In Vivo Architectonic Stability of Fully de Novo Designed Protein-Only Nanoparticles

Abstract

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

“…The different architectonic patterns adopted by GFP oligomers would necessarily be connected to distinct morphometries and biophysical properties of the materials, which have been so far generically identified as planar, toroid nanoparticles [19]. In this context, fine SAXS analyses revealed a broad range of shapes, from rod forms to spherical forms, depending on the SEC population to which they belong (Figure 2 A).…”

“…The de novo designed A5G27-GFP-H6 and T22-GFP-H6 nanoparticles show, by dynamic light scattering (DLS), average size peaks of 14 and 12 nm respectively when produced in the conventional E. coli strains BL21 (DE3) and Origami B respectively [19,20] (Supplementary Figure 1). Any intrinsic morphometric heterogeneity, if existing, has been so far unobserved and eclipsed in the analysis of the raw material.…”

“…The alternative morphometries in SEC peaks identified by SAXS were fully assessed by high resolution TEM and FESEM imaging (Figure 3), confirming the nano-architectonic variability of nanoparticles in a fully visual way. The protein nanoparticles studied here had been conceived as drug carriers for cancer treatments, what was lately encouraged by their good biodistribution when systemically administrated (upon which the material accumulated intracellularly in tumor and metastatic foci but not in liver, kidney, spleen or other nontarget organs) [19,20]. Then, how the morphometry and other physical properties of the oligomeric populations might influence receptor-dependent cell penetration is a critical issue that was addressed in a CXCR4 + cell culture model.…”

“…Driven by electrostatic interactions and with a strong involvement of the histidine-rich tail, these peptides promote the formation of stable oligomers of defined average size in the nanoscale irrespective of the amino acid sequence and origin of the core protein placed in between. The resulting nanoparticles, with a toroid-like shape and usually ranging between 12 and 40 nm, are full stable in vivo [19]. Such stability under physiological conditions, provided by complex forces sustaining protein-protein cross-interactions, enable the material escaping from renal clearance and it also allows prolonged circulation time in blood [19].…”

“…The resulting nanoparticles, with a toroid-like shape and usually ranging between 12 and 40 nm, are full stable in vivo [19]. Such stability under physiological conditions, provided by complex forces sustaining protein-protein cross-interactions, enable the material escaping from renal clearance and it also allows prolonged circulation time in blood [19]. When displaying appropriate peptide ligands of cell surface cancer markers CXCR4 or CD44 (T22 and A5G27 respectively) they specifically accumulate in primary tumor and metastasis in colorectal and mammary cancer models respectively [20,21], being suited for antitumoral drug delivery.…”