Biomedical subjects

Daniel Scherman

Publications and source records attributed to Daniel Scherman.

At least 19 recordsLinked to original sources

PubMed2006

PEGylation of microspheres for therapeutic embolization: preparation, characterization and biological performance evaluation.

In this study, microspheres designed for embolization, defined as GF2000-Trisacryl MS (GF-MS) and DEAE-Trisacryl MS (DEAE-MS), were originally PEGylated using (3-amino propyl) triethoxy silane as coupling agent. Indomethacin was loaded into both PEGylated and non-PEGylated DEAE-MS, displaying ion-exchange ability, through a batch process with a respective capacity of 1.2 and 0.25 g/g. The morphology of naked and PEGylated MS was evaluated by scanning electron microscopy (SEM). Both micosphere resins surface looked like orange skin, although DEAE-MS showed a slightly rougher surface due to the copolymerization process. PEGylated microspheres have a most likely swelling surface owing to the presence of PEG hydrophilic chains. The mean diameters were of about 66 and 60 microm for GF-MS and DEAE-MS, respectively. Data obtained for PEGylated MS by Fourier Transform Infrared spectroscopy (FTIR) confirmed that microspheres were successfully PEGylated. Finally, complement activation in vitro was performed to evaluate the activating capacity of different microspheres. Both PEGylated GF-MS and DEAE-MS activated the complement system of about 33% less than their corresponding naked microspheres, while loading PEGylated DEAE-MS with indomethacin almost suppressed complement activation. This inhibiting role implies that PEGylation as well as loading the microspheres with anti-inflammatory drug has a compact effect on the interaction of microspheres with blood proteins.

Biocompatible Materials↗

PubMed2006

Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents.

Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural requirement of a cis-configuration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series of indolic analogues of CSA-4 was synthesized by means of an efficient strategy. Six compounds (20b, 25b-27b, 32b, and 35b) were identified as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nanomolar level. Both activities were well correlated with the ability to induce morphological changes of EA.hy 926 endothelial cells. In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any isomerization leading to inactive trans compounds.

Angiogenesis Inhibitors↗

PubMed2006

Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experimental arthritis.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is among the most prominent cytokines in rheumatoid arthritis (RA) and is secreted mainly by macrophages. A direct method for restoring the immunologic balance in RA is use of small interfering RNA (siRNA) for silencing the TNFalpha transcript. The aim of this study was to determine the therapeutic effect of systemic administration of TNFalpha siRNA in an experimental model of RA, optimizing its delivery using new liposome formulations. METHODS: Murine macrophages were transfected with siRNA targeting TNFalpha, and expression was measured. The therapeutic effect in collagen-induced arthritis (CIA) was assessed after intravenous delivery of TNFalpha siRNA. Delivery was optimized using a carrier DNA for complexation with the cationic liposome RPR209120/DOPE. Levels of TNFalpha and other cytokines were measured in sera and joint tissue-conditioned media. Biodistribution was determined using a fluorescent siRNA. RESULTS: In vitro, TNFalpha siRNA efficiently and specifically modulated the expression of TNFalpha at both the messenger RNA and protein levels. In vivo, complete cure of CIA was observed when TNFalpha siRNA was administered weekly, complexed with the liposome and combined with carrier DNA. Inhibition (50-70%) of articular and systemic TNFalpha secretion was detected in the siRNA-injected groups, which correlated with a decrease in the levels of interleukin-6 and monocyte chemotactic protein 1. The main organs targeted by siRNA were the liver and spleen; the addition of liposome RPR209120 and carrier DNA significantly increased organ uptake. CONCLUSION: We demonstrated the efficiency of systemic delivery of siRNA designed to silence TNFalpha in CIA, using a liposome carrier system as a way to address the methodologic limitations in vivo.

Animals↗

PubMed2006

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.

Animals↗

PubMed2006

Comparison of promoter region constructs for in vivo intramuscular expression.

BACKGROUND: High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. METHODS: Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. RESULTS: Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). CONCLUSION: For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.

Alkaline Phosphatase↗

PubMed2006

In vivo electrotransfer of interleukin-10 cDNA prevents endothelial upregulation of activated NF-kappaB and adhesion molecules following an atherogenic diet.

OBJECTIVES: Interleukin (IL)-10 has anti-atherogenic properties. However, the molecular mechanisms involved in IL-10 protection against atherosclerosis in vivo remain poorly understood. In this study, we examined the effect of IL-10 cDNA in vivo electrotransfer on diet-induced, endothelial activation. METHODS: C57BL/6J mice were fed an atherogenic diet for 10 days. Expression of VCAM-1 and ICAM-1 was examined in the aortic sinus, a region predisposed to atherogenesis in mice, using immunohistochemistry. NF-kappaB activation was examined using a monoclonal antibody that selectively reacts with the activated form of the p65 subunit. RESULTS: We detected a low basal expression of activated NF-kappaB, VCAM-1 and ICAM-1 in the endothelium of the aortic sinus. Endothelial expression of activated NF-kappaB, VCAM-1 and ICAM-1 was markedly increased after 10 days on the atherogenic diet (p < 0.001). In vivo electrotransfer of a murine IL-10-encoding plasmid completely prevented diet-induced endothelial upregulation of activated NF-kappaB, VCAM-1 and ICAM-1 (p < 0.01). CONCLUSION: In vivo electrotransfer of IL-10 cDNA prevents diet-induced endothelial activation. These results suggest that the protective effects of IL-10 may already occur in the very early stages of atherogenesis.

Animals↗

PubMed2005

Liposome biodistribution by time resolved fluorimetry of lipophilic europium complexes.

The use of conventional fluorophores suffers from some limitations in biological fluids due to low signal/background ratio. Today, this sensitivity issue might be reasonably improved thanks to lanthanide chelates, by selective detection of long decay fluorescence. Use of pulsed light source time-resolved fluorimetry takes into account the fluorescence decay time of the lanthanide chelates to gain sensitivity in biological media. Lipid-DTPA: Eu compounds have been prepared and incorporated into liposomes to evaluate europium based detection of liposomes in biological media. Fluorescence emission was not modified by this incorporation. Europium labelled liposomes were used for biodistribution studies and showed their use in this context.

Animals↗

PubMed2005

Sialyl Lewis(x) analogs based on a quinic acid scaffold as the fucose mimic.

(-)-Quinic acid was used as a starting material for the preparation of sialyl Lewis(x) mimetics in order to target E-selectin. Spatial orientation of the hydroxyl groups of quinic acid could mimic the l-fucose ones. Introduction of a side chain ending with a carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives, incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC(50) within the millimolar range.

Cell Adhesion↗

PubMed2005

Positive in vivo heterologous gene regulation by electric pulses delivery with metallothionein I gene promoter.

BACKGROUND: In vivo electrotransfer is a physical method of gene delivery in various tissues and organs. It is a promising strategy for the systemic secretion of therapeutic proteins and for DNA vaccination. Nevertheless, for the success of gene therapy in clinics, it is essential to develop gene regulation systems. The existing systems described in the literature all rely on the creation of an artificial transcription factor and/or an inducer drug. New strategies based on endogenous regulatable elements are being developed. We have previously identified the murine metallothionein promoter as an endogenous promoter inducible by controlled electric stimuli applied for electrotransfer experiments. We report here a regulation strategy based on this murine metallothionein promoter in a plasmid context using electric pulses delivery as an inducer. METHODS: Plasmids containing different constructions of the murine metallothionein I (mMT-I) promoter were transfected in mice tibialis-cranalis muscles using the simple skeletal muscle electrotransfer method. The regulation system was studied with the murine secreted alkaline phosphatase (MUSEAP) reporter gene. RESULTS: The mMT-I promoter can be transiently induced in vivo by application of electric fields. Its inducibility was analyzed in a plasmid context. We demonstrated that the mechanism of this transcriptional induction is not mediated by the cellular entry of metal ions. The ARE (antioxidant-responsive element) sequence was identified as the element responsive to the electric field stimulation. CONCLUSIONS: This time-control of the expression of a therapeutic gene by physical stimuli could be of value in the context of gene regulation for gene therapy.

Alkaline Phosphatase↗

PubMed2005

[Understanding the cellular and molecular aspects of haematopoietic stem cell emergence and their regulation by RUNX1/AML1].

In the vertebrate embryo, the ventral wall of the aorta is the major site of Haematopoietic Stem Cell (HSC) production. HSC, which are at the basis of the adult blood cells hierarchy, are generated from Endothelial Cells (EC) through a complex cascade of molecular events. The transcription factor RUNX1/AML1 and its cofactor CBFbeta, disrupted in 20 % of acute myeloid leukaemia cases, are thought to control this process. A detailed gene expression analysis of RUNX1 and its associated factors in the chick embryo, prompted us to speculate on the molecular cascades involved in HSC production. The function of RUNX1 is however tightly regulated at several levels, rendering analysis through classical genetic approaches very difficult to manage. To offer new possibilities of investigation, we have designed a technique to target the blood forming system in vivo. Gene transfer was achieved by lipofection following delivery by intra-cardiac injection in the avian embryo. This method was optimised to allow a wide range of functional analysis, either by gain or loss of function, in a simple and efficient manner. In combination with experimental advantages of the avian embryo, this new system of genetic analysis allows us to perform a detailed study of RUNX1 function in HSC production from EC.

Animals↗

PubMed2005

Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer.

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

Animals↗

PubMed2004

pH-sensitive PEG lipids containing orthoester linkers: new potential tools for nonviral gene delivery.

The synthesis and properties of pH-sensitive polyethylene glycol (PEG) lipids are described. The sensitivity of these conjugates to slightly acidic pH was clearly related to the structure of the orthoester linkage involved. It was found that pH-sensitive PEG lipids stabilized cationic lipid/DNA isoelectric complexes as efficiently as their non-pH-sensitive PEG analogs at neutral pH. Lowering the pH resulted in the precipitation of the complexes bearing pH-sensitive PEG lipids as a consequence of their degradation. In contrast, insertion of non-pH-sensitive PEG lipids maintained the complex colloidal stability even at lower pH. In vitro results showed a significant increase in transfection with formulations containing pH-sensitive PEG lipids versus non-pH-sensitive analogs. These conjugates show promising properties as lipoplex-stabilizing agents at neutral pH, which could be triggered by a mild acidic environment such as that occurring in solid tumors, inflammatory tissues, and intracellular endosomal compartments.

Cross-Linking Reagents↗

PubMed2004

Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation.

The clinical application of monoclonal antibodies (mAbs) potentially concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is very often not applicable to long-term treatments because of the restrictive cost of mAbs certified for human use and the side effects associated with injection of massive doses of antibodies. Gene/cell therapies designed for sustained and, possibly, regulatable in vivo production and systemic delivery of mAbs might permit to advantageously replace it. We have already shown that several such approaches allow month- to year-long ectopic antibody production by non-B cells in living organisms. Those include grafting of ex vivo genetically modified cells of various types, in vivo adenoviral gene transfer and implantation of encapsulated antibody-producing cells. Because intramuscular electrotransfer of naked DNA has already been used for in vivo production of a variety of proteins, we have wanted to test whether it could be adapted to that of ectopic mAbs as well. We report here that this is actually the case since both long-term and regulatable production of an ectopic mAb could be obtained in the mouse taken as a model animal. Although serum antibody concentrations obtained were relatively low, these data are encouraging in the perspective of future therapeutical applications of this technology in mAb-based immunotherapies, especially in developing countries where cost-effective and easily implementable technologies would be required for large-scale applications in the context of severe chronic viral diseases such as HIV and HCV infections.

Journal Article↗

PubMed2004

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications.

In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue.

Animals↗

PubMed2004

Physicochemical optimisation of plasmid delivery by cationic lipids.

Non-viral gene therapy is based on the use of plasmid expression vectors and chemical or physical plasmid DNA delivery systems. This review discusses the roles of cationic lipids as vectors for gene transfection, reviews different strategies employed to improve cationic lipids for in vivo use, and provides original results on the physicochemistry of lipoplexes. Cationic lipid/DNA delivery vehicles have evolved considerably since their initial gene transfection experiments. Much work has been carried out to investigate their structure/activity relationships, methods of formulation and physicochemical properties. Further work has also focused on enhancing and prolonging their stability in a physiological environment as well as increasing their site-specific and tissue-specific interactions. Original data presented in this report confirm that cationic lipids associated to DNA form supramolecular lamellar structures, which protect DNA from serum DNAse degradation. The effect of formulation (and hence the size of the particles) on lipoplex in vivo circulation half-life and biodistribution is also discussed. A list of abbreviations can be found at the end of the review.

Animals↗

PubMed2004

Gene therapy of collagen-induced arthritis by electrotransfer of human tumor necrosis factor-alpha soluble receptor I variants.

Electrotransfer is a simple and efficient strategy of nonviral gene delivery. We have used this method to deliver plasmids encoding three human tumor necrosis factor-alpha soluble receptor I variants (hTNFR-Is) a monomeric hTNFR-Is, a chimeric hTNFR-Is/mIgG1, and a dimeric (hTNFR-Is)(2) form. Electrotransfer parameters were studied and because anti-TNF strategies have proven efficient for the treatment of rheumatoid arthritis in clinics, we used a collagen-induced arthritis (CIA) mouse model to assess the efficacy of our constructs in the treatment of the disease. All proteins were proven bioactive, both in vitro and ex vivo. Plasmid intramuscular electrotransfer in mice resulted in a local expression of the three variants for at least 6 months; systemic expression lasted also more than 6 months for the hTNFR-Is/mIgG1 form, while it was shorter for the two other forms. This expression was plasmid dose-dependent. Electrotransfer of 50 microg of hTNFR-Is/mIgG1 at the onset of a CIA induced a clear-cut decrease in both clinical and histologic signs of the disease; the dimeric form also showed some efficacy. Moreover, the long-lasting protective effect was observed for more than 5 weeks. Comparison of this electrotransfer approach with repeated recombinant protein (etanercept) injections highlighted the potential practical interest of gene therapy approach for CIA, which leads to sustained therapeutic effect after single treatment. These results show that electrotransfer may be a useful method to deliver cytokine or anticytokine therapy in rheumatoid arthritis and also illustrate the potentiality of plasmid intramuscular electrotransfer for the rapid screening and assessment of different variant forms of secreted proteins.

Animals↗

PubMed2003

LPS-induced bronchial hyperreactivity: interference by mIL-10 differs according to site of delivery.

When administered to mice systemically or via the airways, LPS induces bronchoconstriction (BC) and/or bronchopulmonary hyperreactivity (BHR), associated with inflammation. Accordingly, a relationship between inflammation and allergic and nonallergic BHR can be hypothesized. We therefore studied the interference of the anti-inflammatory cytokine murine IL-10 (mIL-10) with LPS-induced lung inflammation, BC, and BHR. mIL-10 was administered directly into the airways by intranasal instillation or generated in vivo after muscle electrotransfer of mIL-10-encoding plasmid. Electrotransfer led to high mIL-10 circulating levels for a longer time than after the injection of recombinant mIL-10 (rmIL-10). rmIL-10 administered intranasally reduced lung inflammation and BHR after LPS administration into airways. It also reduced the ex vivo production of TNF-alpha by LPS-stimulated lung tissue explants. Two days after electrotransfer, mIL-10 blood levels were elevated, but lung inflammation, BC, and BHR persisted unaffected. Blood mIL-10 reaches the airways poorly, which probably accounts for the ineffectiveness of mIL-10-encoding plasmid electrotransfer. When LPS was aerosolized 15 days after electrotransfer, lung inflammation persisted but BHR was significantly reduced, an effect that may be related to the longer exposure of the relevant cells to mIL-10. The dissociation between inflammation and BHR indicates that both are not directly correlated. In conclusion, this study shows that mIL-10 is efficient against BHR when present in the airway compartment. Despite this, the muscle electrotransfer with mIL-10-encoding plasmid showed a protective effect against BHR after a delay of 2 wk that should be further investigated.

Administration, Intranasal↗

PubMed2003

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties.

BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1alpha importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin beta, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

Active Transport, Cell Nucleus↗