Salt-Compact Albumin as a New Pure Protein-based Biomaterials: From Design to In Vivo Studies

Abstract

Current biodegradable materials are facing many challenges when used for the design of implantable devices because of shortcomings such as toxicity of crosslinking agents and degradation derivatives, limited cell adhesion, and limited immunological compatibility. Here, a class of materials built entirely of stable protein is designed using a simple protocol based on salt-assisted compaction of albumin, breaking with current crosslinking strategies. Salt-assisted compaction is based on the assembly of albumin in the presence of high concentrations of specific salts such as sodium bromide. This process leads, surprisingly, to water-insoluble handable materials with high preservation of their native protein structures and Young's modulus close to that of cartilage (0.86 MPa). Furthermore, these materials are non-cytotoxic, non-inflammatory, and in vivo implantations (using models of mice and rabbits) demonstrate a very slow degradation rate of the material with excellent biocompatibility and absence of systemic inflammation and implant failure. Therefore, these materials constitute promising candidates for the design of biodegradable scaffolds and drug delivery systems as an alternative to conventional synthetic degradable polyester materials.

Keywords: albumin; biodegradable materials; protein‐based materials; salt‐assisted compaction.

Figure 1

Preparation of albumin materials by salt‐assisted compaction. A) Formulation procedure. The evaporation is carried on in an oven (37 °C) for 7 days until the biomaterial is completely dry. After evaporation, washing, and soaking steps are applied to remove the salt, leaving a water‐insoluble albumin material. B) The listed BSA/salt solutions were screened to identify the formulations leading to the formation of water‐insoluble materials. Each solution (BSA concentration = 100 mg mL⁻¹, pH = 6) was evaporated for 7 days until the formation of a dry solid. The obtained solid was washed in milliQ water for 48 h. Only the formulation “M” produced water‐insoluble and handable membranes. The formulation “S” produced water‐soluble residues. The formulation “F” produced fragmented materials that were fragile and not handable. C) Stability of BSA/NaBr membranes in aqueous media, ethanol, and a solution of trypsin. For each dissolution media, three membranes were placed in 25 mL of media. The experiments were performed at 37 °C and under stirring for 7 days. Physiological saline solution (0.9% NaCl), NaCl solution (1 m), NaBr solution (1 m), acidic solution (pH = 3), alkaline solution (pH = 10), trypsin (0.5 mg mL⁻¹).

Figure 2

Rheological evaluation of the viscoelastic behavior of BSA/NaBr membranes (hydrated in water). A) Amplitude sweep tests (frequency = 0.5 Hz, strain = from 0.01 to 100%, 25 °C). Storage (G’, red curve) and loss (G”, blue curve) modulus are represented as a function of the strain. G” modulus reaches a maximum (Payne effect). B) SEM analysis of the cross‐section of BSA/NaBr membranes. C) AFM observation of BSA/NaBr membrane in the liquid phase. D) Relaxation time assay performed by applying a shear strain of 1% (15 min, 25 °C). The increase in stress to a maximum of 2005 Pa is attributed to the rise in applied force during the first 4 s of the experiment to reach the strain of 1%.

Figure 3

Biological evaluation in vitro of BSA/NaBr membranes (prepared with NaBr, molar ratio NaBr/albumin = 664). A) Viability (n = 4) of Balbc 3T3 cells cultivated for 24 h with dilutions (12.5%, 25%, 50%, and 100% v/v) of extracts prepared by incubating BSA/NaBr membranes in culture media (72 h, 37 °C). The cells of the positive (Ctl+) and negative (Ctl‐) controls were incubated in culture media and 20% DMSO (diluted in culture media) respectively. B) Viability (n = 20) of Balbc 3T3 cells cultivated in direct contact with BSA/NaBr membranes. The controls, Ctl+ and Ctl‐, were incubated in culture media and 20% DMSO (diluted in culture media) respectively. C) Balbc 3T3 cells on the surface of BSA/NaBr membranes. D,E) MDCK cells adhered on the surface of BSA/NaBr membranes and formed a monolayer of epithelial cells. F,G) Macrophage activation assessment (n = 8) by titration of nitrites and TNF‐α in the supernatants after cultivation of RAW macrophages for 48 h with BSA/NaBr membranes. The non‐treated group (NT) was cultivated without membranes and without LPS. LPS was added after 24 h (50 ng mL⁻¹) to all LPS‐treated groups, including the LPS‐treated control (T (LPS)). (*) A statistically significant difference was observed (p < 0.05).

Figure 4

Biological evaluation of albumin materials implanted subcutaneously in NMRI‐Nude mice for 28 days. A) Albumin cylindrical implants (≈5 mm in diameter and ≈10 mm in length). B) Histological evaluation of BSA/NaBr materials after mice sacrifice (the localizations of the implants are marked with red arrows). C) Body weight measurements of Nude mice implanted with albumin materials. D) Implant volume measurements during 28 days of subcutaneous implantation in Nude mice.

Figure 5

Biological evaluation of albumin materials implanted subcutaneously in NZW rabbits for 28 days. Surgical incision was performed on the control rabbits (n = 3) without implantation. Treated groups were implanted with HSA/CaCl₂ (n = 6) and RbSA/AcOK (n = 6) materials. A) Body weight measurements of NZW rabbits. B) Implant volume measurements. C‐D) ALAT and ASAT titration in rabbit serum. The dashed line represents the range of concentrations commonly measured for rabbit controls (ALAT: 13–67 IU, ASAT: 3.8–60.4 IU).