In Vitro Fabrication of Microscale Secretory Granules

Abstract

Advanced medical treatments involving drug delivery require fully biocompatible materials with the ability to release functional drugs in a time-prolonged way. Ideally, the delivered molecules should be self-contained as chemically homogenous entities to prevent the use of potentially toxic scaffolds or hold matrices. In nature, peptidic hormones are self-stored in protein-only secretory granules formed by the reversible coordination of Zn 2+ and histidine residues. Inspired by this concept, an in vitro transversal procedure is developed, analyzed, and comparatively applied for the fabrication of protein-only secretory granules at the microscale. These materials can be produced from any polyhistidine-tagged protein using physiological concentrations of Zn 2+ as a potent and versatile glue-like agent. The screening of granules formed by 12 engineered and nonengineered proteins at different Zn 2+ concentrations revealed optimal fabrication conditions and the consequent release profiles. Moreover, the functional and structural properties of the delivered protein are fully validated using a drug-targeting protein platform in a mouse model of human colorectal cancer. In summary, short histidine tags allow the packaging of structurally and functionally dissimilar polypeptides, which supports the proposed fabrication method as a powerful protocol extensible to diverse clinical scenarios in which slow protein drug delivery is required.

Biparatopic Protein Nanoparticles for the Precision Therapy of CXCR4+ Cancers

“…Interestingly, such cytotoxic activities are not observed in cell culture upon moderate exposure times (Figure S4). In our hands, other pro-apoptotic peptides or protein domains from different origins including PUMA, BAXPORO, GWH1 and BAK, show no cytotoxic effects in cell culture but significant induction of apoptosis and reduction in the number of mitotic cells in vivo [40,46,73]. This contrast between the lack of antitumor activity in vitro and the potent induction of apoptosis in cancer cells in vivo may be based on the different regulation of these cells by the cell culture media as compared to their regulation by tumor microenvironment.…”

“…In the hybrid materials, EPI-X4 appears to be the only ligand responsible for promoting cell death as an efficient CXCR4 antagonist. In this regard, it must be noted that T22, in form of different types of protein-only nanoparticles (including GFP, BFP or iRFP) has been never observed as inducer of cell toxicity or apoptosis in cell culture or in vivo, in different animal models of human cancers [16,[46][47][48][49][50][51]. However, the use of T22-based vehicles to deliver small cytotoxic drugs [17,52,53] or proteins [24,40,45,48,54] result in highly selective tumor tissue destruction.…”

Ion-dependent slow protein release from in vivo disintegrating micro-granules

“…The elements selected for clustering were Zn and Ca, alone or as mixtures with Mg and Mn, namely ZnMn, ZnMg, MnMg, CaMn, and CaZn ( Figure 1(B) ). These cations were selected for their regular presence and relative abundance in living beings (Knape et al., 2017 ; Pilchova et al., 2017 ; Al Alawi et al., 2018 ; Li & Yang, 2018 ; Pazirandeh et al., 2020 ; Santos et al., 2020 ), thus skipping toxicities potentially linked to rarer oligoelements. These ions also cover a wide range in the Irving-Williams (Milicevic et al., 2011 ) series, which predicts differential stability of the complexes formed by those cations through coordination with histidine residues.…”

The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles

“…Histidine-rich peptides, when genetically fused to recombinant proteins, confer them the capability to self-assemble as different kinds of functional protein-only materials including fibers, nanoparticles (NPs) and microparticles, through their crossmolecular coordination with divalent cations [1]. This happens because the interactivity exhibited by the hexahistidine tag (H6) and other His-rich tails with Ni 2+ during the immobilized metal affinity chromatographic (IMAC) purification of recombinant proteins [2,3] can be also exploited for a controlled crossmolecular assembly, when adding defined amounts of Zn 2+ , Ca 2+ , Mn 2+ or other divalent cations to solutions of pure Histagged protein [4][5][6]. In fact, this principle sustains the formation of the secretory granules in the mammalian endocrine system [7,8] and of different types of amyloidal and non-amyloidal protein materials existing in nature [4,7,[9][10][11][12][13][14][15][16].…”