In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers

Cataloged from PDF version of article.In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between -3.1 and +21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190-1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B. However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity. © 2004 Elsevier B.V. All rights reserved

In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions

The aim of this study was to prepare nonwoven materials from poly(-caprolactone) (PCL) and their antibiotic containing forms by electrospinning, so as to prevent postsurgery induced abdominal adhesions in rats. -Caprolactone was first polymerized by ring-opening polymerization, and then it was processed into matrices composed of nanofibers by electrospinning. A model antibiotic (Biteral®) was embedded within a group of PCL membranes. In the rat model, defects on the abdominal walls in the peritoneum were made to induce adhesion. The plain or antibiotic embedded PCL membranes were implanted on the right side of the abdominal wall. No membrane implantation was made on the left side of the abdominal wall that served as control. Macroscopical and histological evaluations showed that using these barriers reduces the extent, type, and tenacity of adhesion. The antibiotic embedded membranes significantly eliminated postsurgery abdominal adhesions, and also improved healing

Covalent and non-covalent strategies for surface modification of different textile materials with antimicrobial properties

New advances have been released in textile industry. Contributions at the level of textile fiber chains engineering has allowed modification of their structure, production of smart polymers responding to changes in environment, and attachment or entrapment of cells and bioactive molecules. Likewise, our society high demand of hygienic patterns, has raised the intensive research and development of antimicrobial textiles. Applications are being extended to underwear, sportswear, home furnishing, protective clothes, wound-dressings and in areas with high risk of microbial infection, as hospitals, schools and hotels. Throughout last decades hospitals have faced tough challenges concerning microbial multi-resistance, especially in immunodepressed patients. The strongest cause for microbial resistance may be due to the abuse of antibiotics uptake, either by humans to treat something non appropriately, as by animals, to earn weight. The development of antimicrobial textiles arise as a promising solution that may significantly decrease the risk of nosocomial infections. Several antimicrobial agents have been applied in textiles, namely quaternary ammonium compounds, silver, polyhexamethylene biguanides and triclosan. However, they have shown a reduced spectrum of microbial inhibition which cause resistances, cytotoxicity causing skin irritation, as well as toxic to the environment. Furthermore, these compounds gradually lose their bioactivity with use and launderings. L-cysteine (L-Cys) that is found in several living organisms is a natural defensive thiolated aminoacid never studied before as a potential antimicrobial agent for textiles, which can grant antibacterial properties without cytotoxicity. Furthermore, antimicrobial peptides (AMPs) belong to innate immune system of multicellular organisms and appear as an alternative to antibiotics. They are small, amphipathic, and strongly cationic which bind to negatively charged phospholipid headgroups of microbial membranes. Although the mechanism of AMPs-microbial killing is still not known, many hypotheses have been proposed: (i) membrane depolarization, (ii) formation of physical holes at the membrane, (iii) programmed bacterial death processes, (iv) phospholipidic redistribution, and (v) internalization of the AMP. They have broad-spectrum antimicrobial activity. Once their target is the bacterial membrane microorganisms hardly develop resistance, otherwise they would have to change all their lipidic composition and/or organization, which is high demanding and not energetically worth it. During this work, non-covalently adsorbed L-Cys to wool (patent PAT 104540 A) and to cotton showed to be non-toxic to human cells, and had antimicrobial effects against Gram-negative and Gram-positive bacteria and its main mechanism of action on cotton was assessed by flow citometry. Antimicrobial peptides (AMPs) will also be immobilized on textiles, in order to find if textile imobilized-AMP can attract and kill bacteria. Natural polymers have shown few adverse reactions, once they have excellent humidity control, biocompatibility and low-allergic responses, due to their similarity to macromolecules which biological environment is prepared to recognize and to deal with metabolically. AMPs will be selected, based on their 3D structure, terminal charge and size. Best-studied AMPs are cationic due to their action on negative surface charged microorganisms. Evaluation of minimal inhibitory concentration (MIC) of AMPs will elucidate the amount of AMPs to be used to functionalize textile substrates and cytotoxicity studies will provide the toxicity of functionalized textiles to human cells. In order to develop long-lasting and washable functionalized textiles we propose the covalent binding of AMPs on textiles through selected chemistries already employed on surface modifying of medical devices elsewhere. Alternatively, we will use plasma treatment, which is usually used to modify many surface properties of polymeric materials. This study may allow the development of innovative antimicrobial textiles, simulating microbial-free microenvironments in order to develop, in the future, antimicrobial fabrics to avoid airborne spreading and improve patient’s quality of life in a hospital context

In vitro and in vivo degradation of non-woven materials made of poly(e-caprolactone) nanofibers prepared by electrospinning at different conditions

The aim of this study was to prepare non-woven materials from a biodegradable polymer, poly(ε-caprolactone) (PCL) by electrospinning. PCL was synthesized by ring-opening polymerization of ε-caprolactone in bulk using stannous octoate as the catalyst under nitrogen atmosphere. PCL was then processed into non-woven matrices composed of nanofibers by electrospinning of the polymer from its solution using a high voltage power supply. The effects of PCL concentration, composition of the solvent (a mixture of chloroform and DMF with different DMF content), applied voltage and tip–collector distance on fiber diameter and morphology were investigated. The diameter of fibers increased with the increase in the polymer concentration and decrease in the DMF content significantly. Applied voltage and tip–collector distance were found critical to control 'bead' formation. Elongation-at-break, ultimate strength and Young's modulus were obtained from the mechanical tests, which were all increased by increasing fiber diameter. The fiber diameter significantly influenced both in vitro degradation (performed in Ringer solution) and in vivo biodegradation (conducted in rats) rates. In vivo degradation was found to be faster than in vitro. Electrospun membranes were more hydrophobic than PCL solvent-casted ones; therefore, their degradation was a much slower process

New strategies for surface modification of cotton and silk textiles with antimicrobial properties

Throughout the past decades hospitals have been facing a major challenge concerning the growing multi-drug microbial resistance, especially in immunodepressed patients. The development of antimicrobial textiles offers a promising solution in the prevention of infections in clinical settings since microbial shedding from our body contributes to microorganism spreading into a textile material, either directly in clothes or on surrounding textiles. The use of some chemical antimicrobial agents in textiles has already been tested, as for example quaternaryammonium- compounds (QACs), biguanidines, silver, triclosan, and N-halamines. However, these have proved to be of limited clinical applicability. They exhibit some cytotoxicity causing some irritation of the skin, toxicity to the environment and, except for silver and N-halamines, exhibit a reduced spectrum of microbial inhibition thus bringing about microbial resistance. Besides, with the exception of QACs, which establish durable bonds with textiles, they gradually lose their bioactivity with use and laundering. Therefore a new strategy to develop non-toxic antimicrobial textiles without microbial resistance side-effects are hereby described. Our results demonstrate the potential of the cotton and silk covalent and non-covalent modification with aminoacids and antimicrobial peptides (AMPs) and openingnewavenues to a world of applications in the area of increased risk microbial infections

Nano- and micro-fiber combined scaffolds : a new architecture for bone tissue engineering

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on postimplantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs’ migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.M.I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

The effect of variation in the ratio of specific heats with temperature and pressure on horsepower requirements of reciprocating compressors

The expansion and compression of fluids is discussed in relation to the design of reciprocating compressors. As part of this thesis, the design equations for reciprocating compressors are developed for both ideal and reel gases. The resulting design equations contain, as one of the parameters, the ratio of the specific heats. Using published componential specific heat data, a correlation was developed relating the dependence of the ratio of specific heat on temperature and pressure for the nore common gases. These correlations were developed so as to be compatible with the compressor design equation and to provide rapid evaluation of the various design equations. As part of the analysis of the applicability of the design equations, it was demonstrated that interstage cooling is always desirable, and that real gases with compressibility factors less than unity have lower power requirements for the same pressure change than the corresponding ideal gas

Ground water investigation in the southern half of Franklin County, Missouri

Increasing population and industrialization in Franklin County, Missouri, has led to increased demand for water. Much of this demand will be satisfied from ground water resources. Although the general geology of Franklin County has been mentioned in several publications, no detailed study of structure and ground water geology had been done. This thesis consists of study of stratigraphy, lithology, and geologic structure, and analysis of well log data to determine occurrence and availability of ground water, in the southern half of Franklin County. Stratigraphy and lithology is compiled from previous literature, modified to agree with well log data. Structural geology is determined from correlation of well logs and field mapping of the central 38 square miles of the area. Structural data, shown on contour maps of the tops of two major aquifers, the Gasconade and Eminence formations, on isopach map of the Gasconade, and three cross-sections, reveal four major and several minor northeasterly plunging folds. No evidence was found for some possible faults shown on previously published maps. Outcrops examined are indicated on a detailed geologic map of the field area. Relation of number of wells drilled in various formations to expanding population is shown graphically. Depth to static and dynamic water levels, drawdown, and production rates are described for several formations. Specific capacities of the major aquifers are computed. Variations in chemical quality of ground water are illustrated by representative analyses within the area. Appendices list surface elevation, total depth, production and specific capacity of all wells studied --Abstract, page ii