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. In the specific treatment of cystinosis, preparations containing cysteamine (CYA) are used. The availability of commercialized eyedrops for the targeted treatment is scarce, and only Cystadrops® are commercially available with strong limitations. Thus, magistral CYA-containing compounded eyedrops (CYA-CED) could have a key role in patient care; however, a rationally designed comprehensive study on the commercialized and magistral products is still missing. This work aims to build up a comprehensive study about commercialized and magistral CYA eye drops, involving pharmacokinetic and physicochemical characterization (applying mucoadhesivity, rheology test, investigation of drug release, and parallel artificial membrane permeability assays), as well as ex vivo tests, well supported by statistical analysis.

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. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

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. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

"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.

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. In addition to identification of known metabolites, several new oxidation products of the drug that was studied were characterized. Fast degradation and poor recovery of the catalyst under batch conditions was overcome by immobilization of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTSPP) on the surface of 3-aminopropyl-functionalized silica by electrostatic interaction. The supported catalyst was successfully applied in a packed-bed reactor under continuous-flow reaction conditions for the large-scale synthesis of amiodarone metabolites.

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-ß-cyclodextrin (HP-ß-CD) influenced the electrospinning process, the properties, and the behavior of the obtained nanofibers. In order to reveal any subtle changes attributable to the applied excipients, the prepared samples were subjected to several state of the art imaging and solid state characterization techniques at both macroscopic and microscopic levels. Atomic force microscopy (AFM) revealed the viscoelastic nature of the fibrous samples. At relatively low forces mostly elastic deformation was observed, while at higher loads plasticity predominated. The use of polysorbate led to about two times stiffer, less plastic fibers than the addition of cyclodextrin. The 1H-13C nuclear magnetic resonance (NMR) cross-polarization build-up curves pointed out that cyclodextrin acts as an inner, while polysorbate acts as an outer plasticizer and, due to its "liquid-like" behavior, can migrate in the polymer-matrix, which results in the less plastic behavior of this formulation. Positron annihilation lifetime spectroscopy (PALS) measurements also confirmed the enhanced mobility of the polysorbate and the molecular packing enhancer properties of the cyclodextrin. Solid-state methods suggested amorphous precipitation of the active ingredient in the course of the electrospinning process; furthermore, the nature of the amorphous systems was verified by NMR spectroscopy, which revealed that the use of the examined additives enabled the development of a molecularly dispersed systems of different homogeneities. An accelerated stability study was carried out to track physical state related changes of the incorporated drug and the polymeric carrier. Recrystallization of the active ingredient could not be observed, which indicated a large stress tolerance capacity, but time-dependent microstructural changes were seen in the presence of polysorbate. Raman mapping verified homogeneous drug distribution in the nanofibrous orally dissolving webs. The performed dissolution study indicated that the drug dissolution from the fibers was rapid and complete, but the formed stronger interaction in the case of the PVA-CD-MH system resulted in a little bit slower drug release, compared to the PS80 containing formulation. The results obviously show that the complex physicochemical characterization of the polymer-based fibrous delivery systems is of great impact since it enables the better understanding of material properties including the supramolecular interactions of multicomponent systems and consequently the rational design of drug-loaded nanocarriers of required stability.

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).

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.

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. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

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. Physicochemical parameters of the 32 compounds were calculated using three independent forms of software (Marvin Sketch, RDKit, and ACD/Percepta). The relationship between the lipid-specific permeabilities and the physicochemical descriptors of the compounds was investigated using linear correlation, Spearman correlation, and PCA analysis. While the results showed only subtle differences between total and polar lipids, permeability through liver lipids highly differed from that of the heart or brain lipid-based models. Correlations between the in silico descriptors (e.g., number of amide bonds, heteroatoms, and aromatic heterocycles, accessible surface area, and H-bond acceptor-donor balance) of drug molecules and permeability values were also found, which provides support for understanding tissue-specific permeability.

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. In vitro transcorneal permeability was studied using a parallel artificial membrane permeability assay (PAMPA). Ex vivo permeation was examined on AA-LLipo-treated porcine cornea by determining the AA content on the ocular surface, in the cornea as well as in the aqueous humor using HPLC, and by Raman-mapping visualizing the AA-distribution. Our results showed that the liposomal formulation improved the chemical stability of AA, while drug release was observed with the same kinetic efficiency as from the free-form of AA solution. Both corneal-PAMPA and porcine corneal permeability studies showed that AA-LLipo markedly improved the corneal absorption kinetics of AA, thus, increasing the AA content in the cornea and aqueous humor. AA-LLipo formulation could potentially increase the bioavailability of AA in corneal tissues.

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.

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. The effect of the metalloporphyrin binding linkers was investigated on the catalytic efficiency and the metabolic profile of chloroquine as a model drug. The length of the amino-substituted linkers affects the chloroquine conversion as well as the ratio of human major and minor metabolites. While testing the immobilized catalysts in the continuous-flow reactor, results showed that the presented biomimetic system could be a promising alternative for the early-stage investigation of drug metabolites regarding analytical or synthetic goals as well.

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., vinyl, propyl, phenyl, or octyl) functions resulted in functional NPs enabling cross-linking agents, such as glutardialdehyde or bisepoxides (glycerol diglycidyl ether, neopentylglycol diglycidyl ether, and poly(propylene glycol) diglycidyl ether), to bind and cross-link enzymes covalently and to form macroporous microparticles. These CLEANs were able to diminish several weaknesses of traditional cross-linked enzyme aggregates as biocatalysts, such as poor mechanical resistance, difficult recovery, and storage, strengthening their use for packed-bed enzyme reactors. Lipase B from Candida antarctica (CaLB) was selected as model enzyme for development of robust CLEANs, which were successfully tested for various industrially relevant applications including a kinetic resolution of a racemic alcohol and the production of various natural fragrance compounds under continuous-flow conditions.

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. In this study, we investigated the formulation of lipases (from Aspergillus oryzae and Burkholderia cepacia) paired with different cyclodextrins in poly(vinyl alcohol) (PVA) nanofibers by electrospinning technique. We examined the effect of the presence of cyclodextrins and nanoformulation on the lipase activity. The rheological and morphological characterizations of precursors and nanofibers were also performed using a viscometer as well as electron and Raman microscope. We found that by selecting the appropriate CD:lipase ratio, the activity of the investigated enzyme could be multiplied, and cyclodextrins can support the homogeneous dispersion of lipases inside the solid formula. In addition, the entrapment of lipases in PVA nanofibers led to a significant increase in activity compared to the preformulated precursor. In this way, the nanofibrous formulation of lipases combining CDs as additives can provide an efficient and sustainable possibility for designing novel solid medicines in ERT.

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. The immobilized enzyme was mixed with the polymer and compression molded films were prepared at 70 °C. Activity measurements proved that the enzyme remains active even after adsorption; in fact, larger activities were measured for the immobilized enzyme than for the neat enzyme preparation. The supported enzyme degraded PCL efficiently, the rate of degradation depended on the amount of enzyme adsorbed. The kinetics of degradation was described quantitatively with an appropriate model accounting for two of the three steps of the process, i.e. degradation and the denaturation of the enzyme. The determination of time constants allows the adjustment of degradation rate. This is the first time that the enzyme, which catalyzes degradation, is incorporated into the polymer, and not into the degradation medium, thus allowing the preparation of resorbable scaffolds with controlled lifetime.

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. In order to determine the best core- and shell-solution combination, a full factorial experimental design is used. A combination of various morphological (scanning electron microscopy and transmission electron microscopy) and microstructural characterisation techniques (X-ray photoelectron spectroscopy and Raman spectroscopy) was applied to non-invasively obtain information about the structure of the fibres and the embedded drugs. The results indicate that core-shell fibres of different compositions could be successfully prepared with various structural homogeneities. The best core-shell structure was obtained using a combination of 15% (w/w) shell concentration and 8% (w/w) PLA solution concentration. In addition to the conventional core-shell structural verification methods, the Raman spectroscopy method was implemented to reveal not only the core-shell structure of the PLA/PVP nanofibers but also the form of the embedded drugs. The Raman mapping of the fibres confirm the above results, and it is shown that an amorphous solid dispersion is formed as a result of the coaxial electrospinning process.

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. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.

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.