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
© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC..
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.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2023 |
|---|---|
| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:120 |
|---|---|
| Enthalten in: |
Biotechnology and bioengineering - 120(2023), 4 vom: 06. Apr., Seite 1038-1054 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Knödler, Matthias [VerfasserIn] |
|---|
| Links: |
|---|
| Anmerkungen: |
Date Completed 16.03.2023 Date Revised 22.03.2023 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1002/bit.28312 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM350510164 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM350510164 | ||
| 003 | DE-627 | ||
| 005 | 20231226045150.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231226s2023 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1002/bit.28312 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1168.xml |
| 035 | |a (DE-627)NLM350510164 | ||
| 035 | |a (NLM)36539373 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Knödler, Matthias |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a 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 |
| 264 | 1 | |c 2023 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 16.03.2023 | ||
| 500 | |a Date Revised 22.03.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a active targeting | |
| 650 | 4 | |a cancer treatment | |
| 650 | 4 | |a plant molecular farming | |
| 650 | 4 | |a protein nanoparticle | |
| 650 | 4 | |a transient expression | |
| 650 | 7 | |a Apoferritins |2 NLM | |
| 650 | 7 | |a 9013-31-4 |2 NLM | |
| 650 | 7 | |a Ferritins |2 NLM | |
| 650 | 7 | |a 9007-73-2 |2 NLM | |
| 650 | 7 | |a Iron |2 NLM | |
| 650 | 7 | |a E1UOL152H7 |2 NLM | |
| 700 | 1 | |a Opdensteinen, Patrick |e verfasserin |4 aut | |
| 700 | 1 | |a Sankaranarayanan, Ramya Ambur |e verfasserin |4 aut | |
| 700 | 1 | |a Morgenroth, Agnieszka |e verfasserin |4 aut | |
| 700 | 1 | |a Buhl, Eva Miriam |e verfasserin |4 aut | |
| 700 | 1 | |a Mottaghy, Felix M |e verfasserin |4 aut | |
| 700 | 1 | |a Buyel, Johannes Felix |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Biotechnology and bioengineering |d 1976 |g 120(2023), 4 vom: 06. Apr., Seite 1038-1054 |w (DE-627)NLM000014702 |x 1097-0290 |7 nnns |
| 773 | 1 | 8 | |g volume:120 |g year:2023 |g number:4 |g day:06 |g month:04 |g pages:1038-1054 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1002/bit.28312 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 120 |j 2023 |e 4 |b 06 |c 04 |h 1038-1054 | ||