Stable Bioactive Enzyme-Containing Multilayer Films Based on Covalent Cross-Linking from Mussel-Inspired Adhesives

The use of immobilized enzymes is mandatory for the easy separation of the enzyme, the unreacted substrates, and the obtained products to allow repeated enzymatic assays without cumbersome purification steps. The immobilization procedure is however critical to obtain a high fraction of active enzyme. In this article, we present an enzyme immobilization strategy based on a catechol functionalized alginate. We demonstrate that alkaline phosphatase (ALP) remains active in multilayered films made with alginate modified with catechol moieties (AlgCat) for long duration, that is, up to 7 weeks, provided the multilayered architecture is cross-linked with sodium periodate. This cross-linking reaction allows to create covalent bonds between the amino groups of ALP and the quinone group carried by the modified alginate. In the absence of cross-linking, the enzymatic activity is rapidly lost and this reduction is mainly due to enzyme desorption. We also show that NaIO₄ cross-linked (AlgCat-Alp)_<i>n</i> films can be freeze-dried and reused at least 3 weeks later without lost in enzymatic activity

Monitoring of CTL released from the gels.

<p>Fluorescence spectrophotometry was used to monitor CTL-Rhodamine released out of AC and AC/PlusbisSH gels over time (MH medium, 37°C).</p

Gelation kinetics of the AC and AC/PlubisSH gels.

<p>Rheological properties of AC (A) and AC/PlubisSH (B) hydrogels recorded at a frequency of 1 Hz at a temperature of 37°C (elastic modulus, G’ and viscous modulus, G”).</p

Antibacterial activity of gels.

<p>Metabolic activity of bacterial supernatant after 24h (A). « Plastic » corresponds to a negative control, <i>i</i>.<i>e</i>. bacteria in medium on the 96-well plate, without any gel. « Medium » corresponds to culture media in the 96-well plate without any bacteria or gel. « Antibiotic » corresponds to the positive control with bacteria in medium with two standard antibiotics (tetracyclin and cefotoxim) as supplements. The asterisk (*) denotes a statistical difference between the metabolic activity of <i>P</i>. <i>gingivalis</i> found in the supernatants of AC and AC-CTL gels, (#) indicates a statistical difference between the metabolic activity of <i>P</i>. <i>gingivalis</i> found in the supernatant of AC/PlubisSH and AC/PlubisSH-CTL (p < 0.05). Inhibition of colonies forming units (CFU) of the supernatants after 24h of seeding (B and C). These supernatants were previously removed from the gels respectively after 5h (B) and 24h (C) of seeding. The control used in these figures (corresponding to 100% CFU) corresponds to colonies on agar plates obtained from supernatant of AC gel without CTL (Tissue Culture Polystyrene was not used as control because it leads to an homogenous growth of bacteria without colonies). Error bars represent means ± SD.</p

<i>In vitro</i> quantitative adhesion of hydrogels on titanium (Ti) and gingiva.

<p>Dissipative energy during detachment of AC and AC/PlubisSH hydrogels from gingiva is measured after 30 min of contact with the substrates (Ti/Ti or Ti/gingiva) at 37°C.</p