Abstract
BackgroundProduction of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials.ResultsWe have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions.DiscussionThe observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.
Highlights
Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product applica‐ tion
Lipopolysaccharide (LPS) removal increases the complexity of protein purification processes, and it is recognized that LPS, even in low amounts, is a common contaminant of complex protein complexes such as bacteriophages [6] and of plain recombinant proteins [7]
We were interested in dissecting the structure and activities of these tumor-homing nanoparticles when produced in endotoxin-free E. coli cells, whose complex genetic development might have resulted in relevant physiological shifts
Summary
Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product applica‐ tion. Contaminant LPS and other cell envelope components are responsible for wrongly attributed biological effects of recombinant proteins tested in biomedical assays, and for poor biocompatibility (some examples can be found in [8,9,10]) In this context, Gram-positive bacteria, yeast and mammalian cells are endotoxin-free alternatives to E. coli, and their prevalence in protein drug production has steadily increased [11, 12], not absent of problems. We wanted here to explore how the production, functionalities and nanoarchitecture of a complex, virus-like protein nanoparticle (T22-GFPH6) designed as drug carrier [16] would be affected by the use of this particular fabrication platform These particles are formed by a self-organizing, CXCR4-targeted polypeptide that accumulates, in the assembled form, in tumoral tissues of colorectal cancer animal models [16, 17]. We were interested in dissecting the structure and activities of these tumor-homing nanoparticles when produced in endotoxin-free E. coli cells, whose complex genetic development might have resulted in relevant physiological shifts
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