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Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring.

ABSTRACT: The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.

SUBMITTER: Fitzgerald S

PROVIDER: S-EPMC9305252 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The use of NIR-fluorescence imaging to demarcate tumour boundaries for real-time guidance of their surgical resection has a huge untapped potential. However, fluorescence imaging using molecular fluorophores, even with a targeting biomolecule attached, has a major shortcoming of signal interference from non-specific background fluorescence outside the region of interest. This poor selectivity necessitates prolonged time delays to allow clearance of background fluorophore and retention within the tumour prior to image acquisition. In this report, an innovative approach to overcome this issue is described in which cancer targeted off to on bio-responsive NIR-fluorophores are utilised to switch-on first within the tumour. Bio-responsive cRGD, iRGD and PEG conjugates have been synthesised using activated ester/amine or maleimide/thiol couplings to link targeting and fluorophore components. Their off to on emission responses were measured and compared with an always-on non-responsive control with each bio-responsive derivative showing large fluorescence enhancement values. Live cell imaging experiments using metastatic breast cancer cells confirmed in vitro bio-responsive capabilities. An in vivo assessment of MDA-MB 231 tumour imaging performance for bio-responsive and always-on fluorophores was conducted with monitoring of fluorescence distributions over 96 h. As anticipated, the always-on fluorophore gave an immediate, non-specific and very strong emission throughout whereas the bio-responsive derivatives initially displayed very low fluorescence. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD and iRGD conjugates both had effective tumour turn-on in the first hour, though the cRGD derivative had superior specificity for tumour over the iRGD conjugate. The pegylated derivative had similar switch-on characteristics but over a much longer period, taking 9 h before a significant emission was observable from the tumour. Evidence for in vivo active tumour targeting was obtained for the best performing cRGD bio-responsive NIR-AZA derivative from competitive binding studies. Overall, this cRGD-conjugate has the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections.

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Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring.

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