Enhanced Stability of Long-Living Immobilized Recombinant β-d-N-Acetyl-Hexosaminidase A on Polylactic Acid (PLA) Films for Potential Biomedical Applications

Abstract

β-d-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase that catalyzes the cleavage of the β-1,4 bond in N-acetyl-d-galactosamine (Gal-NAc) and N-acetyl-d-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates. It is widely expressed in both the prokaryotic and eukaryotic world, where it performs multiple and important functions. Hex has antifungal activity in plants, is capable of degrading many biological substrates, and can play an important role in the biomedical field for the treatment of Tay-Sachs and Sandhoff diseases. With the aim being able to obtain a device with a stable enzyme, a method of covalent immobilization on polylactic acid (PLA) films was developed for the A isoform of the β-d-N-acetyl-hexosaminidase enzyme (HexA), produced in a recombinant way from Human Embryonic Kidney-293 (HEK-293) cells and suitably purified. An in-depth biochemical characterization of the immobilized enzyme was carried out, evaluating the optimal temperature, thermal stability, pH parameters, and Km value. Moreover, the stability of the enzymatic activity over time was assessed. The results obtained showed an improvement in terms of kinetic parameters and stability to heat for the enzyme following immobilization and the presence of HexA in two distinct immobilized forms, with an unexpected ability for one of them to maintain its functionality for a long period of time (over a year). The stability and functionality of the enzyme in its immobilized form are therefore extremely promising for potential biotechnological and biomedical applications.

HexA-Enzyme Coated Polymer Nanoparticles for the Development of a Drug-Delivery System in the Treatment of Sandhoff Lysosomal Storage Disease

“…The immobilization protocol is the object of an Italian Patent n. 102020000003344 and has previously been described elsewhere [ 35 , 45 ]. It involves a first functionalization step in order to cover the material with NH 2 groups and a subsequent activation phase of the NPs with glutaraldehyde.…”

“…Afterward, NPs were activated with a 2.5% glutaraldehyde solution in deionized water, left to act for 3 h at room temperature. After removal of the glutaraldehyde solution and abundant washing of the PLA NPs with deionized water, the previously purified HexA enzyme [ 35 ] was immobilized, leaving 1 mL of the 50 µg/mL enzymatic solution in an overnight incubation at 4 °C. Each step of the functionalization/immobilization process was followed by centrifugation at 14,000× g for 20 min to form an NP pellet.…”

“…These nanosystems ensure greater physico-chemical and biological stability of the transported molecules and in this way, the intravenously injected enzymes reach the body’s organs more efficiently [ 14 , 15 ]. Enzymes’ stability is a major issue when dealing with their delivery and different immobilization techniques offer different advantages, for example, entrapment allows enzyme activity protection, while covalent immobilization, being characterized by strong bonding with the polymer surface, prevents enzyme molecules from leaching and endows them with high thermal stability [ 35 , 36 ]. As part of the enormous potential represented by drug delivery mediated by NPs, we developed a system consisting of polymeric NPs of polylactic acid (PLA) covered with a lysosomal enzyme, β- d - N -acetyl-hexosaminidase A, to evaluate the effectiveness of internalization and functioning of HexA-NP complex in cellular models of Sandhoff disease, one of the most serious disorders among all LSDs [ 37 ].…”

“…PLA was chosen because it is a biocompatible and biodegradable material, but especially by virtue of its long half-life when in contact with biological media. In fact, biocompatibility, biodegradability, and long half-life are the three key features that make PLA perfectly fit for biomedical applications [ 35 ]. Biocompatibility is necessary for a safe administration of the enzyme, biodegradability allows the polymer not to be accumulated in the body after it has carried out its task, and a long half-life is fundamental for the prolonged functionality of the enzyme-NP system.…”

“…There is currently no cure for this pathology, and although ERT has been the most used strategy, it has so far failed due to the instability of the recombinant enzyme administered and its inability to reach the CNS [ 42 , 43 , 44 ]. However, as already fully explained in our previous article [ 35 ], it is possible to develop a system in which the HexA enzyme is covalently immobilized on a solid support to increase its stability. In fact, the immobilization of the enzyme prevents its inactivation, potentially caused by multiple factors, and increases its half-life up to over a year.…”