Hydrophobic oxime ethers: a versatile class of pDNA and siRNA transfection lipids

The manipulation of the cationic lipid structures to increase polynucleotide binding and delivery properties, while also minimizing associated cytotoxicity, has been a principal strategy for developing next-generation transfection agents. The polar (DNA binding) and hydrophobic domains of transfection lipids have been extensively studied; however, the linking domain comprising the substructure used to tether the polar and hydrophobic domains has attracted considerably less attention as an optimization variable. Here, we examine the use of an oxime ether as the linking domain. Hydrophobic oxime ethers were readily assembled via click chemistry by oximation of hydrophobic aldehydes using an aminooxy salt. A facile ligation reaction delivered the desired compounds with hydrophobic domain asymmetry. Using the MCF-7 breast cancer, H1792 lung cancer and PAR C10 salivary epithelial cell lines, our findings show that lipoplexes derived from oxime ether lipids transfect in the presence of serum at higher levels than commonly used liposome formulations, based on both luciferase and green fluorescent protein (GFP) assays. Given the biological compatibility of oxime ethers and their ease of formation, this functional group should find significant application as a linking domain in future designs of transfection vectors.     

用专利制度保护化学化工技术的策略和技巧

化工技术具有发明活动活跃、实用性强、实施快等特点 ,利用专利制度保护化工技术具有重要的意义。本文介绍了我国专利制度的基本特点以及申请化工技术专利保护的策略和技巧。     

omega-Hydrazino linear polyethylenimine: a monoconjugation building block for nucleic acid delivery

Nonviral gene therapy requires efficient vectors that are able to deliver nucleic acids inside the targeted cell nucleus. Developing new tools for the synthesis of supramolecular vectors with improved transfection efficiency and better biodistribution is therefore a crucial issue. Here we describe the synthesis of a 140-mer linear polyethylenimine (L-PEI) terminated at one end by a highly nucleophilic hydrazine residue. This cationic polymer, whose backbone is well known for its remarkable gene-delivery efficiency, constitutes a building block for omega-regioselective conjugation to molecules through the formation of stable linkages such as the hydrazone bonds. To demonstrate the potential of the omega-hydrazino linear polyethylenimine, human serum transferrin, a ligand that is well know to improve gene-delivery systems, was used as a model of sensitive material. The blood protein was oxidized to generate an aldehyde function and was subsequently conjugated to hydrazino PEI. The new polyethylenimine-transferrin (PEI-Tf) vector was purified and was shown to condense plasmid DNA into compact superstructures compatible with cellular uptake. Finally, the cellular-binding and gene-delivery properties of PEI/DNA polyplexes incorporating different quantities of transferrin were evaluated by FACS analysis and luciferase assay.     

Towards a methodology for the systematic analysis and design of efficient chemical processes: Part 1. From unit operations to elementary process functions

A successful intensification of a chemical process requires a holistic view of the process and a systematic debottlenecking, which is obtained by identifying and eliminating the main transport resistances that limit the overall process performance and thus can be considered as rate determining steps on the process level. In this paper, we will suggest a new approach that is not based on the classical unit operation concept, but on the analysis of the basic functional principles that are encountered in chemical processes.A review on the history of chemical engineering in general and more specifically on the development of the unit operation concept underlines the outstanding significance of this concept in chemical and process engineering. The unit operation concept is strongly linked with the idea of thinking in terms of apparatuses, using technology off the shelf. The use of such “ready solutions” is of course convenient in the analysis and design of chemical processes; however, it can also be a problem since it inherently reduces the possibilities of process intensification measures.Therefore, we break with the tradition of thinking in terms of “unit apparatuses” and suggest a new, more rigorous function-based approach that focuses on the underlying fundamental physical and chemical processes and fluxes.For this purpose, we decompose the chemical process into so-called functional modules that fulfill specific tasks in the course of the process. The functional modules itself can be further decomposed and represented by a linear combination of elementary process functions. These are basis vectors in thermodynamic state space. Within this theoretical framework we can individually examine possible process routes and identify resistances in individual process steps. This allows us to analyze and propose possible options for the intensification of the considered chemical process.     

Phospholipases: Occurrence and production in microorganisms,assay for high-throughput screening,and gene discovery from natural and man-made diversity

Various kinds of phospholipids have wide industrial applications such as in food and nutraceuticals, cosmetics, agricultural products, and pharmaceuticals. The demand for reliable biocatalysts for the production of phospholipid products, such as phospholipases A₁, A₂, C, and D, has steadily increased over the past several decades. A large number of microbial phospholipases have been isolated and characterized, and the increasing availability of these enzymes could eventually lead to the sustained development of phospholipid-related biotechnology. Although a number of reactions have been performed using phospholipases, a reliable and efficient supply of superior phospholipases in quantity is still a challenge for their practical application. High-throughput functional assay methods for phospholipases should be devised to develop superior new species from the huge diversity of phospholipases. Recent biotechnological advances in the discovery of new phospholipase genes from natural sources, such as extremophiles of phospholipases by protein engineering, such as directed evolution, can provide valuable means of rapidly developing practical uses of phospholipases for various applications.     

Among the trends for a modern chemical engineering,the third paradigm: The time and length multiscale approach as an efficient tool for process intensification and product design and engineering

To respond to the changing needs of the chemical and related industries in order both to meet today's economy demands and to remain competitive in global trade, a modern chemical engineering is vital to satisfy both the market requirements for specific nano and microscale end-use properties of products, and the social and environmental constraints of industrial meso and macroscale processes. Thus an integrated system approach of complex multidisciplinary, non-linear, non-equilibrium processes and phenomena occurring on different length and time scales of the supply chain is required. That is, a good understanding of how phenomena at a smaller length-scale relates to properties and behaviour at a longer length-scale is necessary (from the molecular-scale to the production-scales). This has been defined as the triplet “molecular Processes-Product-Process (3PE)” integrated multiscale approach of chemical engineering. Indeed a modern chemical engineering can be summarized by four main objectives: (1) Increase productivity and selectivity through intensification of intelligent operations and a multiscale approach to processes control: nano and micro-tailoring of materials with controlled structure. (2) Design novel equipment based on scientific principles and new production methods: process intensification using multifunctional reactors and micro-engineering for micro structured equipment. (3) Manufacturing end-use properties to synthesize structured products, combining several functions required by the customer with a special emphasis on complex fluids and solid technology, necessating molecular modeling, polymorph prediction and sensor development. (4) Implement multiscale application of computational chemical engineering modeling and simulation to real-life situations from the molecular-scale to the production-scale, e.g., in order to understand how phenomena at a smaller length-scale relate to properties and behaviour at a longer length-scale. The presentation will emphasize the 3PE multiscale approach of chemical engineering for investigations in the previous objectives and on its success due to the today's considerable progress in the use of scientific instrumentation, in modeling, simulation and computer-aided tools, and in the systematic design methods.     

Organometallic compounds with biological activity: a very selective and highly potent cellular inhibitor for glycogen synthase kinase 3

A chiral second-generation organoruthenium half-sandwich compound is disclosed that shows a remarkable selectivity and cellular potency for the inhibition of glycogen synthase kinase 3 (GSK-3). The selectivity was evaluated against a panel of 57 protein kinases, in which no other kinase was inhibited to the same extent, with a selectivity window of at least tenfold to more than 1000-fold at 100 microM ATP. Furthermore, a comparison with organic GSK-3 inhibitors demonstrated the superior cellular activity of this ruthenium compound: wnt signaling was fully induced at concentrations down to 30 nM. For comparison, the well-established organic GSK-3 inhibitors 6-bromoindirubin-3'-oxime (BIO) and kenpaullone activate the wnt pathway at concentrations that are higher by around 30-fold and 100-fold, respectively. The treatment of zebrafish embryos with the organometallic inhibitor resulted in a phenotype that is typical for the inhibition of GSK-3. No phenotypic change was observed with the mirror-imaged ruthenium complex. The latter does not, in fact, show any of the pharmacological properties for the inhibition of GSK-3. Overall, these results demonstrate the potential usefulness of organometallic compounds as molecular probes in cultured cells and whole organisms.     

Human gene control by vital oncogenes: revisiting a theoretical model and its implications for targeted cancer therapy

An important assumption of our current understanding of the mechanisms of carcinogenesis has been the belief that clarification of the cancer process would inevitably reveal some of the crucial mechanisms of normal human gene regulation. Since the momentous work of Bishop and Varmus, both the molecular and the biochemical processes underlying the events in the development of cancer have become increasingly clear. The identification of cellular signaling pathways and the role of protein kinases in the events leading to gene activation have been critical to our understanding not only of normal cellular gene control mechanisms, but also have clarified some of the important molecular and biochemical events occurring within a cancer cell. We now know that oncogenes are dysfunctional proto-oncogenes and that dysfunctional tumor suppressor genes contribute to the cancer process. Furthermore, Weinstein and others have hypothesized the phenomenon of oncogene addiction as a distinct characteristic of the malignant cell. It can be assumed that cancer cells, indeed, become dependent on such vital oncogenes. The products of these vital oncogenes, such as c-myc, may well be the Achilles heel by which targeted molecular therapy may lead to truly personalized cancer therapy. The remaining problem is the need to introduce relevant molecular diagnostic tests such as genome microarray analysis and proteomic methods, especially protein kinase identification arrays, for each individual patient. Genome wide association studies on cancers with gene analysis of single nucleotide and other mutations in functional proto-oncogenes will, hopefully, identify dysfunctional proto-oncogenes and allow the development of more specific targeted drugs directed against the protein products of these vital oncogenes. In 1984 Willis proposed a molecular and biochemical model for eukaryotic gene regulation suggesting how proto-oncogenes might function within the normal cell. That model predicted the existence of vital oncogenes and can now be used to hypothesize the biochemical and molecular mechanisms that drive the processes leading to disruption of the gene regulatory machinery, resulting in the transformation of normal cells into cancer.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of thebacterial phosphoenol-pymvate-dependent phosphotransferase systemof Staphylococcus aureus was modified by sitespecific mutagenesison the corresponding lacE gene in order to replace the histidineresidues 245, 274 and 510 and the cysteine residue 476 of theamino acid sequence with a serine residue. The wild-type andmutant genes were expressed in Escherichia coli and the geneproducts were characterized in different in vitro test systems.In vitro phosphorylation studies on mutant derivatives of thelactose-specific enzyme II led to the conclusion that cysteinersidue 476 is the active-site for phosphorylation of this enzymeII by a phospho-enzyme III of the same sugar specificity. Acysteine residue phosphor) lated intermediate was first postulatedfor the mannitol-specific enzyme II of E.coli and studies performedindependently concerning the lactose-specific enzyme II of Lactobacilluscasei are in agreement with the above results.     

Modeling of a membrane reactor system for crude palm oil transesterification. Part I: Chemical and phase equilibrium

Using a membrane reactor for reversible transesterification reaction involves reaction and product separation within a single unit. However, a pseudohomogeneous reaction and heterogeneous separation must be maintained for successful membrane reactor operation. Present research is aimed to develop an integrated model of chemical and phase equilibrium (CPE) and modified Maxwell–Stefan equation that describes the simultaneous CPE and mass transport phenomena of biodiesel production from crude palm oil (CPO) using a membrane reactor. In the first part of this work, a systematic approach describing simultaneous CPE of CPO transesterification in the membrane reactor was developed with the reconciliation of transesterification reaction and phase equilibrium that involves six‐component. The results revealed that regressed apparent equilibrium constant, value of 17.557 1.51% were higher than the literatures. This indicates that forward reaction of the reversible CPO transesterification is much favored in the membrane reactor than the conventional reactor. © 2015 American Institute of Chemical Engineers AIChE J, 61: 1968–1980, 2015     

Surface modification of TPGS-b-(PCL-ran-PGA) nanoparticles with polyethyleneimine as a co-delivery system of TRAIL and endostatin for cervical cancer gene therapy

The efficient delivery of therapeutic genes into cells of interest is a critical challenge to broad application of non-viral vector systems. In this research, a novel TPGS-b-(PCL-ran-PGA) nanoparticle modified with polyethyleneimine was applied to be a vector of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and endostatin for cervical cancer gene therapy. Firstly, a novel biodegradable copolymer, TPGS-b-(PCL-ran-PGA), was synthesized and characterized. The nanoparticles were fabricated by an emulsion/solvent evaporation method and then further modified with polyethyleneimine (PEI) carrying TRAIL and/or endostatin genes. The uptake of pIRES2-EGFP and/or pDsRED nanoparticles by HeLa cells were observed by fluorescence microscopy and confocal laser scanning microscopy. The cell viability of TRAIL/endostatin-loaded nanoparticles in HeLa cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Severe combined immunodeficient mice carrying HeLa tumor xenografts were treated in groups of six including phosphate-buffered saline control, blank TPGS-b-(PCL-ran-PGA) nanoparticles, blank TPGS-b-(PCL-ran-PGA)/PEI nanoparticles, and three types of gene nanoparticles. The activity was assessed using average increase in survival time, body weight, and solid tumor volume. All the specimens were then prepared as formalin-fixed and paraffin-embedded tissue sections for hematoxylin-eosin staining. The data showed that the nanoparticles could efficiently deliver plasmids into HeLa cells. The cytotoxicity of the HeLa cells was significantly increased by TRAIL/endostatin-loaded nanoparticles when compared with control groups. The use of TPGS in combination with TRAIL and endostatin had synergistic antitumor effects. In conclusion, the TRAIL/endostatin-loaded nanoparticles offer considerable potential as an ideal candidate for in vivo cancer gene delivery.