Mass Spectrometry, Structural Analysis, and Anti-Inflammatory Properties of Photo-Cross-Linked Human Albumin Hydrogels

Abstract

Albumin-based hydrogels offer unique benefits such as biodegradability and high binding affinity to various biomolecules, which make them suitable candidates for biomedical applications. Here, we report a non-immunogenic photocurable human serumbased (HSA) hydrogel synthesized by methacryloylation of human serum albumin by methacrylic anhydride (MAA). We used matrix-assisted laser desorption ionization-time-offlight mass spectrometry, liquid chromatography-tandem mass spectrometry, as well as size exclusion chromatography to evaluate the extent of modification, hydrolytic and enzymatic degradation of methacrylated albumin macromer and its cross-linked hydrogels. The impacts of methacryloylation and cross-linking on alteration of inflammatory response and toxicity were evaluated in vitro using brain-derived HMC3 macrophages and Ex-Ovo chick chorioallantoic membrane assay. Results revealed that the lysines in HSA were the primary targets reacting with MAA, though modification of cysteine, threonine, serine, and tyrosine, with MAA was also confirmed. Both methacrylated HSA and its derived hydrogels were nontoxic and did not induce inflammatory pathways, while significantly reducing macrophage adhesion to the hydrogels; one of the key steps in the process of foreign body reaction to biomaterials. Cytokine and growth factor analysis showed that albumin-based hydrogels demonstrated anti-inflammatory response modulating cellular events in HMC3 macrophages. Ex-Ovo results also confirmed the biocompatibility of HSA macromer and hydrogels along with slight angiogenesismodulating effects. Photocurable albumin hydrogels may be used as a non-immunogenic platform for various biomedical applications including passivation coatings.

Amyloid‐Based Albumin Hydrogels

“…While our experiments were performed with albumin from bovine sources, the high sequence homology with human albumin and previous work comparing the behavior of different albumins suggests that similar hydrogels could be prepared by human serum albumin. [11,56] Previous albumin-based hydrogels were chemically modified to introduce reactive groups on the protein for its subsequent cross-linking into 3D networks. [7][8][9]11] While such approaches result in more robust hydrogels due to covalent bond formation, they are associated with extra reaction and purification steps, concerns of side effects and extra costs.…”

“…[11,56] Previous albumin-based hydrogels were chemically modified to introduce reactive groups on the protein for its subsequent cross-linking into 3D networks. [7][8][9]11] While such approaches result in more robust hydrogels due to covalent bond formation, they are associated with extra reaction and purification steps, concerns of side effects and extra costs. Amyloid-like fibril formation of non-modified albumin was previously considered, but usually entailed a heating step that most likely causes protein degradation, as shown for similar proteins undergoing amyloidogenesis.…”

“…While our experiments were performed with albumin from bovine sources, the high sequence homology with human albumin and previous work comparing the behavior of different albumins suggests that similar hydrogels could be prepared by human serum albumin. [ 11,56 ]…”

“…Hence unsurprisingly, it has been previously considered by many as the building block for creating albumin-based hydrogels. [6][7][8][9][10][11] In most cases however, the use of chemical reagents to either chemically modify the albumin, or induce cross-linking of side chains, introduces concerns related to cytotoxicity, reproducibility, and undesired side products. An alternative approach that we explored in this study, is to denature albumin and drive its assembly into amyloid-like fibrils, which would then physically interact to create a free-standing hydrogel material.…”

Mussel-Inspired Surface Functionalization of Porous Albumin Cryogels Supporting Synergistic Antibacterial/Antioxidant Activity and Bone-Like Apatite Formation

“…It is a desired biomaterial because it stabilizes important proteins, hormones, metal ions, nanoparticles, and pharmaceuticals in addition to acting as a mild pH buffer [ 8 , 9 ]. Albumin hydrogels have gained increasing interest in biomedical applications ( Figure 1 and Table 1 ), particularly in treating numerous diseases, and tissue engineering due to the excellent qualities of albumin such as sustainability, non-immunogenicity, low cost, excellent biocompatibility, and biodegradability [ 7 , 10 , 11 , 12 , 13 , 14 ]. Hydrogels made from albumin can be created using a photo-triggered gelation process, chemicals, heat, or pH.…”