Recombinant Protein-Based Nanoparticles: Elucidating Their Inflammatory Effects In Vivo and Their Potential as a New Therapeutic Format

Abstract

Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties—intrinsic to IBs—with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.

Potential of MMP-9 based nanoparticles at optimizing the cow dry period: pulling apart the effects of MMP-9 and nanoparticles

“…3d), as they are the first immune cell to arrive to the inflammatory site, and in agreement with the behaviour previously observed in mice after the administration of rMMP-9 IBs 14 . However, in the mouse model, the inactive rMMP-9 nanoparticles only induced a slightly and transitory inflammatory effect, whereas the active form had a clear and sustained effect over time 14 . Thus, the performance of mutMMP-9 nanoparticles was different in the mouse intra-dermis model compared with the bovine mammary gland.…”

“…Thus, these results confirm that the obtained effects in the bovine mammary gland were mainly due to the format (i.e., IB) but not due to the activity of the protein embedded in these nanoparticles. Again, this was an unexpected outcome because, in mice, the effect of MMP-9 could be clearly differentiated already at day 1 after injections from the inflammatory consequences of the nanoparticle format 14 . The question now is what is causing such different effects between the two animal models?…”

“…Aiming to determine whether the detected immune response triggered in the mammary gland by rMMP-9 IBs 8 ( Fig. 1) was only due to the nanoparticle format or whether the activity of the MMP-9 embedded in such nanoparticles was also relevant, as observed in mice 14 , an Exp. 3 was conducted.…”

The future of recombinant host defense peptides

“…IBs are protein nanoparticles or aggregates whose formation has been widely described in E. coli [56,57], but also in other microbial expression systems such as lactic acid bacteria [66][67][68][69][70] and yeast [70,71]. These aggregates can be easily purified [72] and offer interesting features not available in a soluble form.…”

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies

“…In this study, two endotoxin-free prokaryotic expression systems, Clearcoli ® BL21(DE3) and Lactococcus lactis were transformed with an expression vector containing a His-tagged version of the bovine MMP-9 gene to compare for the ability of the corresponding protein folding machinery to cope with the product of the overexpressed gene. MMP-9 was produced mostly in the form of IBs in both expression systems, and mild detergent treatment was performed to release entrapped protein, as reported [ 18 ]. The results showed that in both cases, four different peaks were obtained after affinity chromatography analysis indicating the presence of several subpopulations of conformers with variable ability to interact and coordinate to the Ni 2+ of the resin.…”