A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery.
| Autor: | Vaughan HJ; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Est-Witte S; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Dockery LT; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Urello MA; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Boyd J; Discovery Sciences, BioPharma R&D, AstraZeneca, Gaithersburg, MD, USA., Keyser BD; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Zhuang L; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Marelli M; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA., Christie RJ; Biologics Engineering, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA. |
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| Jazyk: | angličtina |
| Zdroj: | Science and technology of advanced materials [Sci Technol Adv Mater] 2024 May 13; Vol. 25 (1), pp. 2351791. Date of Electronic Publication: 2024 May 13 (Print Publication: 2024). |
| DOI: | 10.1080/14686996.2024.2351791 |
| Abstrakt: | Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand. Competing Interests: All authors are past or current employees of AstraZeneca. (© 2024 AstraZeneca. Published by National Institute for Materials Science in partnership with Taylor & Francis Group.) |
| Databáze: | MEDLINE |
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