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

Supercritical phase inversion of starch-poly(e-caprolactone) for tissue engineering applications

In this work, a starch-based polymer, namely a blend of starch-poly(ε-caprolactone) was processed by supercritical assisted phase inversion process. This processing technique has been proposed for the development of 3D structures with potential applications in tissue engineering applications, as scaffolds. The use of carbon dioxide as non-solvent in the phase inversion process leads to the formation of a porous and interconnected structure, dry and free of any residual solvent. Different processing conditions such as pressure (from 80 up to 150 bar) and temperature (45 and 55°C) were studied and the effect on the morphological features of the scaffolds was evaluated by scanning electron microscopy and micro-computed tomography. The mechanical properties of the SPCL scaffolds prepared were also studied. Additionally, in this work, the in vitro biological performance of the scaffolds was studied. Cell adhesion and morphology, viability and proliferation was assessed and the results suggest that the materials prepared are allow cell attachment and promote cell proliferation having thus potential to be used in some for biomedical applications.Ana Rita C. Duarte is grateful for financial support from Fundacao para a Ciencia e Tecnologia through the grant SFRH/BPD/34994/2007

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

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

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

Stable amorphous georgeite as a precursor to a high-activity catalyst .

Copper and zinc form an important group of hydroxycarbonate minerals that include zincian malachite, aurichalcite, rosasite and the exceptionally rare and unstable—and hence little known and largely ignored1—georgeite. The first three of these minerals are widely used as catalyst precursors2, 3, 4 for the industrially important methanol-synthesis and low-temperature water–gas shift (LTS) reactions5, 6, 7, with the choice of precursor phase strongly influencing the activity of the final catalyst. The preferred phase2, 3, 8, 9, 10 is usually zincian malachite. This is prepared by a co-precipitation method that involves the transient formation of georgeite11; with few exceptions12 it uses sodium carbonate as the carbonate source, but this also introduces sodium ions—a potential catalyst poison. Here we show that supercritical antisolvent (SAS) precipitation using carbon dioxide (refs 13, 14), a process that exploits the high diffusion rates and solvation power of supercritical carbon dioxide to rapidly expand and supersaturate solutions, can be used to prepare copper/zinc hydroxycarbonate precursors with low sodium content. These include stable georgeite, which we find to be a precursor to highly active methanol-synthesis and superior LTS catalysts. Our findings highlight the value of advanced synthesis methods in accessing unusual mineral phases, and show that there is room for exploring improvements to established industrial catalysts

Analysis of the Plant bos1 Mutant Highlights Necrosis as an Efficient Defence Mechanism during D. dadantii/Arabidospis thaliana Interaction

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection