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

Abstract

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 bestperforming 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

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco

“…It seemed implausible to us that these proteins would form aggregates due to the base salinity of the extraction buffer (~ 20 mS cm −1 ) at 22 °C. Instead, unspecific binding of proteins to depth filters containing diatomaceous earth has been reported before (Knödler et al 2023 ; Opdensteinen et al 2021 ), and we then assumed that electrostatic interactions between the proteins and the charged components of the depth filter caused the product losses as previously observed for other molecules (Menzel et al 2018 ). We therefore tested a set of depth filters that contained less diatomaceous earth (Fig.…”

Killer to cure: Expression and production costs calculation of tobacco plant‐made cancer‐immune checkpoint inhibitors

“…Therefore, we have augmented our previous cost model (Buyel and Fischer, 2012; Fischer and Buyel, 2020; Knödler et al ., 2023) with various options in upstream plant production and downstream biomass processing (Table S4). Two general production scenarios were derived: (1) fully‐automated transient expression using a vertical farm in a middle‐ to high‐income country for the production of emergency or personalized medicines (automated/transient; e.g .…”

“…ICIs are revolutionary in cancer treatments (Tang et al, 2018), but efforts have to be made to improve accessibility, especially to LMICs, by lowering costs. Combination therapy to improve their efficacy (Khair et al, 2019;Ma et al, 2021) also necessitates separate manufacturing lines for each component, which can increase manufacturing costs. We believe ICIs can be made more economically feasible using plants, a relatively new but attractive alternative production platform to the current mammalian cellbased bioreactors (Moon et al, 2020;Paul et al, 2013).…”

“…Despite the huge clinical success of ICIs, response rates only reach a maximum of 40%, and many responsive patients can go on to acquire resistance (Borghaei et al, 2015;Garon et al, 2015;Larkin et al, 2015). For this reason, a combination of ICIs may be beneficial in increasing patient response rates (Khair et al, 2019;Ma et al, 2021). A major barrier to this is the price of therapy, with a course per patient costing ~£70 000 (Centers for Medicare and Medicaid Services (CMS), 2021; Doyle, 2016; National Institute for Health Care and Excellence, 2021; Verma et al, 2018).…”

Nanodrug Hijacking Blood Transferrin for Ferroptosis-Mediated Cancer Treatment

“…As cell death featured by the accumulation of lethal lipid peroxides (LPO), ferroptosis can overcome the apoptosis resistance of classical therapeutics and holds great promise for cancer treatment. − Since the production of LPO is derived from the iron-mediated Fenton reaction, which converts endogenous H 2 O 2 to hydroxyl radicals (·OH) that oxidize lipids, ferroptosis heavily relies on the intracellular iron ion level. , Currently, two strategies have been developed to supplement iron ions: one is the induction of intracellular ferritin degradation, − and the other is the delivery of iron-based materials, such as zero-valent iron, iron oxide, , and iron-organic frameworks (MOFs). , However, there is a low content of intracellular ferritin (141.3–587.9 ng per mg of cytosol protein), and the iron-based materials easily cause adverse effects such as liver damage and chronic kidney disease at a dose as low as 5 mg/kg. − Although the exogenous iron-containing proteins have superior biocompatibility and have been employed to supplement intracellular iron, their extraction, purification, and storage are complicated and costly, seriously hindering their clinical application. − Therefore, the development of a new iron-supplement strategy to induce ferroptosis is urgent.…”