Preserving Catalytic Activity and Enhancing Biochemical
Stability of the Therapeutic Enzyme Asparaginase by Biocompatible
Multilayered Polyelectrolyte Microcapsules
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Abstract
The present study focuses on the
formation of microcapsules containing
catalytically active l-asparaginase (L-ASNase), a protein
drug of high value in antileukemic therapy. We make use of the layer-by-layer
(LbL) technique to coat protein-loaded calcium carbonate (CaCO<sub>3</sub>) particles with two or three poly dextran/poly-l-arginine-based bilayers. To achieve high loading efficiency, the
CaCO<sub>3</sub> template was generated by coprecipitation with the
enzyme. After assembly of the polymer shell, the CaCO<sub>3</sub> core
material was dissolved under mild conditions by dialysis against 20
mM EDTA. Biochemical stability of the encapsulated l-asparaginase
was analyzed by treating the capsules with the proteases trypsin and
thrombin, which are known to degrade and inactivate the enzyme during
leukemia treatment, allowing us to test for resistance against proteolysis
by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability,
the stability at the physiological temperature, and the long-term
storage stability of the encapsulated enzyme were investigated. We
show that encapsulation of l-asparaginase remarkably improves
both proteolytic resistance and thermal inactivation at 37 °C,
which could considerably prolong the enzyme’s in vivo half-life
during application in acute lymphoblastic leukemia (ALL). Importantly,
the use of low EDTA concentrations for the dissolution of CaCO<sub>3</sub> by dialysis could be a general approach in cases where the
activity of sensitive biomacromolecules is inhibited, or even irreversibly
damaged, when standard protocols for fabrication of such LbL microcapsules
are used. Encapsulated and free enzyme showed similar efficacies in
driving leukemic cells to apoptosis