Simple plant-based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor-targeted drug delivery and bioimaging in cancer therapy.

Autor: Knödler M; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., Aachen, Germany.; Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany., Opdensteinen P; Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., Aachen, Germany.; Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany., Sankaranarayanan RA; Department of Nuclear Medicine, University Hospital Aachen, RWTH Aachen University, Aachen, Germany., Morgenroth A; Department of Nuclear Medicine, University Hospital Aachen, RWTH Aachen University, Aachen, Germany., Buhl EM; Electron Microscopy Facility, Institute for Pathology, University Hospital Aachen, RWTH Aachen University, Aachen, Germany., Mottaghy FM; Department of Nuclear Medicine, University Hospital Aachen, RWTH Aachen University, Aachen, Germany.; Department of Radiology and Nuclear Medicine, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands., Buyel JF; Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.; Department of Biotechnology (DBT), Institute of Bioprocess Science and Engineering (IBSE), University of Natural Resources and Life Sciences, Vienna (BOKU), Vienna, Austria.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2023 Apr; Vol. 120 (4), pp. 1038-1054. Date of Electronic Publication: 2023 Jan 02.
DOI: 10.1002/bit.28312
Abstrakt: Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g -1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5'-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg -1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.
(© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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