Functionalizing cryogels with the GPGKLVFF peptide for amyloid-β binding: A comparative study of two synthetic pathways

Macroporous cryogels (Cry) represent a versatile class of scaffolds with broad applicability in biomedical fields, including biomolecular immobilization, diagnostic sensing, and tissue engineering. More recently, their application in neuroscience has gained increasing interest as a viable alternative to conventional systems for drug delivery and neural tissue regeneration. A novel aspect of this work is the design and synthesis of cryogels functionalized with a peptide sequence capable of selectively binding β-amyloid (Aβ) peptides, key biomarkers of Alzheimer's Disease (AD). The peptide GPGKLVFF (KL), was covalently linked into the cryogel matrix using two distinct chemical protocols. The first, a one-step procedure, involves the simultaneous polymerization of a 2-hydroxyethyl methacrylate (HEMA)-N,N'-methylenebisacrylamide (MBAA) and different amounts of a methacrylate-conjugate peptide (maa-KL), enabling precise control over the degree of functionalization. The second, a two-step procedure, involves peptide grafting onto a preformed poly-methacrylic acid (pMAA) cryogel, resulting in significantly higher functionalization yields, albeit with less homogeneity in the material. Successful peptide incorporation was confirmed by infrared (IR) and Raman spectroscopy. A straightforward quantification protocol based on comparing the intensities of selected Raman marker bands was developed, allowing detection of grafted peptides even at low concentrations. The morphology of the cryogels was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The ability of the functionalized cryogels to reversibly bind and release Aβ40 was assessed by electrospray-ionization mass spectrometry (ESI-MS). This study represents a crucial first step toward developing a diagnostic platform for the early detection of β-amyloid proteins in AD.