No abstract

Spherical biopolymer particles have been fabricated, applying coprecipitation with calcium carbonate, followed by cross-linking of the macromolecules and dissolution of the inorganic support. Particles made of roughly 80% horseradish peroxidase (HRP) as well as glucose oxidase (GOX) were prepared and enzyme activities were confirmed, applying the Amplex Red assay. The enzyme particles were reusable for at least six times, with a remaining activity of 30-50% from the initial one. When multiple coprecipitation steps and one or several cross-linking procedures were applied, multicompartment particles were obtained. Each of the resulting concentric compartments could be independently loaded with biomolecules. Three coupled enzymes, beta-glucosidase (beta-Glu), GOX, and HRP have been incorporated stepwise in such particles. Each of these enzymes was located in a separate compartment, in a desired sequence, and at a defined position. The distance between the enzyme containing compartments was also varied, including spacing compartments consisting of bovine serum albumin (BSA). When fluorogenic substrates for beta-Glu and HPR were used, the start and the end of the coupled enzyme reaction were visualized and recorded inside of individual particles, applying confocal laser scanning microscopy. A strong influence of the spacing on the reaction kinetics of the last enzyme was observed, suggesting an impaired diffusion of the intermediate products of the chain reaction through the spacing compartments made of BSA. The influence of the spacing between compartments containing different enzymes on the reaction kinetics was demonstrated on the microscopic scale within one microparticle, which to the best of our knowledge was not achieved until now.

Rhodamin 6G were precipitated within the capsules.

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.