Preserving Catalytic Activity and Enhancing Biochemical Stability of the Therapeutic Enzyme Asparaginase by Biocompatible Multilayered Polyelectrolyte Microcapsules

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 (CaCO₃) particles with two or three poly dextran/poly-l-arginine-based bilayers. To achieve high loading efficiency, the CaCO₃ template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO₃ 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 CaCO₃ 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

Nanoplasmonics for Dual-Molecule Release through Nanopores in the Membrane of Red Blood Cells

A nanoplasmonics-based opto-nanoporation method of creating nanopores upon laser illumination is applied for inducing diffusion and triggered release of small and large molecules from red blood cells (RBCs). The method is implemented using absorbing gold nanoparticle (Au-NP) aggregates on the membrane of loaded RBCs, which, upon near-IR laser light absorption, induce release of encapsulated molecules from selected cells. The binding of Au-NPs to RBCs is characterized by Raman spectroscopy. The process of release is driven by heating localized at nanoparticles, which impacts the permeability of the membrane by affecting the lipid bilayer and/or trans-membrane proteins. Localized heating and temperature rise around Au-NP aggregates is simulated and discussed. Research reported in this work is relevant for generating nanopores for biomolecule trafficking through polymeric and lipid membranes as well as cell membranes, while dual- and multi-molecule release is relevant for theragnostics and a wide range of therapies