Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli

Abstract

Background: 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 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.

Results: We 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.

Discussion: The 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.

Keywords: Biodistribution; Biomaterials; E. coli; Endotoxin-free strains; Nanomedicine; Nanoparticles; Protein engineering; Recombinant proteins.

Fig. 1

Protein production and purification. a Scheme of the modular T22-GFP-H6 building block. Sizes of boxes are only approximate and do not precisely correspond to the length of primary aa sequence. b Western blot of crude cell extracts of T22-GFP-H6-producing strains. MW indicates the molecular weight of selected markers. c His-tag affinity chromatography of T22-GFP-H6 produced in the different E. coli strains. Purification was performed using an imidazole concentration gradient. Buffer B contains 500 mM imidazole. d Maldi-TOF identification of T22-GFP-H6 produced in different E. coli strains and purified by affinity chromatography (P2)

Fig. 2

Quantitative analyses of T22-GFP-H6 production levels and activity. a Yield of T22-GFP-H6 produced in E. coli strains Origami B, MC4100, KPM335 and BW30270, upon purification, from 500 ml of original protein producing cultures. P1 and P2 indicate the main elution peaks 1 and 2. b Specific fluorescence of T22-GFP-H6 calculated by using the above data. Symbols mean significant differences: **p < 0.001; *p < 0.05

Fig. 3

Physical properties of protein nanoparticles. a Size of the self-assembled nanoparticles purified in two elution peaks and measured by DLS. b Surface charge distribution of self-assembled nanoparticles measured as zeta potential. Symbols mean significant differences: **p < 0.001; *p < 0.05

Fig. 4

Morphometric characterization of nanoparticles ultrastructure. Representative TEM and FESEM (insets) images of all strains and main elution peaks 1 and 2. Scale bars represent 50 nm

Fig. 5

Cell penetrability of nanoparticles. a Internalization of self-assembling nanoparticles analyzed by intracellular fluorescence of HeLa cells incubated with 25 nM of T22-GFP-H6 for 1 h. Internalize fluorescence (determined by flow cytometry) is corrected by specific fluorescence (determined by fluorimetry). The quotient represents relative protein amounts. b Internalization of nanoparticles visualized by confocal microscopy. Nucleus (blue) of HeLa cells were labeled using Hoechst 33342, membranes (red) were labeled with CellMask Deep Red. Cells were incubated with 25 nM of T22-GFP-H6 (green) for 1 h. Bars indicate 15 µm. c Three dimensional 3D reconstruction of T22-GFP-H6 nanoparticles (P2) from KPM335 into HeLa cells. Images were obtained using 35 layers per image. Three different fields of confocal microscopy as well as three angles were viewed through Imaris Bitplane Software. Scale bars indicate 15 µm and symbols mean significant differences: **p < 0.001; *p < 0.05

Fig. 6

In vivo biodistribution of nanoparticles in tumor tissue. a Protein accumulation determined by ex vivo imaging of GFP-emitted fluorescence by representative subcutaneous colorectal cancer tumors grown in mice (measurements performed 5 h after the intravenous administration of a 500 μg nanoparticle dose). b Quantitation of GFP-emitted fluorescence in tumors of the compared groups, expressed as total radiant efficiency (photon/s/cm²/sr/μW/cm²). c Representative micrographs of nanoparticle internalization, as detected by IHC with an anti-GFP antibody, in the tumor cell cytosol and CXCR4 expression observed in tumor cells before nanoparticle injection in representative tumors. d Mean ± S.E. of CXCR4 expression H-score in tumor tissue (measured prior to nanoparticle administration) and GFP H-score (a measure of nanoparticle internalization in tumor cells) of mice belonging to the Origami B, MC4100, KPM335, BW30270 or buffer-treated groups. Note the similar level of CXCR4 expression in tumors among groups and the correlation between tumor-emitted fluorescence and the amount of nanoparticle internalized in tumor cells

Fig. 7

Nanoparticle biodistribution in normal tissues. a Representative ex vivo images of T22-GFP-H6 nanoparticles uptake in mouse brain, liver, kidney, lung or heart tissue after injection as measured by GFP-emitted fluorescence. b Quantitation of GFP fluorescence expressed as radiant efficiency. Note the negligible level of detected fluorescence, being undistinguishable from the background fluorescence observed in vehicle-treated mice. This was in contrast to the high level of nanoparticle accumulation observed in tumor tissues in the same animals (see Fig. 6)

Fig. 8

Conformational protein status and activity of the cell’s quality control. a Smoothed CD spectra from 250 to 205 nm of T22-GFP-H6 produced by different E. coli strains and purified at higher imidazol concentration (P2). Beta sheet structure signal is detected at 218 nm. b Expression of chaperone genes in E. coli KPM335 compared to their expression levels in E. coli BW30270. Relative changes in expression of target genes as quantified by RT-qPCR using the comparative 2^−∆∆Ct method [25] and the ihfB gene for normalization were converted into log2 values to display the fold changes in log2 scale. The values represent the means and standard deviations based on duplicate RT-qPCR runs for each of the three independent biological replicates per strain. The statistical significance of the differential expression patterns was analyzed using the paired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Genes labelled with a dash are those whose expression change (either up- or down-regulation) is coincident with the proteomic analysis of a conventional E. coli strain when entering into the stationary phase (according to data from [26]). The tig, hscB, hchA and cbpA gene products were not monitored in this previous study