Immobilization of human tyrosine hydroxylase onto magnetic nanoparticles - A novel formulation of a therapeutic enzyme.

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking.

A Comparative Pharmacokinetic Study for Cysteamine-Containing Eye Drops as an Orphan Topical Therapy in Cystinosis.

Cystinosis is a low-prevalence lysosomal storage disease. The pathomechanism involves abnormal functioning of the cystinosine lysosomal cystine transporter (CTNS), causing intraliposomal accumulation of the amino acid cysteine disulfide, which crystallizes and deposits in several parts of the body. The most common ophthalmic complication of cystinosis is the deposition of "gold dust" cystine crystals on the cornea, which already occurs in infancy and leads to severe photosensitivity and dry eyes as it gradually progresses with age.

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.

Natural Lipid Extracts as an Artificial Membrane for Drug Permeability Assay: In Vitro and In Silico Characterization.

In vitro non-cellular permeability models such as the parallel artificial membrane permeability assay (PAMPA) are widely applied tools for early-phase drug candidate screening. In addition to the commonly used porcine brain polar lipid extract for modeling the blood-brain barrier's permeability, the total and polar fractions of bovine heart and liver lipid extracts were investigated in the PAMPA model by measuring the permeability of 32 diverse drugs. The zeta potential of the lipid extracts and the net charge of their glycerophospholipid components were also determined.

Effect of liposomal formulation of ascorbic acid on corneal permeability.

Ascorbic acid (AA) has a pivotal role in corneal wound healing via stimulating the biosynthesis of highly organized extracellular matrix components, but its rapid degradation and low corneal permeability limits its therapeutic effects. In this paper, we present the pharmacokinetic properties of a liposomal-based formulation of AA in terms of corneal permeation. Chemical stability, shelf-life, and drug release rate of lyophilized liposome (AA-LLipo) formulation was determined in comparison to free-form of AA solution using high-performance liquid chromatography (HPLC) and rapid equilibrium dialysis.

Novel Approach for the Isolation and Immobilization of a Recombinant Transaminase: Applying an Advanced Nanocomposite System.

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers.

Effect of Binding Linkers on the Efficiency and Metabolite Profile of Biomimetic Reactions Catalyzed by Immobilized Metalloporphyrin.

The investigation of liver-related metabolic stability of a drug candidate is a widely used key strategy in early-stage drug discovery. Metalloporphyrin-based biomimetic catalysts are good and well-described models of the function of CyP450 in hepatocytes. In this research, the immobilization of an iron porphyrin was performed on nanoporous silica particles via ionic interactions.

Immobilization of the Aspartate Ammonia-Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli.

Cross-Linked Enzyme-Adhered Nanoparticles (CLEANs) for Continuous-Flow Bioproduction.

Nanostructured but micro-sized biocatalysts were created by bottom-up technology using multi-functionalized silica nanoparticles (NPs) as nano-sized building blocks to form cross-linked enzyme-adhered nanoparticles (CLEANs) as robust micro-sized particles with beneficial internal structure and good mechanical properties. Systematic surface modification of NPs with a grafting mixture consisting of organosilanes with reactive (aminopropyl) and inert (e. g.

Nanofibrous Formulation of Cyclodextrin Stabilized Lipases for Efficient Pancreatin Replacement Therapies.

Enzyme replacement therapies (ERT) have been of great help over the past 30 years in the treatment of various lysosomal storage disorders, including chronic pancreatitis and its common complication, exocrine pancreatic insufficiency. Research shows that difficulties in designing such drugs can be overcome by using appropriate additives and various enzyme immobilization techniques. Cyclodextrins (CDs) can be considered as a promising additive for enzyme replacement therapies, as they are known to enhance the activity of enzymes in a complex process due to their specific binding.

Magnetic Nanoparticles with Dual Surface Functions-Efficient Carriers for Metalloporphyrin-Catalyzed Drug Metabolite Synthesis in Batch and Continuous-Flow Reactors.

The dual functionalization of magnetic nanoparticles with inert (methyl) and reactive (aminopropyl) groups enables efficient immobilization of synthetic metalloporphyrins (such as 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin) via covalent or ionic interactions. The proportion of reactive function on the surface has significant effect on the biomimetic activity of metalloporphyrins. The optimized magnetic nanocatalyst containing porphyrin was successfully applied for biomimetic oxidation of antihypertensive drug Amlodipine in batch and continuous-flow reactors as well.

Design and application of a bi-functional redox biocatalyst through covalent co-immobilization of ene-reductase and glucose dehydrogenase.

An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E.

Novel combination of non-invasive morphological and solid-state characterisation of drug-loaded core-shell electrospun fibres.

In recent years, core-shell nanofibrous drug delivery systems have received increasing attention due to their ability to incorporate two or more active pharmaceutical ingredients (APIs) individually into the desired layer (either core or sheath) and thereby finely tune the release profiles of even incompatible drugs in one system. This study aims to perform formulation and solid-state characterisation of levofloxacin-loaded polylactic acid (PLA) - naproxen-sodium-loaded polyvinyl pyrrolidone (PVP) bicomponent core-shell fibrous sheets and examine the electro spinnability of the precursor combinations. The selected drugs have potential therapeutic relevance in similar systems intended for wound healing; however, in this study, they are used as model drugs to understand the physicochemical properties of a drug loaded system.

Controlled degradation of poly-ε-caprolactone for resorbable scaffolds.

A lipase from Burkholderia cepacia was successfully adsorbed on the surface of halloysite nanotubes and the coated tubes were incorporated into poly-ε-caprolactone (PCL). The efficiency of the halloysite in the adsorption of the enzyme was characterized by the total protein content determined with the Bradford method. The activity of the adsorbed enzyme was estimated by the kinetic resolution of racemic 1-phenylethanol.

"Fishing and Hunting"-Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media.

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.

Liver-on-a-Chip‒Magnetic Nanoparticle Bound Synthetic Metalloporphyrin-Catalyzed Biomimetic Oxidation of a Drug in a Magnechip Reactor.

Biomimetic oxidation of drugs catalyzed by metalloporphyrins can be a novel and promising way for the effective and sustainable synthesis of drug metabolites. The immobilization of 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin (FeTPFP) and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin (FeTSPP) via stable covalent or rapid ionic binding on aminopropyl-functionalized magnetic nanoparticles (MNPs-NH) were developed. These immobilized catalysts could be efficiently applied for the synthesis of new pharmaceutically active derivatives and liver related phase I oxidative major metabolite of an antiarrhythmic drug, amiodarone integrated in a continuous-flow magnetic chip reactor (Magnechip).

Green synthesis and immobilization of gold nanoparticles and their application for the reduction of -nitrophenol in continuous-flow mode.

A green and facile method has been developed for the preparation of immobilized gold nanoparticles (AuNPs) using agarose as a reducing and stabilizing agent. The size of the synthesized AuNPs ranges between 10 and 100 nm, and their average size can be controlled by the concentrations of the agarose and gold salt. The agarose matrix as a mild and green reaction medium can provide a good dispersion environment for forming AuNPs, and the hydrogel can be well homogenized with polyacrylic macroporous microbeads as well, which can adsorb and stabilize the particles leading to the simultaneous synthesis and immobilization of AuNPs avoiding harmful inorganic compounds or organic solvents.

Smart Nanoparticles for Selective Immobilization of Acid Phosphatases.

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non-specific acid phosphatases bearing a His-tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni as the most appropriate metal ion were applied as support.

Microstructural Distinction of Electrospun Nanofibrous Drug Delivery Systems Formulated with Different Excipients.

The electrospun nanofiber-based orally dissolving webs are promising candidates for rapid drug release, which is due to the high surface area to volume ratio of the fibers and the high amorphization efficacy of the fiber formation process. Although the latter is responsible for the physical and/or chemical instability of these systems. The primary aim of the present study was to elucidate how the addition of polysorbate 80 (PS80) and hydroxypropyl-β-cyclodextrin (HP-β-CD) influenced the electrospinning process, the properties, and the behavior of the obtained nanofibers.

Co-immobilized Whole Cells with ω-Transaminase and Ketoreductase Activities for Continuous-Flow Cascade Reactions.

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst.

Biomimetic Synthesis of Drug Metabolites in Batch and Continuous-Flow Reactors.

A medium-throughput screening (MTS) of biomimetic drug metabolite synthesis is developed by using an iron porphyrin catalyst. The microplate method, in combination with HPLC-MS analysis, was shown to be a useful tool for process development and parameter optimization in the production of targeted metabolites and/or oxidation products of forty-three different drug substances. In the case of the biomimetic oxidation of amiodarone, the high quantity and purity of the isolated products enabled detailed HRMS and NMR spectroscopic studies.