L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain.

L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

Interfacial Effects and the Nano-Scale Disruption in Adsorbed-Layer of Acrylate Polymer-Tween 80 Fabricated Steroid-Bearing Emulsions: A Rheological Study of Supramolecular Materials.

The effect of polymer adsorption on the stability and viable shelf life of 55 µm diameter oil-in-water (O/W) emulsions containing the steroid, betamethasone 21-phosphate was investigated. Two acrylate polymers, Carbopol® 971P and 974P, were added in the role of emulsion stabilizers to a model system, representing a non-ionic low molecular weight surfactant-stabilized emulsion (topically applied medicinal cream). For the purposes of this study the dosage of the viscosifier was maintained below 1% w/v and consequently, the consistency of the emulsion was measured in the diluted form. One of the polymers was responsible for elevated degrees of droplet creaming and coalescence and this was closely linked to its surface tension lowering capacity. This lowering was seen at 62 mN/m compared to the routine values at equivalent concentrations of 68 mN/m and 35 mN/m for the betamethasone drug and non-ionic surfactant-Tween 80, respectively. The same polymer also demonstrated a predisposition to form low-micron and greater sized aggregates of nanoparticles that led to extensive flocculation and the formation of a sedimentary precipitate, formed from an amalgam of the components found in the creamed droplet layer.

Offensive waste valorisation in the UK: Assessment of the potentials for absorbent hygiene product (AHP) recycling.

Offensive human waste refers to non-hazardous waste that contains body fluids from non-infectious humans, comprised of post-consumer Absorbent Hygiene Products (AHPs), swabs, dressings, bedding, gloves, and other materials. While this waste category requires more stringent handling, storage and disposal measures than general waste, its non-hazardous nature suggests that there are opportunities for waste valorisation. An inventory of 200 offensive human waste bags collected from various healthcare institutions in South-Eastern England show that about 76% of the waste is comprised of AHPs, most of which are adult incontinence pads and child nappies. Mixed plastics comprised of predominantly HDPE represent 9% of the waste. To evaluate the potentials for offensive human waste valorisation, small-scale separation tests involving artificially-soiled nappies and associated mixed plastic packaging wastes have been performed. Findings suggest that about 50% of the total superabsorbent polymer is recoverable from fluff pulp fractions, recoveries of which are unaffected by the presence of ionic species typically present in human waste. On the other hand, recovery of mixed plastic packaging is more challenging. Overall, however, findings suggest that viable AHP recycling is possible if recyclate materials are targeted towards non-food related markets outlets such as the construction and land remediation sectors.

Architectures and Mechanical Properties of Drugs and Complexes of Surface-Active Compounds at Air-Water and Oil-Water Interfaces.

BACKGROUND: Drugs can represent a multitude of compounds from proteins and peptides, such as growth hormones and insulin and on to simple organic molecules such as flurbiprofen, ibuprofen and lidocaine. Given the chemical nature of these compounds two features are always present. A portion or portions of the molecule that has little affinity for apolar surfaces and media and on the contrary a series of part or one large part that has considerable affinity for hydrophilic, polar or charged media and surfaces. A series of techniques are routinely used to probe the molecular interactions that can arise between components, such as the drug, a range of surface- active excipients and flavor compounds, for example terpenoids and the solvent or dispersion medium. RESULTS: Fifty-eight papers were included in the review, a large number (16) being of theoretical nature and an equally large number (14) directly pertaining to medicine and pharmacy; alongside experimental data and phenomenological modelling. The review therefore simultaneously represents an amalgam of review article and research paper with routinely used or established (10) and well-reported methodologies (also included in the citations within the review). Experimental data included from various sources as diverse as foam micro-conductivity, interferometric measurements of surface adsorbates and laser fluorescence spectroscopy (FRAP) are used to indicate the complexity and utility of foams and surface soft matter structures for a range of purposes but specifically, here for encapsulation and incorporation of therapeutics actives (pharmaceutical molecules, vaccines and excipients used in medicaments). Techniques such as interfacial tensiometry, interfacial rheology (viscosity, elasticity and visco-elasticity) and nanoparticle particle size (hydrodynamic diameter) and charge measurements (zeta potential), in addition to atomic force and scanning electron microscopy have proven to be very useful in understanding how such elemental components combine, link or replace one another (competitive displacement). They have also proven to be both beneficial and worthwhile in the sense of quantifying the unseen actions and interplay of adsorbed molecules and the macroscopic effects, such as froth formation, creaming or sedimentation that can occur as a result of these interactions. CONCLUSION: The disclosures and evaluations presented in this review confirm the importance of a theoretical understanding of a complex model of the molecular interactions, network and present a framework for the understanding of really very complex physical forms. Future therapeutic developers rely on an understanding of such complexity to garner a route to a more successful administration and formulation of a new generation of therapeutic delivery systems for use in medicine.

Preparation of liposomes containing small gold nanoparticles using electrostatic interactions.

The development of liposome-nanoparticle colloid systems offers a versatile approach towards the manufacture of multifunctional therapeutic platforms. A strategy to encapsulate small metallic nanoparticles (<4nm) within multilamellar vesicles, effected by exploiting electrostatic interactions was investigated. Two liposome-gold nanoparticle (lipo-GNP) systems were prepared by the reverse-phase evaporation method employing cationic or anionic surface functionalised particles in combination with oppositely charged lipid compositions with subsequent post-formulation PEGylation. Structural characterisation using electron microscopy and elemental analysis revealed a regular distribution of GNPs between adjacent lipid bilayers of intact liposomes. Nanoparticle encapsulation efficacy of the two lipo-GNP systems was revealed to be significantly different (p=0.03), evaluated by comparing the ratio of measured lipid to gold concentration (loading content) determined by a colorimetric assay and atomic emission spectroscopy, respectively. It was concluded that the developed synthetic strategy is an effective approach for the preparation of liposome-nanoparticle colloids with potential to control the relative concentration of encapsulated particles to lipids by providing favourable electrostatic interactions.

Ozone decomposition on Ag/SiO2 and Ag/clinoptilolite catalysts at ambient temperature.

Silver modified zeolite (Bulgarian natural clinoptilolite) and Ag/silica catalysts were synthesized by ion exchange and incipient wet impregnation method respectively and characterized by different techniques. DC arc-AES was used for Ag detection. XRD spectra show that Ag is loaded over the surface of the SiO(2) sample and that after the ion-exchange process the HEU type structure of clinoptilolite is retained. UV-VIS (specific reflection at 310 nm) and IR (band at 695 cm(-1)) spectroscopy analysis proved that silver is loaded as a T-atom into zeolite channels as Ag(+), instead of Na(+), Ca(2+), or K(+) ions, existing in the natural clinoptilolite form. The samples Ag/SiO(2) and Ag-clinoptilolite were tested as catalysts for decomposition of gas phase ozone. Very high catalytic activity (up to 99%) was observed and at the same time the catalysts remained active over time at room temperature.

Nanoemulsion encapsulation and in vitro SLN models of delivery for cytotoxic methotrexate.

The complementary mixing and de-mixing of phases is seen as a methodology for use in 'smart' drug delivery systems. Surface tensions were routinely lowered to 50-55 mN/m in drug mixtures and this is compared to low molecular weight emulsifiers such as Tween 80. In the research reported here, mixing of a heterocyclic drug and an amphiphilic polymer is responsible for structural inconsistency at the air-water (A/W) interface and presents itself as a means of release of drug from a series of complex drug formulations. The work was extended to look at the behavior of both "soft" and "hard" particle hydrophobic and hydrophilic particle stabilization of emulsions of 20 and 0.7 µm. The work is based on spread interfacial monolayers, thin liquid films emulsion droplets and model latex nanoparticle beads (60 and 500 nm) in the presence and absence of poloxamer (Pluronic) and methotrexate. The interfacial rheological characteristics and AFM-based nano-rheology were used to aid the elucidation of surface structure and nano-architectures within the plane of the adsorbed interfacial layer and for use in the fabrication of solid nano-particle stabilized micron and nano-sized oil-inwater (O/W) emulsions.

Sculpted amphiphilic liposomal particles for modifiable medicinal applications.

The surface coverage and leaflet anchoring of polymeric surfactants and other amphiphiles can be used to provide a particle with extensive modifiable 'stealth' properties. This is not a new conceptual tool but one which has significant strategic advantage and recent proven clinical application in a "drug-delivery-package". This has been established and widely reported as a fundamental basis for optimal utility with long-circulation drug nanoparticles. The indirect "disguising" of the medicinal payload presents itself as a form of "pro-drug" system that can be used to extend circulation lifetime and that is key to a number of essential current and future risk-bearing therapies. Appropriate particle sizing and leaflet chemical structuring, in terms of number of strata, complexity, extent and curvature can also be used as the means to "drug anchoring" for the incorporation of "hydrophobic" drugs. Actives that are chemo- or solvent labile can also be housed, often routinely, within the inner-protected chamber of such capsular systems. This has frequently formed the basis of a number of recently developed commercial preparations that are used ubiquitously in now standard chemotherapy. Additionally, the use of complementary and non-complementary phospholipid or other vesicle ingredient mixtures below, at or above the lipid "gel transition" temperature or within a "glassy" crystalline packing configuration can provide a particle of varied physical and mechanical composition that provides a possible route to the production of fusogenic nano-drugs or control over the drug release profile. Successive layering of the nanoparticle can in principle provide the means to a step-wise or timed release of the particle contents and one that can be adapted to the site of intended use.

Molecular modeling directed by an interfacial test apparatus for the evaluation of protein and polymer ingredient function in situ.

A simplified apparatus is described that measures the damping of a suspended measuring device. The movement of the device (bob) is damped by the properties of the air-water surface adsorbed material. Its value lies in describing the surface chemomechanical properties of ingredients and excipients used in food, nutraceutical, cosmetic (cosmeceutical), and natural drug-food product formulations that traverse the food sciences. Two surfactants, two food and drug-grade polymers, and five naturally occurring food and serum proteins were tested and used to estimate and model interfacial viscoelasticity. Equilibration times of >15 min were found to give sufficiently stable interfaces for routine assessment. The viscoelasticity of the air-water interface was estimated with reference to model solutions. These model solutions and associated self-assembled interfacial nanostructured adsorbed layers were fabricated using a preliminary screening process with the aid of a specialized foaming apparatus ( C(300) values), surface tension measurements (23-73 mN/m), and referential surface shear and dilation experiments. The viscoelasticity measured as a percentage of surface damping ( D) of a pendulum was found to range from 1.0 to 22.4% across the samples tested, and this represented interfacial viscosities in the range of 0-4630 microNs/m. The technique can distinguish between interfacial compositions and positions itself as an easily accessible valuable addition to tensiometric and analytical biochemistry-based techniques.

Effects of poly (ethylene glycol) chains conformational transition on the properties of mixed DMPC/DMPE-PEG thin liquid films and monolayers.

Foam thin liquid films (TLF) and monolayers at the air-water interface formed by DMPC mixed with DMPE-bonded poly (ethylene glycol)s (DMPE-PEG(550), DMPE-PEG(2000) and DMPE-PEG(5000)) were obtained. The influence of both (i) PEG chain size (evaluated in terms of Mw) and mushroom-to-brush conformational transition and (ii) of the liposome/micelle ratio in the film-forming dispersions, on the interfacial properties of mixed DMPC/DMPE-PEG films was compared. Foam film studies demonstrated that DMPE-PEG addition to foam TLFs caused (i) delayed kinetics of film thinning and black spot expansion and (ii) film stabilization. At the mushroom-to-brush transition, due to steric repulsion increased DMPE-PEG films thickness reached 25 nm while pure DMPC films were only 8 nm thick Newton black films. It was possible to differentiate DMPE-PEG(2000/5000) from DMPE-PEG(550) by the ability to change foam TLF formation mechanism, which could be of great importance for "stealth" liposome design. Monolayer studies showed improved formation kinetics and equilibrium surface tension decrease for DMPE-PEG monolayers compared with DMPC pure films. SEM observations revealed "smoothing" and "sealing" of the defects in the solid-supported layer surface by DMPE-PEGs adsorption, which could explain DMPE-PEGs ability to stabilize TLFs and to decrease monolayer surface tension. All effects in monolayers, foam TLFs and solid-supported layers increased with the increase of PEG Mw and DMPE-PEG concentration. However, at the critical DMPE-PEG concentration (where mushroom-to-brush conformational transition occurred) maximal magnitude of the effects was reached, which only slightly changed at further DMPE-PEG content and micelle/liposome ratio increase.

Administration of drug and nutritional components in nano-engineered form to increase delivery ratio and reduce current inefficient practice.

The article critically discusses parenteral delivery of self-assembled lipid or amphiphile nanoparticles, in the form of aggregated clusters or particles (capsules). The end-product or drug form is for application by administration of the medicine active in encapsulated form. This is used for site-specific cell manipulation and clinical therapy and introduces this directly to the body via the systemic route. The technology discussed represents a platform formulation that can be modified for a range of specific cellular targets. The components of the nanoparticle are assembled piece-by-piece and this provides an element of design flexibility, with the core particle being built-up in a succession of layers to ensure circulatory longevity and storage stability. This strategy excludes a more generalised delivery and widespread lack of active targeting and thus low dosage rather than avoidance of target, which is at best detrimental and at worst catastrophic in terms of non-targeted cell death. However, in some cases such as the AmBisome nanoparticle this drug delivery approach can work. This "better focussing" is achieved by a dual use of i) biocompatible particle coating chemistry and a ii) cell-ligand imprinted nanoparticle surface that furnishes the engineered nanoparticle with a recognition element to form a complex but more efficacious dispersible fusogenic pro-drug moiety. Non-targeted delivery of drugs such as those commonly forming the basis of transdermal delivery have been generically based on topical or adhesive patch-based delivery (emulsions) systems. This procedure even with recent advancements and patents is customarily inefficient in dosage and payload delivery, inconsistent in terms of product potency and inflexible to further modification or purpose-related enhancement. The assembly and delivery methodologies discussed here take the new experimental medicine and review them in a more focused and purposeful therapy-constructed manner. It is the use of a smart platform technology that is responsible for increased efficacy when applied to the disorder of interest.

Engineering of nanoemulsions for drug delivery.

Nanoemulsions, usually spherical, are a group of dispersed particles used for pharmaceutical and biomedical aids and vehicles that show great promise for the future of cosmetics, diagnostics, drug therapies and biotechnologies. They exist in a wide variety of forms that are dictated by the particle components. Nanoemulsions are generally considered to be in the size range of less than and around 100 nm in diameter. The particles can exist as water-in-oil and oil-in-water forms, where the core of the particle is either water or oil, respectively. More complex variations also exist but these are often larger. The longer-term properties of the particle are dependent on the composition of the adsorbed material lying at the dispersed droplet interface with the dispersion medium. This has an impact on the partitioning and extraction of droplet contents. Thermodynamically stable particles are characterized by having a very low surface tension and this produces a very large surface area. Nanoemulsions can also include small meta-stable very small-scale emulsions; here the surface properties and chemistry can strongly influence behaviour. Processing, storage and formulation composition can also have an impact on the longevity of a pharmaceutical preparation. Some revolutionary new nanoemulsion droplets based on fluorinated compounds are finding a number of widespread biomedical roles and applications. Developments in nanoemulsion technology are likely to lead to a much greater use of this medium in future pharmaceuticals.

Developments in microarray technologies.

The focus of high-throughput drug discovery has progressed through the genome and the transcriptome and is now moving towards more difficult problems in assessing the proteome, glycome and metabolome. Microarrays are currently the major tool in the assessment of gene expression via cDNA or RNA analysis; however, they are also used to screen libraries of proteins and small molecules. Microarrays have helped to extract more information from smaller sample volumes and enabled the incorporation of low-cost high-throughput assays in the drug discovery process. The technology continues to develop and is being rapidly transferred into more challenging areas, with the potential to further aid and enhance the drug discovery process through the development of, for example, proteomic, glycomic and tissue arrays.

Methods to quantify the biotin-binding capacity of streptavidin-coated polypropylene PCR plates.

The biotin-binding capacity in the wells of a streptavidin-coated PCR plate were quantified by means of a fluorescence intensity assay utilising biotin-labelled fluorescein and a colorimetric assay using biotin-labelled alkaline phosphatase. The biotin binding capacities were determined to be 59 and 58 pmol respectively.

Developments in microarray technologies.

The focus of high-throughput drug discovery has progressed through the genome and the transcriptome and is now moving towards more difficult problems in assessing the proteome, glycome and metabolome. Microarrays are currently the major tool in the assessment of gene expression via cDNA or RNA analysis; however, they are also used to screen libraries of proteins and small molecules. Microarrays have helped to extract more information from smaller sample volumes and enabled the incorporation of low-cost high-throughput assays in the drug discovery process. The technology continues to develop and is being rapidly transferred into more challenging areas, with the potential to further aid and enhance the drug discovery process through the development of, for example, proteomic, glycomic and tissue arrays.

Stability of soap films: hysteresis and nucleation of black films.

We study the stability of soap films of a nonionic surfactant under different applied capillary pressures on the film. Depending on the pressure, either a thick common black film (CBF), or a micro-scopically thin Newton black film (NBF) is formed as a (metastable) equilibrium state, with a first-order (discontinuous) transition between the two. Studying the dynamics of the CBF-NBF transition, it is found that under certain conditions a hysteresis for the transition is observed: for a given range of pressures, either of the two states may be observed. We quantify the nucleation process that is at the basis of these observations both experimentally and theoretically.