Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins

Abstract

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Hybrid Micro-/Nanoprotein Platform Provides Endocrine-like and Extracellular Matrix-like Cell Delivery of Growth Factors

“…It must be noted that more than one single bioactive protein could be combined in a multiple display system, which might allow the fast exploration of new synergies (or the full exploitation of the known ones) in the protein-based control of cell growth and differentiation. − Even the proposed platform is highly promising, the right selection of the particular GF, its ability to aggregate as His-tagged forms as leaking material, and the insolubility of the SGs leading to background adsorption might represent potential limitations for a straightforward application, which would involve a tailored design for specific purposes. Apart from that, the simplicity of the SG fabrication, the lack of in vivo toxicity, the full biological activity retailed by the building block polypeptide, even structurally complex, and the time-sustained release of the embedded proteins (combined with the mechanical stability of SGs) make the properties of the SG platform transversal and especially suited as a novel tool to control and regulate mammalian cell growth.…”

Zinc-Assisted Microscale Granules Made of the SARS-CoV-2 Spike Protein Trigger Neutralizing, Antivirus Antibody Responses

“…Since the success of vaccination is at least partially observed because of a prolonged exposure to antigens, we speculated that secretory granules might be good immunostimulatory agents. In fact, the slow release of eRDB from Zn-assisted depots had been already demonstrated in vitro, as well as their lack of toxicity . Being the amount of Zn in the granules (∼5 mg per dose) much lower than the recommended daily intake for humans (with an upper limit of ∼40 mg/day) and much far below the range of the metal in the human body (2–4 g), the presence of the metal should not be a matter of concern.…”

“…Even though some prototypes have reached the market, they have generally resulted in disappointing levels of immune activation and in limited effectiveness, that require repeated administrations and incorporation of adjuvants, resulting in high economic costs and logistic complexity. , Among other reasons, the failure in selecting or presenting the antigens in an appropriate way might be the cause for moderate immune responses. − In previous studies, we have developed Zn-supported protein-releasing microscale granular materials that, upon subcutaneous injection, act as intriguing drug-delivery systems . These protein granules are formed by pure protein preparations in which the individual polypeptide chains are clustered together into insoluble aggregates by the fast coordination between histidine residues of soluble proteins and divalent cations. , In the absence of local or systemic toxicities, the resulting micrometer-scale particles disassemble, through a self-disintegrating process in vivo, into functional building block polypeptides, during prolonged time periods . Both the structure and mechanics of the synthetic granular materials are similar to those of natural protein depots of protein/peptide hormones in the mammalian endocrine system, − Therefore, the observed protein leakage from the artificial materials, based on Zn chelation can be considered a secretion-like event within an endocrine-like process.…”