Chemical modification of α-chymotrypsin enabling its release from alginate hydrogel by electrochemically generated local pH change.

This study investigates the controlled release of α-chymotrypsin from an alginate hydrogel matrix. When protein molecules entrapped in the hydrogel matrix have a size smaller than the hydrogel pores, their hold/release in/from the polymer matrix is controlled by the electrostatic interaction between the guest molecules and host polymer. α-Chymotrypsin, as a model protein, was chemically modified with negatively charged species to change its pI and to convert its attractive interaction with a negatively charged alginate hydrogel matrix to a repulsion interaction allowing its release by pH-triggered signal. Then, bulk pH changes and electrochemically controlled local pH changes resulting from oxygen reduction were used for the controlled release of the enzyme from the alginate hydrogel. Three batches of modified α-chymotrypsin with different linker/enzyme ratios were synthesized, and their release profiles were investigated. The activity of both unmodified and modified α-chymotrypsin was evaluated using a UV-visible spectrophotometer following the standard procedure for the enzymatic assay of α-chymotrypsin (EC 3.4.21.1) and compared across all batches. Direct infusion electrospray ionization mass spectrometry (DI ESI-MS) was used to analyze the protein modifications and their impact on the isoelectric point values.

Time-Separated Pulse Release-Activation of an Enzyme from Alginate-Polyethylenimine Hydrogels Using Electrochemically Generated Local pH Changes.

ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.

Electrochemical and Biocatalytic Signal-Controlled Payload Release from a Metal-Organic Framework.

A metal-organic framework (MOF), ZIF-8, which is stable at neutral and slightly basic pH values in aqueous solutions and destabilized/dissolved under acidic conditions, is loaded with a pH-insensitive fluorescent dye, rhodamine-B isothiocyanate, as a model payload species. Then, the MOF species are immobilized at an electrode surface. The local (interfacial) pH value is rapidly decreased by means of an electrochemically stimulated ascorbate oxidation at +0.4 V (Ag/AgCl/KCl). Oxygen reduction upon switching the applied potential to -0.8 V allows to return the local pH to the neutral/basic pH, then stopping rapidly the release process. The developed method allows electrochemical control over stimulated or inhibited payload release processes from the MOF. The pH variation proceeds in a thin film of the solution near the electrode surface. The switchable release process is realized in a buffer solution and undiluted human serum. As the second option, the pH decrease stimulating the release process is achieved upon an enzymatic reaction using esterase and ester substrate. This approach potentially allows the release activation controlled by numerous enzymes assembled in complex biocatalytic cascades. It is expected that related electrochemical or biocatalytic systems can represent novel signal-responding materials with switchable features for delivering (bio)molecules within biomedical applications.

Stimulation-Inhibition of Protein Release from Alginate Hydrogels Using Electrochemically Generated Local pH Changes.

The electrochemically controlled release of proteins was studied in a Ca2+-cross-linked alginate hydrogel deposited on an electrode surface. The electrochemical oxidation of ascorbate or reduction of O2 was achieved upon applying electrical potentials +0.6 or -0.8 V (vs Ag/AgCl/KCl 3 M), respectively, resulting in decreasing or increasing pH locally near an electrode surface. The obtained local acidic solution resulted in the protonation of carboxylic groups in the alginate hydrogel and, as a result, the formation of a hydrophobic shrunken hydrogel film. Conversely, the produced alkaline local environment resulted in a hydrophilic swollen hydrogel film. The release of the proteins was effectively inhibited from the shrunk hydrogel and activated from the swollen hydrogel film. Overall, the electrochemically produced local pH changes allowed control over the biomolecule release process. While the release inhibition by applying +0.6 V was always effective and could be maintained as long as the positive potential was applied, the release activation was different depending on the protein molecular size, being more effective for smaller species, and molecule charge, being more effective for negatively charged species. The repetitive change from the inhibited to stimulated state of the biomolecule release process was obtained upon cyclic application of oxidative and reductive potentials (+0.6 V ↔ -0.8 V). The alginate hydrogel film shrinking-swelling as well as the protein release process were studied and visualized using a confocal fluorescent microscope. In order to be observed, an external surface of the alginate film and the loaded protein molecules were labeled with different fluorescent dyes, which then produced colored fluorescent images under a confocal microscope.

Multiple pH waves generated electrochemically and propagated from an electrode surface.

Local pH changes were produced upon electrochemical reactions. Cyclic application of reductive and oxidative potentials resulted in the formation of pH waves in the form of distinct solution layers. Multiple layers with basic and acidic pH values were visualized with a fluorescence confocal microscope following fluorescence of pH-dependent dyes.