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Poly(N-methylvinylamines) with secondary amines can form complexes with plasmid DNA (pDNA) and provide transfection efficiency in HeLa cells in the same order as linear polyethyleneimine but with higher cell viability. Chemical modifications of poly(N-methylvinylamine) backbones are performed to further improve transfection efficiency while maintaining low degree of cytotoxicity. In a first type of polymer, primary amino groups are incorporated via a copolymerization strategy. In a second one, primary amino and imidazole groups are incorporated also via a copolymerization strategy. In a third one, secondary amino groups are substituted with methylguanidine functions through a postpolymerization reaction. Thus, novel polymers of various molecular masses are synthesized, characterized, and their interaction with pDNA studied. Then, their transfection efficiency and cytotoxicity are tested in HeLa cells. Two polymethylvinylamine-based copolymers, one containing 20% of imidazole moieties and another one composed of 12% of guanidinyl units allow remarkable transfection efficiency of HeLa, pulmonary (16HBE), skeletal muscle (C2C12), and dendritic (DC2.4) cells. Overall, this work thus identifies new promising DNA carriers and chemical modifications that improve the transfection efficiency while maintaining low degree of cytotoxicity.
Gene transfection with polymeric carrier remains a challenge ; particularly, high transfection levels combined with low toxicity are hard to achieve. We herein revisit polyvinylamines, an old and neglected family of cationic polymers. They can be readily obtained by controlled hydrolysis of polyvinylamides prepared through (controlled) radical polymerization. A series of tailor-made and well-defined polyvinylamines bearing primary amino groups, and poly(N-methylvinylamine) bearing secondary amines, were evaluated for the transfection of cells with pDNA as a function of their molar mass, molar mass distribution, and degree of deacetylation. Unexpected high transfection levels, in combination with low cytotoxicity were recorded for both series. Surprisingly, a great impact of the molar mass was observed for the primary amine polyvinylamine series, whereas the results were mostly independent of molar mass or dispersity for the polymer bearing secondary amine. It was further established that a certain percentage of acetamide groups increased the transfection level, while maintaining low cytotoxicity. These results highlight for the first time the real potential of polyvinylamines as gene carriers, and make these polymers very attractive for further development in gene therapy.
Synthetic vectors such as cationic polymers and cationic lipids remain attractive tools for non-viral gene transfer which is a complex process whose effectiveness relies on the ability to deliver a plasmid DNA (pDNA) into the nucleus of non-dividing cells. Once in the cytosol, the transport of pDNAs towards the nuclear envelope is strongly impaired by their very low cytosolic mobility due to their large size. To promote their movement towards the cell nucleus, few strategies have been implemented to exploit dynein, the microtubule's (MT's) motor protein, for propagation of cytosolic pDNA along the MTs towards the cell nucleus. In the first part of this review, an overview on MTs, dynein, dynein/virus interaction feature is presented followed by a summary of the results obtained by exploitation of LC8 and TCTEL1 dynein light chain association sequence (DLC-AS) for non-viral transfection. The second part dedicated to the adenoviral protein E3-14.7K, reports the transfection efficiency of polyplexes and lipoplexes containing the E3-14.7K-derived P79-98 peptide linked to pDNA. Here, several lines of evidence are given showing that dynein can be targeted to improve cytosolic pDNA mobility and accumulate pDNA near nuclear envelope in order to facilitate its transport through the nuclear pores. The linkage of various DLC-AS to pDNA carriers led to modest transfection improvements and their direct interaction with MTs was not demonstrated. In contrast, pDNA linked to the P79-98 peptide interacting with TCTEL1 via a cytosolic protein (fourteen seven K-interacting protein-1 (FIP-1)), interaction with MTs is evidenced in cellulo and transfection efficiency is improved.
Cationic lipid/co-lipid combinations have been extensively explored in gene delivery as alternatives to viral vectors. To be established as a gold standard of chemical vectors, considerable improvement in their transfection efficiency is however required. Herein, we report a simple procedure to synthesize new cationic and co-lipids for the DNA transfection of dendritic cells (DCs). Seven α-amino-lipophosphonates featuring two aza-heterocycles with protonable sites (imidazole or pyridine) were synthesized and used as co-lipids in liposomes with cationic lipid. For each liposome, the cationic lipid is either the imidazolium lipophosphoramidate (Lipid 2) or α-amino-lipophosphonate containing basic tertiary aliphatic amine in the polar head group (Lipid 3b). The cationic lipids either with new co-lipids or DOPE formed positively charged nano-sized stable liposomes that effectively interact with plasmide DNA (pDNA) to produce lipoplexes. Membrane fusion studies showed that α-amino-phosphonates featuring imidazole moiety in the polar head group exhibited higher fusion at pH 5.5 than pH 7.4. This study suggests that the best formulations for the transfection of DCs (based on the % transfected cells and the intensity of EGFP-based fluorescence) are Lipid 2 associated with either 3a, 3d or DOPE and the cationic lipid 3b formulated with 3a or DOPE as helper lipid. Furthermore, Lipid 3a could be used as an alternative to DOPE as a helper lipid. Overall, these results indicate that novel imidazole containing α-amino-phosphonates can serve as effective transfection agents for the DC-based vaccines.
Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx6-THF19-MeOx6) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and DeltaF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on DeltaF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl- selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx6-THF19-MeOx6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOxn-THFm-MeOxn copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients.
When administrated in the blood circulation, plasmid DNA (pDNA) complexed with synthetic vectors must pass through a vascular endothelium to transfect underlying tissues. Under inflammatory condition, cytokines can modify the endothelium integrity. Here, the trans-endothelial passage (TEP) of DNA complexes including polyplexes, lipoplexes and lipopolyplexes was investigated in the presence of tumor necrosis factor-a (TNF-alpha), interleukin-1 beta (IL-1 beta) or insulin-like growth factor-1 (IGF-1). The experiments were performed by using an in vitro model comprising a monolayer of mouse cardiac endothelial cells (MCEC) seeded on a trans-well insert and the transfection of C2C12 myoblasts cultured on the lower chamber as read out of TEP. We report that polyplexes made with a histidinylated derivative of lPEI (His-lPEI) exhibit the highest capacity (10.5 mu g cm(-2) h versus 0.324 mu g cm(-2) h) to cross TNF-alpha-induced inflamed endothelium model, but this positive effect is counterbalanced by the presence of IL-1 beta. His-lPEI polyplex TEP is also increased in the presence of IGF-1 (2.58 mu g cm(-2) h). TEP of lipid-based DNA complexes including lipoplexes and lipopolyplexes was lowest compared with polymer-based DNA complexes. Overall, the results indicate that under inflammation, His-lPEI polyplexes have a good profile to cross a vascular endothelium of striated muscle with low cytotoxicity and high transfection efficiency of C2C12 myoblasts. These data provide insights concerning the endothelial passage of vectors in inflammatory conditions and can serve as a basis towards in vivo studies.
The self-assembly of a plasmid DNA (pDNA) with cationic polymers or cationic liposomes forms nanosized supramolecular structures called lipoplexes, polyplexes and lipopolyplexes. Here, we report that when two polyplex preparations made using the same polymer and the same pDNA but labelled with two different fluorophores are mixed together, pDNA molecules are exchanged. Indeed, when Flu-pDNA complexed with histidinylated lPEI (Flu-pDNA/His-lPEI) polyplexes are mixed with Cy5-pDNA complexed with histidinylated lPEI (Cy5-pDNA/His-lPEI) polyplexes, a high quantity of polyplexes emitting dual fluorescence is observed and FRET indicates that one single polyplex contains two kinds of fluorescent pDNA molecules. This phenomenon depends on the polymer-type and the strength of the pDNA/polymer interaction. No exchange is observed with polylysine polyplexes, caged His-lPEI polyplexes, lipoplexes, lipopolyplexes or when His-lPEI polyplexes are mixed with lipoplexes. Our results suggest that aggregation or collapse of polyplexes occurs after their interaction leading to their unpackaging followed by the formation of new polyplexes with the exchange of pDNA.
Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid-based nanoplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with histidinylated l-polyethylenimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-N-histamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, His-lPEI polyplex showed high intracellular availability of DNA and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport toward the nucleus.
An archaeal diether lipid possessing a tri-antenna of α-D-mannopyranoside linked via an oligoethylene spacer to a (2S)-2-(phytanyloxy)-3-(hexadecyloxy)propanoic acid backbone (TriMan-Diether) was designed and synthesized. This new mannosylated lipid inserted in liposomes would show both DC-targeting and adjuvant properties thanks to the TriMan structure and the diether tail part, respectively.
Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes. (C) 2014 Elsevier B.V. All rights reserved.
Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.
Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 x 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 mug/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000, and gene expression is higher than observed with FreeStyle and JetPEI(R). Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.
We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (IPEI) and histidinylated IPEI (His(16)-IPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His(16)-IPEI polyplexes. Cryo-TEM analyses revealed that His(16)-IPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of IPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His(16)-IPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.
Lipopolyplexes formulations resulting from association of nucleic acid, cationic liposomes and a cationic polymer are attracting formulations for siRNA delivery. Herein, imidazole- and imidazolium-based liposomes in association with histidinylated polymers are studied to produce siRNA lipopoplyplexes (LPRi) subsequently used for gene silencing. Several kinds of imidazole/histidine liposomes and cationic polymers are tested. The gene silencing effect is evaluated with synthetic siRNA directed against EGFP or luciferase mRNA, in HeLa cells stably expressing EGFP or B16F10 melanoma cells stably expressing luciferase, respectively. SiRNA formulations are compared with those prepared using some commercial transfection reagents. One formulation called His-lPEI LPRi100 comprising siRNA, histidinylated lPEI (His-lPEI) and liposomes 100 made with O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate and O,O-dioleyl-N-histamine phosphoramidate appears to give the best specific inhibition of gene expression at 10nM siRNA in a dose-dependent manner with low cytotoxicity. This formulation exhibits a size and a zeta potential of 60nm and +84mV, respectively. According to our previous works, histidinylated lipopolyplexes appears as a versatile formulation for DNA, mRNA and siRNA transfection.
A series of linear polyethylenimine (lPEI) substituted with histidine residue (His-lPEI) was synthesized using the Michael reaction in order to provide new highly efficient vectors for gene therapy applications (up to 95% of transfected cells) with remarkable low cytotoxicity compared to lPEI-based polyplexes.
The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in nondividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was similar to 20-fold higher when the luciferase mRNA was capped with 3'-O-methyl-m7(5')GpPP(5')G (anti-reverse cap analogue ; ARCA) than with m7(5')Gppp(5')G (CAP).
Background : The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn2+ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His-pLK) complexes.
Background : Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway.
Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes.
Ingénieur d'études , Thérapies innovantes et nanomédecine


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subset: commoncrawl


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VLSI Design
Volume 12, Issue 2, Pages 275-300
Integrated Area-power Optimal State Assignment
Department of Electrical and Computer Engineering, Coover Hall, Iowa State University, Ames 50011-3060, IA, USA
Received 20 June 2000; Revised 3 August 2000
Copyright © 2001 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper presents a state assignment algorithm with the objective of lower energy along with area comparable to the area-targeting state assignments such as JEDI. The underlying framework is MUSTANG's complete weighted graph with weights representing state affinity. The weight computation phase estimates the computation energy of potential common cubes using steady state probabilities for transitions. The weight computation phase also identifies a large set of potential state cliques, which are incorporated into a recursive bipartitioning based state assignment procedure. Reuse of cliques identified by the weight computation phase results in a faster and efficient state assignment. The energy targeting weights result in ≈9% lower area and 18% lower power than area targeting weights in JEDI over 29 MCNC Logic Synthesis 93 benchmarks. The clique based state assignment performs almost as well as the annealing based state assignment in JEDI, and takes only about half as much time.


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Major Nelson announced that the classic side scrolling shoot-'m-up game Defender will be today's new Xbox Live Arcade game. The game was first released in 1980 and will now make its debute onto the Xbox 360.
Xbox turns 5 today and we get defender!
Like I said, GAAAAAAAY!
This wasn't released as some happy anniversary present.. this is just the typical Wednsday Arcade game... why are you guys pissed?
I've been waiting for some quick Defender action in hi-def... Soon to download and play... I wonder if/how the on-line is... gCM