characterized by both expression of CAIX and a hypoxic tumor microenvironment.</p><p><strong>Results: </strong>First, a family of CAIX-targeting probes was prepared by functionalizing a novel glucamine-bearing heptamethine cyanine (Dye1) with a modified acetazolamide moiety, whose acetyl group was replaced with i) aminooctanoic acid C8, ii) phenylalanine, iii) amino-PEG2-acid and iv) the longer linker 4a, or 2 commercially available benzenesulfonamides. From the in vitro screening of this first group of compounds, the C8-AZA targeting moiety was selected due to its highest affinity. Indeed, Dye1-C8-AZA exhibited the lowest KD values for both bCAII (6.1 ± 1.6 nM) and CAIX-expressing HT-29 cells (58 ± 9 nM), even lower than HypoxyFluor-1 (HF-1), a CAIX-targeted dye already reported in the literature. Then, other heptamethine cyanines (Dye2-Dye5, linear or cyclic, with different substituents on the indolenines and different conjugation position of the targeting vector) were functionalized with C8-AZA and fully characterized both in vitro and in vivo, to evaluate the combinatory effect of vector and fluorophore on the performance of the resulting probes. The different chemical features of the cyanines influenced the optical properties, solubility, binding with albumin, biodistribution, and imaging efficacy of the probes, while leaving unaffected the high affinity to the target. When tested in vivo for the visualization of CAIX-expressing HT-29 tumors, all C8-AZA probes showed high and specific tumor accumulation, often superior to HF-1.</p><p><strong>Conclusions: </strong>Several CAIX-targeting probes were synthesized to test the combinatory effect of different molecular vectors and dyes on the biological properties. All probes containing the C8-AZA targeting moiety displayed higher affinity and specificity to the target, while imaging efficacy in vivo was strongly influenced also by the structure of the labelling dye. All probes, and among them especially Dye1-C8-AZA, displayed efficient in vivo tumor accumulation. These results support further studies toward clinical testing of CAIX as suitable target for tumor fluorescence imaging and pave the way for future clinical applications.</p>\",\"PeriodicalId\":14486,\"journal\":{\"name\":\"Investigative Radiology\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":8.0000,\"publicationDate\":\"2025-05-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Investigative Radiology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1097/RLI.0000000000001204\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"RADIOLOGY, NUCLEAR MEDICINE & MEDICAL IMAGING\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Investigative Radiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1097/RLI.0000000000001204","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"RADIOLOGY, NUCLEAR MEDICINE & MEDICAL IMAGING","Score":null,"Total":0}
Novel Near Infrared Dyes Targeting Carbonic Anhydrase IX for Fluorescence Imaging Applications.
Objectives: Fluorescence tumor-targeted imaging is a sensitive technique that may assist surgeons to remove residual cancer tissue during resection. Carbonic anhydrase IX (CAIX) is a tumor-associated cell-surface glycoprotein, upregulated in hypoxic environments, and a suitable biomarker to develop targeted dyes for fluorescence-guided surgery. This study describes design, synthesis, in vitro and in vivo assessment of novel CAIX-targeting fluorescent probes based on the well-known drug acetazolamide, addressing the contribution of both targeting moiety and fluorophore structure on imaging efficacy.
Materials and methods: All the CAIX-targeting heptamethine cyanines synthesized and described in the present work were characterized in terms of their optical properties in different media. The affinity to human serum albumin was evaluated by UV-VIS spectrophotometry. The affinity to the CA catalytic site was determined on a recombinant bovine CAII enzyme (bCAII), with a fluorescent-based assay. Human colon adenocarcinoma HT-29 cells, highly expressing CAIX, were used for the in vitro characterization, including cell binding, uptake and competition assays, by flow cytometry. Finally, the in vivo tumor targeting efficacy of a selected group of probes was assessed by Optical Imaging in a mouse subcutaneous tumor from HT-29 cells, characterized by both expression of CAIX and a hypoxic tumor microenvironment.
Results: First, a family of CAIX-targeting probes was prepared by functionalizing a novel glucamine-bearing heptamethine cyanine (Dye1) with a modified acetazolamide moiety, whose acetyl group was replaced with i) aminooctanoic acid C8, ii) phenylalanine, iii) amino-PEG2-acid and iv) the longer linker 4a, or 2 commercially available benzenesulfonamides. From the in vitro screening of this first group of compounds, the C8-AZA targeting moiety was selected due to its highest affinity. Indeed, Dye1-C8-AZA exhibited the lowest KD values for both bCAII (6.1 ± 1.6 nM) and CAIX-expressing HT-29 cells (58 ± 9 nM), even lower than HypoxyFluor-1 (HF-1), a CAIX-targeted dye already reported in the literature. Then, other heptamethine cyanines (Dye2-Dye5, linear or cyclic, with different substituents on the indolenines and different conjugation position of the targeting vector) were functionalized with C8-AZA and fully characterized both in vitro and in vivo, to evaluate the combinatory effect of vector and fluorophore on the performance of the resulting probes. The different chemical features of the cyanines influenced the optical properties, solubility, binding with albumin, biodistribution, and imaging efficacy of the probes, while leaving unaffected the high affinity to the target. When tested in vivo for the visualization of CAIX-expressing HT-29 tumors, all C8-AZA probes showed high and specific tumor accumulation, often superior to HF-1.
Conclusions: Several CAIX-targeting probes were synthesized to test the combinatory effect of different molecular vectors and dyes on the biological properties. All probes containing the C8-AZA targeting moiety displayed higher affinity and specificity to the target, while imaging efficacy in vivo was strongly influenced also by the structure of the labelling dye. All probes, and among them especially Dye1-C8-AZA, displayed efficient in vivo tumor accumulation. These results support further studies toward clinical testing of CAIX as suitable target for tumor fluorescence imaging and pave the way for future clinical applications.
期刊介绍:
Investigative Radiology publishes original, peer-reviewed reports on clinical and laboratory investigations in diagnostic imaging, the diagnostic use of radioactive isotopes, computed tomography, positron emission tomography, magnetic resonance imaging, ultrasound, digital subtraction angiography, and related modalities. Emphasis is on early and timely publication. Primarily research-oriented, the journal also includes a wide variety of features of interest to clinical radiologists.