Operative parameters optimization production of liposomes for the encapsulation of hydrophilic compounds using a new supercritical process

Liposomes are spherical vesicles formed by a inner aqueous core and a double lipidic layer around it. Conventional techniques for the production of liposomes are characterized by several drawbacks, like the production of micrometric vesicles, a difficult control of the Particle Size Distribution (PSD) and low encapsulation efficiencies (EE) of hydrophilic compounds. Many supercritical semi-continuous techniques were proposed in literature. They are successful in the intent of producing liposomes of smaller diameter, but the EE of hydrophilic compounds and the reproducibility are still a challenge. For this reason, it was recently proposed a new supercritical process whose aim is to invert the steps of production of liposomes, by first creating water droplets and then to fast surround them by phospholipids. We discovered that the high diffusion coefficient of phospholipids in supercritical carbon dioxide allows a fast coverage of water droplets preserving the drug content into the liposome core. In this work, hydrophilic compounds were encapsulated in the vesicles produced using SuperLip, such as Fluorescein, Bovine Serum Albumin (BSA) and Ampicillin, obtaining monodispersed spherical vesicles with a mean size from 100 to 300 nm. Operative parameters like water flow rate and lipid to water mass ratio were optimized. The EEs were evaluated with UV-Vis spectroscopy according to methods reported in literature, and obtaining high values up to 99 % for the three investigated compounds

Biopolymer Particles for Proteins and Peptides Sustained Release Produced by Supercritical Emulsion Extraction

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Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders using Supercritical or Near-Critical Fluids

Supercritical or near-critical fluid processes for generating microparticles have enjoyed considerable attention in the past decade or so, with good success for substances soluble in supercritical fluids or organic solvents. In this review, we survey their application to the production of protein particles. A recently developed process known as CO2-assisted nebulization with a Bubble Dryer® (CAN-BD) has been demonstrated to have broad applicability to small-molecule as well as macromolecule substances (including therapeutic proteins). The principles of CAN-BD are discussed as well as the stabilization, micronization and drying of a wide variety of materials. More detailed case studies are presented for three proteins, two of which are of therapeutic interest: anti-CD4 antibody (rheumatoid arthritis), α1-antitrypsin (cystic fibrosis and emphysema), and trypsinogen (a model enzyme). Dry powders were formed in which stability and activity are maintained and which are fine enough to be inhaled and reach the deep lung. Enhancement of apparent activity after CAN-BD processing was also observed in some formulation and processing conditions

Operating Parameters Optimization for the Production of Liposomes Loaded with Antibodies Using a Supercritical Fluid-Assisted Process

Encapsulation of antibodies represents a significant advance to protect and deliver these therapeutics in a controlled manner, increasing the stability requested to cover the temporal gap between particle production and their administration. Furthermore, using encapsulation, extracellular, cell surface, and intracellular targets can be reached. This work examines the feasibility of encapsulating mouse IgG isotype control antibodies within phosphatidylcholine-based liposomes using a supercritical fluid-based process called SuperLip (Supercritical-assisted Liposome formation). This process allows a continuous production of both nano- and micrometric liposomes with high encapsulation efficiency working under mild operative conditions. The effect of some operative parameters has been studied on liposome mean diameter, particle size distribution, and antibody entrapment efficiency, comparing these data with those collected working with liposomes obtained by the thin-layer hydration technique. In particular, the effect of water flow rate and of the antibody loading were studied. Antibody-loaded liposomes with mean diameters in the range between 205 and 501 nm have been obtained by using a supercritical fluid-assisted process. High entrapment efficiencies up to 94% have been calculated

Current concepts: tissue engineering and regenerative medicine applications in the ankle joint

Tissue engineering and regenerative medicine (TERM) has caused a revolution in present and future trends of medicine and surgery. In different tissues, advanced TERM approaches bring new therapeutic possibilities in either general population as in young patients and high-level athletes, improving restoration of biological functions and rehabilitation. The mainstream components required to obtain a functional regeneration of tissues may include biodegradable scaffolds, drugs or growth factors and different cell types (either autologous or heterologous) that can be cultured in bioreactor systems (in vitro) prior implantation into the patient. Particularly in the ankle, that is subject of many different injuries (e.g. acute, chronic, traumatic, degenerative), there is still no definitive and feasible answer to “conventional” methods. This review aims to provide current concepts of TERM applications to ankle injuries under pre-clinical and/or clinical research applied to skin, tendon, bone and cartilage problems. A particular attention has been given to biomaterials design and scaffolds processing with potential use in osteochondral ankle lesions.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programmes, including Project OsteoCart (grant no. PTDC/CTM-BPC/115977/2009) for providing funds

Why short-term biochar application has no yield benefits; evidence from three field-grown crops

This study determines the impact of biochar, as a supplement, on soil nutrient availability and yields for three crops within commercial management systems in a temperate environment. Central to the suggestion of biochar benefits is an increase in soil nutrient availability and here we test this idea by examining crop nutrient uptake, growth and yields of field-grown spring barley, strawberry and potato. Biochar produced from Castanea sativa wood, was incorporated into a sandy loam soil at 0, 20 and 50 t ha-1 as a supplement to standard crop management practice. Fertiliser was applied normally for each of the three crops. The biochar contained substantial concentrations of Ca, Mg, K, P, but only K occurred at high concentration in water soluble analysis. The large concentration of extractable K resulted in a significant increase of extractable K in soil. The increased availability of K in biochar-treated soil, with the exception of spring barley grain and the leaves of strawberry during the second year, did not induce greater tissue concentrations. In general, biochar application rate had little influence on the tissue concentration of any nutrient, irrespective of crop or sampling date. There was, however, evidence of a biochar-induced increase in tissue Mo and a decrease in Mn, in strawberry, which could be linked to soil alkalinisation as could the reduction in extractable soil P. These experiments show a single rotational application of biochar to soil had no effect on the growth or harvest yield of any of these field-grown crops. Heavy metal analysis revealed small concentrations in the biochar (i.e. <10 µg g-1 biochar), with the largest levels for Ni, V and Cu

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. [br/] Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. [br/] Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria