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

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

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

Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia-lyase (PAL), found in many organisms, catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in-line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts-type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)-pent-2-ene-4-ynoate yielding enantiopure l-PG, contradicts the proposed highly exothermic single-step mechanism. Computations with the QM/MM models of the N-MIO intermediates from l-PG and l-Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N-MIO intermediate

Combined Nanofibrous Face Mask: Co-Formulation of Lipases and Antibiotic Agent by Electrospinning Technique

The application of enzyme-based therapies has received significant attention in modern drug development. Lipases are one of the most versatile enzymes that can be used as therapeutic agents in basic skin care and medical treatment related to excessive sebum production, acne, and inflammation. The traditional formulations available for skin treatment, such as creams, ointments or gels, are widely applied; however, their use is not always accompanied by good drug penetration properties, stability, or patient adherence. Nanoformulated drugs offer the possibility of combining enzymatic and small molecule formulations, making them a new and exciting alternative in this field. In this study polymeric nanofibrous matrices made of polyvinylpyrrolidone and polylactic acid were developed, entrapping lipases from Candida rugosa and Rizomucor miehei and antibiotic compound nadifloxacin. The effect of the type of polymers and lipases were investigated, and the nanofiber formation process was optimized to provide a promising alternative in topical treatment. Our experiments have shown that entrapment by electrospinning induced two orders of magnitude increase in the specific enzyme activity of lipases. Permeability investigations indicated that all lipase-loaded nanofibrous masks were capable of delivering nadifloxacin to the human epidermis, confirming the viability of electrospinning as a formulation method for topical skin medications

Combined Nanofibrous Face Mask: Co-Formulation of Lipases and Antibiotic Agent by Electrospinning Technique

The application of enzyme-based therapies has received significant attention in modern drug development. Lipases are one of the most versatile enzymes that can be used as therapeutic agents in basic skin care and medical treatment related to excessive sebum production, acne, and inflammation. The traditional formulations available for skin treatment, such as creams, ointments or gels, are widely applied; however, their use is not always accompanied by good drug penetration properties, stability, or patient adherence. Nanoformulated drugs offer the possibility of combining enzymatic and small molecule formulations, making them a new and exciting alternative in this field. In this study polymeric nanofibrous matrices made of polyvinylpyrrolidone and polylactic acid were developed, entrapping lipases from Candida rugosa and Rizomucor miehei and antibiotic compound nadifloxacin. The effect of the type of polymers and lipases were investigated, and the nanofiber formation process was optimized to provide a promising alternative in topical treatment. Our experiments have shown that entrapment by electrospinning induced two orders of magnitude increase in the specific enzyme activity of lipases. Permeability investigations indicated that all lipase-loaded nanofibrous masks were capable of delivering nadifloxacin to the human epidermis, confirming the viability of electrospinning as a formulation method for topical skin medications

Preparation and Characterization of Biocompatible Electrospun Nanofiber Scaffolds

Nanoscale fibers were prepared for the fabrication of scaffolds by using a strong electrostatic field on the polymer solution. Electrospinning is widely applied for production of drug delivery, tissue engineering, and regenerative medicine systems as well as biosensors and enzyme immobilization. Nanofibers, thanks to their high surface area to volume ratio, can also mimic the extracellular matrix, thus it has been recognized as a suitable technique for the fast fabrication of scaffolds. This article demonstrates the fabrication of several nanofibrous scaffolds from biopolymers such as polycaprolactone, poly(lactic acid), poly(lactide-co-glycolide), poly(lactide-co-caprolactone) and poly(hydroxybutyrate-co-hydroxy valerate). The characterization and comparison of the scaffolds were achieved based on the morphology and surface characteristic of the nanofibers. The samples showed hydrophobic characteristic, thus a plasma surface treatment was applied successfully to increase hydrophilicity and the effect of the treatment was evaluated based on the wettability and the change in elemental composition of the surface based on X-ray photoelectron spectroscopy