Entrapment of Enzymes within Sol–Gel-Derived Magnetite

Abstract

Magnetically controlled enzymatic composites have received much attention for both therapeutic and industrial applications. Until now, such materials have been composed of at least four components: the enzyme, magnetic nanoparticles, their stabilizing components, and an organic or inorganic (or hybrid) matrix as a carrier. However, such compositions affect the magnetic response and the enzymatic activity, and also pose obstacles for intravenous administration, because of regulatory restrictions. Here, we present a methodology for the creation of magnetic bioactive nanocomposites composed of only two biocompatible components: an enzyme and magnetite nanoparticles. A series of magnetic biocomposites with a full set of therapeutical and industrial proteins (carbonic anhydrase, ovalbumin, horseradish peroxidase, acid phosphatase, proteinase, and xylanase) were successfully created by the direct entrapment of the proteins within a sol–gel magnetite matrix specially developed for these aims. The activity of the entrapped enzymes was studied at different temperatures and concentrations, and it was found that they showed remarkable thermal stabilization induced by the ferria matrix. For instance, entrapped carbonic anhydrase catalyzed the decomposition of p-nitrophenylacetate at a temperature of 90 °C, while free enzyme completely loses activity and denaturates already at 70 °C. Magnetic characterization of the obtained biomaterials is provided.

Thrombin@Fe3O4 nanoparticles for use as a hemostatic agent in internal bleeding

“…The key feature of the hydrosols is the excellent colloidal stability at neutral pH level and absence of any extraneous molecules on the surface of the nanoparticles. Due to synthetic conditions, the surface of the used magnetite nanoparticles contains a large number of hydroxyl groups, so that their isoelectric point is shifted to pH 8.2 and zeta potential at neutral pH values is sufficient to form a stable colloidal system (ESI Figs S3 and S4 ) Due to the lack of strong acids and bases in the hydrosol composition, it is compatible with enzyme molecules and does not cause their inactivation upon mixing 21 . Human thrombin was selected as a bioactive agent for producing a composite because of its structure and mechanism of action 26 .…”

“…Under the influence of this factor, strong covalent bonds between γ - and also between α -polypeptide chains of fibrin-aggregate molecules are formed, as a result of which it is stabilized into fibrin-polymer insoluble in media with high ionic strengths. Immobilization of thrombin was performed in a manner it was described earlier 21 (for details see experimental part). For this purpose thrombin was entrapped within magnetite sol-gel matrix in a course of irreversible room-temperature sol-gel process.…”

“…Subsequent solvent removal led to formation of interparticle bonds between magnetite nanoparticles and formation of inorganic matrix doped by enzyme molecules immobilized in micropores of the material. The enzyme is immobilized rather physically than chemically, since no covalent bonds are formed between enzyme and inorganic matrix 21 , 29 , 30 . Entrapped enzyme is homogeneously distributed in the composite and can be located both inside the matrix and in its near-surface layer.…”

“…In combination with fibrinogen, it enables skipping most stages of the coagulation mechanism, leading the process to the final stage of the coagulation cascade, so that two drugs quickly form a fibrin clot in the bleeding zone. As regards a method for creating a styptic magnetic colloid, we propose the use of entrapment methodology developed by us previously 21 and successfully tested using the example of injectable thrombolytic drugs 22 .…”

Leach-proof magnetic thrombolytic nanoparticles and coatings of enhanced activity

“…Nanocomposites were obtained (for details, see experimental part) via co-condensation of urokinase-type plasminogen activator (uPA), which is commonly used in clinical practice, and magnetite sol prepared by the recently published method implying the production of high purity magnetite sol without using stabilizers and additives 19 . As a result of irreversible sol-gel transition, a xerogel nanoporous matrix forms, whose nanopores entrap an enzyme, and such a matrix can either be used as an enzymatically active coating or crushed to yield bioactive nanocomposite particles 20 . The matrix itself is made up of regular-shaped truncated octahedra with an average diameter of 10 nm ( Fig.…”