Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 1: Cysteine Residues and Glycans

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities

A Pretargeted Approach for the Multimodal PET/NIRF Imaging of Colorectal Cancer

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans-cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64Cu-labeled tetrazine radioligand (64Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool

Hybrid luminescent nanoparticles for the prostate cancer diagnosis

Ce travail de thèse a consisté en la conception et la réalisation d’une nano-sonde hybride luminescente visant à permettre la détection précoce du cancer de la prostate. La première partie de ce projet a été consacré à la synthèse, par une voie de chimie click, d’une bibliothèque d’acides 4-triazolyl dipicoliniques substitués en position 4 du triazole par une large gamme de substituants. Ces diacides ont permis d’obtenir les complexes d’europium(III) et de terbium(III) correspondant, qui ont montré d’excellentes propriétés optiques, avec des rendements quantiques de luminescence sous excitation UV pouvant atteindre 60% et 36%, pour les complexes d’europium(III) et deterbium(III) respectivement. D’autre-part, ces fluorophores ont pu être excités efficacement en régime biphotonique, à la fois au travers des transitions S0 ®S1 et S0 ®T1. Sur la base de ces résultats, certains de ces chélates ont été sélectionnés afin de les incorporer dans des nanoparticules de silice. Le procédé d’élaboration par microémulsion inverse s’est révélé efficace pour l’incorporation des complexes électriquement neutres, mais n’a pas permis celle de nanohybrides incorporant des complexes chargés négativement. Ces nanohybrides présentent des propriétés optiques caractéristiques des lanthanides, avec des rendements quantiques allant jusqu’à 30%. La surface de ces nano-objets a ensuite été fonctionnalisée par des groupements amino, qui ont permis le greffage de bras espaceur et d’un vecteur ciblant la PSMA, l’un des signaux du cancer de la prostate, nous donnant ainsi accès à un modèle de nano-sonde luminescente. Un autre volet de ce travail a été dédié à l’étude de nouveaux analogues du NAAG, substrat naturel de la PSMA. Bien que la synthèse des deux composés cibles, sélectionnés parmi une vingtaine de structures par modélisation moléculaire, n’ait pu aboutir, elle a été largement avancée. Enfin, la dernière partie de ce travail décrit les premiers résultats obtenus in vitro et in vivo avec les nanosondes. Ces études ont porté sur l’évaluation de la cytotoxicité des nanoparticules ainsi que sur leur biodistribution chez la souris saine et chez la souris porteuse d’une tumeur prostatique. Cette étude a révélé une élimination rapide des nanoparticules par l’organisme, mais n’a malheureusement pas pu mettre en évidence un marquage des zones tumorales par les nanosondes.The aim of this project was the design of a luminescent nanoprobe allowing the early prostate cancer detection.The first part of this project was the synthesis, through a click chemistry approach, of a library of 4-triazolyl dipicolinic acid substituted in position 4 of the triazole by a wide range of substitutive groups. These diacids were used to synthesise the corresponding europium(III) and terbium(III) complexes, which showed excellent optical properties, with photoluminescence quantum yield under UV excitation reaching 60% for europium(III) complexes and 36% for terbium(III) complexes. Moreover, these phosphores have been efficiently excited in biphotonic regime through the transitions S0 ®S1 and S0 ®T1. The more interesting chelates were selected for their further embedding into silica nanoparticles. The water-in-oil emulsion process showed a great efficiency for the incorporation of electrically neutral complexes, but did not allow the embedding of negatively charged ones. The resulting nanohybrids showed optical properties typical of lanthanides, presenting photoluminescence quantum yields up to 30%. The nanoparticles surface was then functionalised by amino groups, which were used to graft a spacer then a prostate tumour cells vector, giving us a luminescent nanoprobe model. The third section of this work was devoted to the study of new analogues of NAAG, the natural substrate of the PSMA, one of the prostatic cancer signals. Although the synthesis of the two target compounds, selected from more than 20 structures by molecular modelling, was uncompleted, they are now within easy reach. The last part describes the preliminary results obtained in vitro and in vivo with the nanoprobes. These studies were focused on the nanoparticles cytotoxitcity assessment, depending on the surface functionalisation, and on their distribution, in healthy and in prostatic tumour bearing mice. This study revealed the fast elimination of the nanoparticles by the organism, but did not show any concentration of nanoprobes in tumoral area

Nano-sondes hybrides luminescentes pour la détection du cancer de la prostate

The aim of this project was the design of a luminescent nanoprobe allowing the early prostate cancer detection.The first part of this project was the synthesis, through a click chemistry approach, of a library of 4-triazolyl dipicolinic acid substituted in position 4 of the triazole by a wide range of substitutive groups. These diacids were used to synthesise the corresponding europium(III) and terbium(III) complexes, which showed excellent optical properties, with photoluminescence quantum yield under UV excitation reaching 60% for europium(III) complexes and 36% for terbium(III) complexes. Moreover, these phosphores have been efficiently excited in biphotonic regime through the transitions S0 ®S1 and S0 ®T1. The more interesting chelates were selected for their further embedding into silica nanoparticles. The water-in-oil emulsion process showed a great efficiency for the incorporation of electrically neutral complexes, but did not allow the embedding of negatively charged ones. The resulting nanohybrids showed optical properties typical of lanthanides, presenting photoluminescence quantum yields up to 30%. The nanoparticles surface was then functionalised by amino groups, which were used to graft a spacer then a prostate tumour cells vector, giving us a luminescent nanoprobe model. The third section of this work was devoted to the study of new analogues of NAAG, the natural substrate of the PSMA, one of the prostatic cancer signals. Although the synthesis of the two target compounds, selected from more than 20 structures by molecular modelling, was uncompleted, they are now within easy reach. The last part describes the preliminary results obtained in vitro and in vivo with the nanoprobes. These studies were focused on the nanoparticles cytotoxitcity assessment, depending on the surface functionalisation, and on their distribution, in healthy and in prostatic tumour bearing mice. This study revealed the fast elimination of the nanoparticles by the organism, but did not show any concentration of nanoprobes in tumoral area.Ce travail de thèse a consisté en la conception et la réalisation d’une nano-sonde hybride luminescente visant à permettre la détection précoce du cancer de la prostate. La première partie de ce projet a été consacré à la synthèse, par une voie de chimie click, d’une bibliothèque d’acides 4-triazolyl dipicoliniques substitués en position 4 du triazole par une large gamme de substituants. Ces diacides ont permis d’obtenir les complexes d’europium(III) et de terbium(III) correspondant, qui ont montré d’excellentes propriétés optiques, avec des rendements quantiques de luminescence sous excitation UV pouvant atteindre 60% et 36%, pour les complexes d’europium(III) et deterbium(III) respectivement. D’autre-part, ces fluorophores ont pu être excités efficacement en régime biphotonique, à la fois au travers des transitions S0 ®S1 et S0 ®T1. Sur la base de ces résultats, certains de ces chélates ont été sélectionnés afin de les incorporer dans des nanoparticules de silice. Le procédé d’élaboration par microémulsion inverse s’est révélé efficace pour l’incorporation des complexes électriquement neutres, mais n’a pas permis celle de nanohybrides incorporant des complexes chargés négativement. Ces nanohybrides présentent des propriétés optiques caractéristiques des lanthanides, avec des rendements quantiques allant jusqu’à 30%. La surface de ces nano-objets a ensuite été fonctionnalisée par des groupements amino, qui ont permis le greffage de bras espaceur et d’un vecteur ciblant la PSMA, l’un des signaux du cancer de la prostate, nous donnant ainsi accès à un modèle de nano-sonde luminescente. Un autre volet de ce travail a été dédié à l’étude de nouveaux analogues du NAAG, substrat naturel de la PSMA. Bien que la synthèse des deux composés cibles, sélectionnés parmi une vingtaine de structures par modélisation moléculaire, n’ait pu aboutir, elle a été largement avancée. Enfin, la dernière partie de ce travail décrit les premiers résultats obtenus in vitro et in vivo avec les nanosondes. Ces études ont porté sur l’évaluation de la cytotoxicité des nanoparticules ainsi que sur leur biodistribution chez la souris saine et chez la souris porteuse d’une tumeur prostatique. Cette étude a révélé une élimination rapide des nanoparticules par l’organisme, mais n’a malheureusement pas pu mettre en évidence un marquage des zones tumorales par les nanosondes

Nano-sondes hybrides luminescentes pour la détection du cancer de la prostate

Ce travail de thèse a consisté en la conception et la réalisation d une nano-sonde hybride luminescente visant à permettre la détection précoce du cancer de la prostate. La première partie de ce projet a été consacré à la synthèse, par une voie de chimie click, d une bibliothèque d acides 4-triazolyl dipicoliniques substitués en position 4 du triazole par une large gamme de substituants. Ces diacides ont permis d obtenir les complexes d europium(III) et de terbium(III) correspondant, qui ont montré d excellentes propriétés optiques, avec des rendements quantiques de luminescence sous excitation UV pouvant atteindre 60% et 36%, pour les complexes d europium(III) et deterbium(III) respectivement. D autre-part, ces fluorophores ont pu être excités efficacement en régime biphotonique, à la fois au travers des transitions S0 ®S1 et S0 ®T1. Sur la base de ces résultats, certains de ces chélates ont été sélectionnés afin de les incorporer dans des nanoparticules de silice. Le procédé d élaboration par microémulsion inverse s est révélé efficace pour l incorporation des complexes électriquement neutres, mais n a pas permis celle de nanohybrides incorporant des complexes chargés négativement. Ces nanohybrides présentent des propriétés optiques caractéristiques des lanthanides, avec des rendements quantiques allant jusqu à 30%. La surface de ces nano-objets a ensuite été fonctionnalisée par des groupements amino, qui ont permis le greffage de bras espaceur et d un vecteur ciblant la PSMA, l un des signaux du cancer de la prostate, nous donnant ainsi accès à un modèle de nano-sonde luminescente. Un autre volet de ce travail a été dédié à l étude de nouveaux analogues du NAAG, substrat naturel de la PSMA. Bien que la synthèse des deux composés cibles, sélectionnés parmi une vingtaine de structures par modélisation moléculaire, n ait pu aboutir, elle a été largement avancée. Enfin, la dernière partie de ce travail décrit les premiers résultats obtenus in vitro et in vivo avec les nanosondes. Ces études ont porté sur l évaluation de la cytotoxicité des nanoparticules ainsi que sur leur biodistribution chez la souris saine et chez la souris porteuse d une tumeur prostatique. Cette étude a révélé une élimination rapide des nanoparticules par l organisme, mais n a malheureusement pas pu mettre en évidence un marquage des zones tumorales par les nanosondes.The aim of this project was the design of a luminescent nanoprobe allowing the early prostate cancer detection.The first part of this project was the synthesis, through a click chemistry approach, of a library of 4-triazolyl dipicolinic acid substituted in position 4 of the triazole by a wide range of substitutive groups. These diacids were used to synthesise the corresponding europium(III) and terbium(III) complexes, which showed excellent optical properties, with photoluminescence quantum yield under UV excitation reaching 60% for europium(III) complexes and 36% for terbium(III) complexes. Moreover, these phosphores have been efficiently excited in biphotonic regime through the transitions S0 ®S1 and S0 ®T1. The more interesting chelates were selected for their further embedding into silica nanoparticles. The water-in-oil emulsion process showed a great efficiency for the incorporation of electrically neutral complexes, but did not allow the embedding of negatively charged ones. The resulting nanohybrids showed optical properties typical of lanthanides, presenting photoluminescence quantum yields up to 30%. The nanoparticles surface was then functionalised by amino groups, which were used to graft a spacer then a prostate tumour cells vector, giving us a luminescent nanoprobe model. The third section of this work was devoted to the study of new analogues of NAAG, the natural substrate of the PSMA, one of the prostatic cancer signals. Although the synthesis of the two target compounds, selected from more than 20 structures by molecular modelling, was uncompleted, they are now within easy reach. The last part describes the preliminary results obtained in vitro and in vivo with the nanoprobes. These studies were focused on the nanoparticles cytotoxitcity assessment, depending on the surface functionalisation, and on their distribution, in healthy and in prostatic tumour bearing mice. This study revealed the fast elimination of the nanoparticles by the organism, but did not show any concentration of nanoprobes in tumoral area.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

Luminescent Thin Films and Nanoparticles of Europium Doped Hybrids Based on Organosilyl β-Diketonate

International audienceNovel organic-inorganic hybrid materials shaped as transparent films and core-shell nanoparticles were prepared by the sol-gel process from a new organo-alkoxysilane based on a b-diketonate (DBM-OH) and doped with trivalent europium ions. The resultant hybrid materials were characterized by Raman spectroscopy and electronic microscopy. Films thickness and nanoparticles size were respectively measured around 1 lm and 30 nm. The photoluminescence study, including the recording of excitation and emission spectra as well as the determination of the decay times, was investigated at roomtemperature.The results have pointed out an efficient ligand-to-Eu3? intramolecular energy transfer resulting in a high quantum yield as determined by using an integrating sphere

Colloidal chemistry with patchy silica nanoparticles

We report a new route to synthesize clusters, or so-called colloidal molecules (CMs), which mimic the symmetry of molecular structures made of one central atom. We couple site-specifically functionalized patchy nanoparticles, i.e., valence-endowed colloidal atoms (CAs), with complementary nanospheres through amide bonds. By analogy with the Gillespie formalism, we show that AX4, AX3E1 and AX2E2 CMs can be obtained from tetravalent sp3-like CAs when the relative amount of both building units is varied in a controlled manner. We obtain AX2 CMs from divalent sp-like CAs. We also show that it is possible to covalently attach two different types of satellites to the same central patchy nanoparticle to create more complex CMs, opening the way to the fabrication of new multifunctional nanostructures with well-controlled shape and composition.Dilater le système atomique conventionnel à l'échelle colloïdale grâce à des particules préprogrammées pour une valence donnéeInitiative d'excellence de l'Université de Bordeau

Two-photon absorption properties of Eu3+-DPA-triazolyl complexes and derived silica nanoparticles embedding covalently these complexes.

International audienceSeveral complexes and silica-based nanohybrids of rare-earth ions (Eu3+, Gd3+) have been synthesized from dimethyl 4-azidopyridine-2,6-dicarboxylate (4) following the Click chemistry approach. A complete spectroscopic study indicates that such compounds exhibit strong sensitization by the antenna effect from both UV and NIR excitations. The Gd3+-based materials show phosphorescence under ambient conditions, which originates from the lowest-energy intra-ligand triplets. Fine analysis of the NIR excitation spectra using time resolved photoluminescence spectroscopy (TRS) indicates that the spectral repartition of the triplet T1 state differs notably between the complexes and the NPs embedding the complexes. Moreover the dependence of Eu3+ luminescence vs. incident beam power in the NIR region diverges from pure quadratic dependence expected in the framework of the two-photon absorption process. The results are discussed considering the occurrence of a direct singlet-to-triplet optical absorption transition (S0[RIGHTWARDS ARROW]T1) upon NIR excitation