Microfluidic Multiple Chamber Chip Reactor Filled with Enzyme-Coated Magnetic Nanoparticles

In this chapter, a novel microfluidic device (MagneChip) is described which comprises microliter volume reaction chambers filled with magnetically fixed enzyme-coated magnetic nanoparticles (ecMNPs) and with an in-line UV detector. In the experiments, MNPs with phenylalanine ammonia-lyase (PAL)—an enzyme which catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate in many organisms—immobilized on the surface were applied as biocatalyst to study the characteristics of the MagneChip device. In the reaction chambers of this microfluidic device, the accurate in situ quantization of the entrapped MNPs was possible using a resonant coil magnetometer integrated below the chambers. Computational fluid dynamics (CFD) calculations were used to simulate the flow field in the chambers. The enzyme-catalyzed biotransformations could be performed in the chip with excellent reproducibility and of repeatability. The platform enabled fully automatic multiparameter measurements with a single biocatalyst loading of about 1 mg PAL-ecMNP in the chip. A study on the effect of particle size and arrangement on the catalytic activity revealed that the mass of ecMNPs fixed in the chamber is independent of the particle diameter. Decreasing the particle size resulted in increasing catalytic activity due to the increased area to volume ratio. A binary mixture of particles with two different particle sizes could increase the entrapped particle mass and further the catalytic activity compared to the best uniform packing. The platform enabled a study of biotransformation of l-phenylalanine and five unnatural substrates by consecutive reactions using same PAL-ecMNP loading. With the aid of the platform, we first demonstrated that PAL can catalyze the ammonia elimination from the noncyclic propargylglycine as substrate

Biomimetikus katalizátorok fejlesztése mikrofluidikai reaktorokhoz: Development of Biomimetic catalysts for microfluidic reactors

During discovery of drug molecules, metabolism studies are important topic, which are usually carried out in vivo or in vitro using cell based systems. Instead of using living organism-based methods biomimetic systems can offer a promising alternative. Synthetic metalloporphyrins as biomimetic catalysts have strong structural similarity to the active site of the CYP enzymes responsible for the oxidative metabolism of drugs. The applicability and robustness of the porphyrin catalysts can be improved by immobilization techniques involving rationally functionalized solid carriers. The use of magnetic nanoparticles (MNPs) as catalyst carrier provides unique benefits, while the trapping, isolation or separation of the particles from the reaction mixture can be achieved with magnetic field. During my research, MNPs were prepared, modified with reactive function groups for immobilization of porphyrin catalyst, and inert groups which can influence the function group density. The fine-tuned immobilized catalyst was chosen and applied for continuous-flow microfluidical experiment. By the application of the developed biomimetic catalyst and reactor system, drug metabolites can be produced in very rapid way for further stages of drug discovery.   Kivonat A gyógyszervegyületek kifejlesztése során az egyik kritikus fontosságú terület a metabolizmus kutatás, mely során jellemzően in vivo és in vitro májsejt alapú rendszereket használnak, melyek számos hátránnyal rendelkeznek. Az ún. biomimetikus eljárások ígéretes alternatívát jelenthetnek, melyek a kiemelkedő katalitikus hatással bíró szintetikus metalloporfirinekkel megvalósíthatók. A porfirinek alkalmazása a szerkezeti hasonlóságukon alapszik a metabolizmusban részvevő CYP enzimek aktív helyén található hem csoporttal. Az érzékeny porfirin katalizátor alkalmazhatóságát, stabilitását nagyban javíthatjuk, ha valamilyen szilárd hordozó felületére rögzítjük. Mágneses nanorészecskék alkalmazása előnyös katalizátor hordozóként, mivel helyhez rögzítésük vagy elválasztásuk a reakcióelegytől mágneses erőtérrel megvalósítható. Munkám során mágneses nanorészecskék szintézisét és felületmódosítását valósítottam meg. A részecskék felületén a porfirin rögzítésére szolgáló reaktív funkcióscsoportokat, valamint a funkcióscsoport sűrűségét befolyásoló inert csoportokat alakítottam ki. Az ily módon finomhangolt katalizátort sikeresen alkalmaztam folyamatos áramú mikrofluidikai reaktorokban gyógyszermetabolitok szintézisére

Nanoformulation of Therapeutic Enzymes: A Short Review

Enzyme replacement therapy (ERT) is a therapeutic approach that involves the administration of specific enzymes to the patient in order to correct metabolic defects caused by enzyme deficiency. The formulation of ERTs involves the production, purification, and formulation of the enzyme into a stable and biologically active drug product, often using recombinant DNA technology. Non-systemic ERTs often involve the immobilization of the enzyme on a carrier, such as hydrogels, liposomes, or nanoparticles. ERT holds great promise for the treatment of a wide range of genetic disorders, and its success regarding lysosomal storage diseases, such as Fabry disease, Gaucher disease, and Pompe disease has paved the way for the development of similar therapies for other genetic disorders too

Electrospun Nanofibers for Entrapment of Biomolecules

This chapter focuses on nanofiber fabrication by electrospinning techniques for the effective immobilization of biomolecules (such as enzymes or active pharmaceutical ingredients—APIs). In this chapter, the development of precursor materials (from commercial polymer systems to systematically designed biopolymers), entrapment protocols, and precursor-nanofiber characterization methods are represented. The entrapment ability of poly(vinyl alcohol) and systematically modified polyaspartamide nanofibers was investigated for immobilization of two different lipases (from Candida antarctica and Pseudomonas fluorescens) and for formulation of the antibacterial and antiviral agent, rifampicin. The encapsulated biomolecules in electrospun polymer fibers could be promising nanomaterials for industrial biocatalysis to produce chiral compound or in the development of smart drug delivery systems

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases

Electrospinning was applied to create easy-to-handle and high- surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac- 2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB- T2-150). © 2016 Springer-Verlag Berlin Heidelber

Novel biomimetic nanocomposite for investigation of drug metabolism

In vitro mimicking of hepatic drug metabolism is a key issue in early-stage drug discovery. Synthetic metalloporphyrins show structural similarity with the heme type prosthetic group of cytochrome P450 as primary hepatic enzyme in oxidative drug biotransformation. Therefore, they can catalyze these oxidations. Concerning economical aspects and the poor stability of metalloporphyrin, their immobilization onto or into solid carriers can be promising solution. This study presents a novel immobilized metalloporphyrin nanocomposite system and its potential use as biomimetic catalysts. The developed two-step immobilization procedure consists of two main steps. First, the ionic binding of meso-tetra (parasulphonatophenyl) iron porphyrin onto functionalized magnetic nanoparticles is established, followed by embedding the nanoparticles into polylactic acid nanofibers by electrospinning technique. Due to the synergistic morphological and chemo-structural advantages of binding onto nanoparticles and embedding in polymeric matrices the biomimetic efficiency of metalloporphyrin can be remarkably enhanced, while substrate conversion value was tenfold larger than which could be achieved with classic human liver microsomal system

Conservation of the Biocatalytic Activity of Whole Yeast Cells by Supported Sol – Gel Entrapment for Efficient Acyloin Condensation

In this study, an efficient and generally applicable 2nd generation sol – gel entrapment method was developed for immobilization of yeastcells. Cells of Lodderomyces elongisporus, Candida norvegica, Debaryomyces fabryi, Pichia carsonii strains in admixture with hollow silica microspheres support were immobilized in sol – gel matrix obtained from polycondensation of tetraethoxysilane. As biocatalysts in theselective acyloin condensation of benzaldehyde catalyzed by pyruvate decarboxylase of the yeast, the novel immobilized whole-cell preparations were compared to other states of the cells such as freshly harvested wet cell paste, lyophilized cells and sol – gel entrapped preparations without hollow silica microspheres support. Reusability and storability studies designated this novel 2nd generation sol – gel method as a promising alternative for solid formulation of whole-cells bypassing expensive and difficult downstream steps while providing easy-to-handle and stable biocatalysts with long-term preservation of the biocatalytic activity