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Midoux Patrick


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2016   Références trouvées : 3

Barbeau J., Lemiègre L., Quelen A., Malard V., Gao H., Gonçalves C., Berchel M., Jaffrès P.-A., Pichon C., Midoux P. and Benvegnu T.  (2016)

Synthesis of a trimannosylated-equipped archaeal diether lipid for the development of novel glycoliposomes

Carbohydrate Research (2016) 435, 142-148 - doi : 10.1016/j.carres.2016.10.003
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.

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.

Demoulins T., Milona P., Englezou P. C., Ebensen T., Schulze K., Suter R., Pichon C., Midoux P., Guzman C. A., Ruggli N. and McCullough K. C.  (2016)

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Nanomedicine-Nanotechnology, Biology and Medicine (2016) 12 (3) 711-722 - doi : 10.1016/j.nano.2015.11.001
Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb) ; their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity ; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR : The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb) ; their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity ; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR : The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Vassaux G., Angelova A. L., Rommelaere J., Baril P., Midoux P. and Cordelier P.  (2016)

The promise of gene therapy for pancreatic cancer

Human Gene Therapy Methods (2016) 27 (2) 127-133 - doi : 10.1089/hum.2015.141
Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.

Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.


2015   Références trouvées : 6

Delalande A., Leduc C., Midoux P., Postema M. and Pichon C.  (2015)

Efficient Gene Delivery by Sonoporation Is Associated with Microbubble Entry into Cells and the Clathrin-Dependent Endocytosis Pathway

Ultrasound in medicine & biology (2015) 41 (7) 1913-1926 - doi : 10.1016/j.ultrasmedbio.2015.03.010
Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation ; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings ; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation ; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings ; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Delalande A., Gosselin M.-P., Suwalski A., Guilmain W., Leduc C., Berchel M., Jaffres P.-A., Baril P., Midoux P. and Pichon C.  (2015)

Enhanced Achilles tendon healing by fibromodulin gene transfer

Nanomedicine (2016) 11 (7) 1735-1744 - doi : 10.1016/j.nano.2015.05.004
Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR : Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.

Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR : Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.

Belmadi N., Midoux P., Loyer P., Passirani C., Pichon C., Le Gall T., Jaffres P.-A., Lehn P., Montier T.  (2015)

Synthetic vectors for gene delivery : An overview of their evolution depending on routes of administration

Biotechnology Journal (2015) first published online : Jun 3, 2015 - doi : 10.1002/biot.201400841
Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein ; the replacement of a missing or inactive gene ; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein ; the replacement of a missing or inactive gene ; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

Wytrwal M., Leduc C., Sarna M., Goncalves C., Kepczynski M., Midoux P., Nowakowska M., Pichon C.  (2015)

Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives

International Journal of Pharmaceutics (2015) 478 (1) 372-382 - doi : 10.1016/j.ijpharm.2014.11.053
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.

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.

Rasolonjatovo B., Gomez J. P., Même W., Goncalves C., Huin C., Bennevault-Celton V., Le Gall T., Montier T., Lehn P., Cheradame H., Midoux P., Guegan P.  (2015)

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) Amphiphilic Triblock Copolymers : Synthesis, Physicochemical Characterizations, and Hydrosolubilizing Properties

Biomacromolecules (2015) 16 (3) 748-756 - doi : 10.1021/bm5016656
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.

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.

Midoux P., Pichon C.  (2015)

Lipid-based mRNA vaccine delivery systems

Expert Review of Vaccines (2015) 14 (2) 221-234 - doi : 10.1586/14760584.2015.986104
Synthetic mRNAs can become biopharmaceutics allowing vaccination against cancer, bacterial and virus infections. Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Although immune responses are generated by naked mRNAs, their formulations with chemical carriers are expected to provide more specificity and internalization in dendritic cells (DCs) for better immune responses and dose reduction. This review reports lipid-based formulations (LBFs) that have proved preclinical efficacy. The selective delivery of mRNA LBFs to favor intracellular accumulation in DCs and reduction of the effective doses is discussed, notably to decorate LBFs with carbohydrates or glycomimetics allowing endocytosis in DCs. We also report how smart intracellular delivery is achieved using pH-sensitive lipids or polymers for an efficient mRNA escape from endosomes and limitations regarding cytosolic mRNA location for translation.

Synthetic mRNAs can become biopharmaceutics allowing vaccination against cancer, bacterial and virus infections. Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Although immune responses are generated by naked mRNAs, their formulations with chemical carriers are expected to provide more specificity and internalization in dendritic cells (DCs) for better immune responses and dose reduction. This review reports lipid-based formulations (LBFs) that have proved preclinical efficacy. The selective delivery of mRNA LBFs to favor intracellular accumulation in DCs and reduction of the effective doses is discussed, notably to decorate LBFs with carbohydrates or glycomimetics allowing endocytosis in DCs. We also report how smart intracellular delivery is achieved using pH-sensitive lipids or polymers for an efficient mRNA escape from endosomes and limitations regarding cytosolic mRNA location for translation.


2014   Références trouvées : 8

Goncalves C., Gross F., Guegan P., Cheradame H., Midoux P.  (2014)

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1

Biotechnology journal (2014) 9 (11) 1380-1388 - doi : 10.1002/biot.201400324
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.

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.

Montier, T., Pichon, C., Blondel, M. and Midoux, P.  (2014)

Editorial : Current status and prospects on nucleic acid transfer

Biotechnology Journal (2014à 9 (11) 1363-1364 - doi : 10.1002/biot.201300510

Mahindhoratep, S., Bouda, H. A., El Shafey, N., Scherman, D., Kichler, A., Pichon, C., Midoux, P., Mignet, N. and Bureau, M. F.  (2014)

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer

Biochim Biophys Acta 1840 (11) 3257-3263 - doi : 10.1016/j.bbagen.2014.06.013
BACKGROUND : When activated, NF-kappaB can promote the nuclear import and transcription of DNA possessing NF-kappaB consensus sequences. Here, we investigated whether NF-kappaB is involved in the plasmid electrotransfer process. METHODS : Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappaB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBalpha were studied as a marker of NF-kappaB activation. RESULTS : Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-kappaB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IkappaBalpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IkappaBalpha and thus of free NF-kappaB in the absence of any stimulation. GENERAL SIGNIFICANCE : pDNA electrotransfer can increase transgene expression independently of NF-kappaB. The insertion of NF-kappaB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-kappaB in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappaB consensus sequences.

BACKGROUND : When activated, NF-kappaB can promote the nuclear import and transcription of DNA possessing NF-kappaB consensus sequences. Here, we investigated whether NF-kappaB is involved in the plasmid electrotransfer process. METHODS : Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappaB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBalpha were studied as a marker of NF-kappaB activation. RESULTS : Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-kappaB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IkappaBalpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IkappaBalpha and thus of free NF-kappaB in the absence of any stimulation. GENERAL SIGNIFICANCE : pDNA electrotransfer can increase transgene expression independently of NF-kappaB. The insertion of NF-kappaB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-kappaB in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappaB consensus sequences.

Maury, B., Goncalves, C., Tresset, G., Zeghal, M., Cheradame, H., Guegan, P., Pichon, C. and Midoux, P.  (2014)

Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells

Biomaterials (2014) 35 (22) 5977-5985 - doi : 10.1016/j.biomaterials.2014.04.007
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.

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.

Kasparkova, J. Thibault, T.Kostrhunova, H., Stepankova, J., Vojtiskova, M., Muchova, T., Midoux, P., Malinge, J.M. and Brabec, V.  (2014)

Different affinity of nuclear factor-kappa B proteins to DNA modified by antitumor cisplatin and its clinically ineffective trans isomer

Febs Journal (2014) 281 (5) 1393-1408 - doi : 10.1111/febs.12711
Nuclear factor-kappa B (NF-kB) comprises a family of protein transcription factors that have a regulatory function in numerous cellular processes and are implicated in the cancer cell response to antineoplastic drugs, including cisplatin. We characterized the effects of DNA adducts of cisplatin and ineffective transplatin on the affinity of NF-kB proteins to their consensus DNA sequence (kB site). Although the kB site-NF-B protein interaction was significantly perturbed by DNA adducts of cisplatin, transplatin adducts were markedly less effective both in cell-free media and in cellulo using a decoy strategy derivatized-approach. Moreover, NF-B inhibitor JSH-23 [4-methyl-N-1-(3-phenylpropyl)benzene-1,2-diamine] augmented cisplatin cytotoxicity in ovarian cancer cells and the data showed strong synergy with JSH-23 for cisplatin. The distinctive structural features of DNA adducts of the two platinum complexes suggest a unique role for conformational distortions induced in DNA by the adducts of cisplatin with respect to inhibition of the binding of NF-kB to the platinated kB sites. Because thousands of B sites are present in the DNA, the mechanisms underlying the antitumor efficiency of cisplatin in some tumor cells may involve downstream processes after inhibition of the binding of NF-B to B site(s) by DNA adducts of cisplatin, including enhanced programmed cell death in response to drug treatment.

Nuclear factor-kappa B (NF-kB) comprises a family of protein transcription factors that have a regulatory function in numerous cellular processes and are implicated in the cancer cell response to antineoplastic drugs, including cisplatin. We characterized the effects of DNA adducts of cisplatin and ineffective transplatin on the affinity of NF-kB proteins to their consensus DNA sequence (kB site). Although the kB site-NF-B protein interaction was significantly perturbed by DNA adducts of cisplatin, transplatin adducts were markedly less effective both in cell-free media and in cellulo using a decoy strategy derivatized-approach. Moreover, NF-B inhibitor JSH-23 [4-methyl-N-1-(3-phenylpropyl)benzene-1,2-diamine] augmented cisplatin cytotoxicity in ovarian cancer cells and the data showed strong synergy with JSH-23 for cisplatin. The distinctive structural features of DNA adducts of the two platinum complexes suggest a unique role for conformational distortions induced in DNA by the adducts of cisplatin with respect to inhibition of the binding of NF-kB to the platinated kB sites. Because thousands of B sites are present in the DNA, the mechanisms underlying the antitumor efficiency of cisplatin in some tumor cells may involve downstream processes after inhibition of the binding of NF-B to B site(s) by DNA adducts of cisplatin, including enhanced programmed cell death in response to drug treatment.

Brevet, D., Hocine, O., Delalande, A., Raehm, L., Charnay, C., Midoux, P., Durand, J.O. and Pichon, C.  (2014)

Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles

International Journal of Pharmaceutics (2014) 471 (1-2) 197-205 - doi : 10.1016/j.ijpharm.2014.05.020
Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then l-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo.

Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then l-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo.

Maurel, D. B. Benaitreau, D. Jaffre, C. Toumi, H. Portier, H. Uzbekov, R. Pichon, C. Benhamou, C. L. Lespessailles, E. Pallu, S.  (2014)

Effect of the alcohol consumption on osteocyte cell processes : a molecular imaging study

Journal of cellular and molecular medicine (2014) 969 247-274 - doi : 10.1007/978-1-62703-260-5_16
We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

Goncalves C., Berchel M., Gosselin M.P., Malard V., Cheradame H., Jaffres P.A., Guegan P., Pichon C. and Midoux P.  (2014)

Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection

International journal of pharmaceutics (2014) 460 (1-2) 264-272 - doi : 10.1016/j.ijpharm.2013.11.005
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.

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.


2013   Références trouvées : 9

Pichon C. and Midoux P.  (2013)

Mannosylated and histidylated LPR technology for vaccination with tumor antigen mRNA

Methods in molecular biology (Clifton, N.J.) (2013) 969 247-274 - doi : 10.1007/978-1-62703-260-5_16
mRNA-based vaccines are currently being developed for treating various diseases including cancers. For this purpose, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen offers several advantages over plasmid DNA encoding tumor antigen including better delivery and security. In this chapter, we report the preparation of mannosylated mRNA nanoparticles termed mannosylated lipopolyplexes or Man-LPR loaded with mRNA encoding a melanoma antigen. This formulation enhances the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The mRNA is formulated with histidylated liposomes and a histidylated polymer. Those pH-sensitive vectors promote membrane destabilization in endosomes upon the protonation of their histidine groups, allowing nucleic acid delivery in the cytosol. To favor DCs targeting via the mannose receptor, a mannose lipid is incorporated in the liposomes. Here, we provide protocols for the preparation of mannosylated liposomes, the synthesis of mRNA, mice immunization based on systemic injection, measurement of the cellular immune response and determination of the number of transfected splenic DC.

mRNA-based vaccines are currently being developed for treating various diseases including cancers. For this purpose, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen offers several advantages over plasmid DNA encoding tumor antigen including better delivery and security. In this chapter, we report the preparation of mannosylated mRNA nanoparticles termed mannosylated lipopolyplexes or Man-LPR loaded with mRNA encoding a melanoma antigen. This formulation enhances the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The mRNA is formulated with histidylated liposomes and a histidylated polymer. Those pH-sensitive vectors promote membrane destabilization in endosomes upon the protonation of their histidine groups, allowing nucleic acid delivery in the cytosol. To favor DCs targeting via the mannose receptor, a mannose lipid is incorporated in the liposomes. Here, we provide protocols for the preparation of mannosylated liposomes, the synthesis of mRNA, mice immunization based on systemic injection, measurement of the cellular immune response and determination of the number of transfected splenic DC.

Dubois A.V., Midoux P., Gras D., Si-Tahar M., Brea D., Attucci S., Khelloufi M.K., Ramphal R., Diot P., Gauthier F. and Herve V.  (2013)

Poly-L-Lysine compacts DNA, kills bacteria, and improves protease inhibition in cystic fibrosis sputum

American journal of respiratory and critical care medicine (2013) 188 (6) 703-709 - doi : 10.1164/rccm.201305-0912OC
RATIONALE : Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES : We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS : We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS : Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS : Poly-L-lysine may be an alternative to dornase-alpha to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

RATIONALE : Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES : We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS : We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS : Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS : Poly-L-lysine may be an alternative to dornase-alpha to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

Delalande A., Kotopoulis S., Postema M., Midoux P. and Pichon C.  (2013)

Sonoporation : mechanistic insights and ongoing challenges for gene transfer

Gene (2013) 525 (2) 191-199 - doi : 10.1016/j.gene.2013.03.095
Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Berchel M., Le Corre S., Le Gall T., Couthon-Gourvès H., Haelters J.P., Midoux P., Montier T., Lehn P. and Jaffrès P.A.  (2013)

Trimethylarsonium-Based Cationic Phospholipids for Gene Delivery

Phosphorus, Sulfur, and Silicon and the Related Elements (2013) 188 (1-2) 91-94 - doi : 10.1080/10426507.2012.741161
Lipophosphoramide-based cationic lipids are a class of synthetic vectors used for gene delivery that can be produced in multigram scale. The use of trimethylarsonium moiety as a cationic polar head was beneficial to produce efficient gene delivery vectors for in vivo applications. Moreover, this type of cationic lipid can also exhibit some bactericidal effects.

Lipophosphoramide-based cationic lipids are a class of synthetic vectors used for gene delivery that can be produced in multigram scale. The use of trimethylarsonium moiety as a cationic polar head was beneficial to produce efficient gene delivery vectors for in vivo applications. Moreover, this type of cationic lipid can also exhibit some bactericidal effects.

Bire S., Gosset D., Jegot G., Midoux P., Pichon C. and Rouleux-Bonnin F.  (2013)

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition

BMC Biotechnology (2013) 13 (1) 75 - doi : 10.1186/1472-6750-13-75
Background

Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage : stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components : a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. 

Results

The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. 

Conclusion

Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Background
Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage : stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components : a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability.

Results
The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window.

Conclusion
Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Gomez J.P., Pichon C. and Midoux P.  (2013)

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers

Gene (2013) 525 (2) 182-190 - doi : 10.1016/j.gene.2013.03.055
DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

Sedletska, Y. Culard, F. Midoux, P.and Malinge, J. M.  (2013)

Interaction studies of muts and mutl with DNA containing the major cisplatin lesion and its mismatched counterpart under equilibrium and nonequilibrium conditions

Biopolymers 99 (9) 636-647
The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.

The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.

Ezzine, S., Vassaux, G., Pitard, B., Barteau, B., Malinge, J. M., Midoux, P., Pichon, C. and Baril, P.  (2013)

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression

Nucleic Acids Res - 1-14 - doi : 10.1093/nar/gkt797
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Pigeon L., Gonçalves C., Gosset D., Pichon C., Midoux P.  (2013)

An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency

Small 2013 - doi : 10.1002/smll.201300217
Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.


2012   Références trouvées : 4

Berchel, M., Le Gall, T., Couthon-Gourvès, H., Haelters, J.P., Montier, T., Midoux, P., Lehn, P. and Jaffrès, PA.   (2012)

Lipophosphonate/lipophosphoramidates : A family of synthetic vectors efficient for gene delivery

Biochimie 94 (1) 33-41
Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).

Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).

Delalande, A., Postema, M., Mignet, N., Midoux, P., and Pichon, C.  (2012)

Ultrasound and microbubble-assisted gene delivery : recent advances and ongoing challenges

Ther Deliv. 3 (10) 1199-1215
Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Billiet L., Gomez J.P., Berchel M., Jaffrès P.A., Le Gall T., Montier T., Bertrand E., Cheradame H., Guégan P., Mével M., Pitard B., Benvegnu T., Lehn P., Pichon C. and Midoux P.   (2012)

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line

Biomaterials 33 (10) 2980-2990
Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy.We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy.We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

Perche, F. Lambert, O. Berchel, M. Jaffrès, P.A. Pichon, C. Midoux, P.  (2012)

Gene transfer by histidylated lipopolyplexes : A dehydration method allowing preservation of their physicochemical parameters and transfection efficiency

International Journal of Pharmaceutics 423 (1) 144-150
Lipid-Polycation-DNA complexes (LPD) is a promising non-viral system for nucleic acids delivery. Usually, LPD are prepared just before their use. In the present work, we have examined whether dehydration of a new type of LPD (named LPD100) might be a storage option. LPD100 comprises PEGylated histidylated polylysine/pDNA polyplexes and a liposomal formulation made with lipophosphoramidates containing N-methylimidazolium and histamine polar heads. LPD100 were dehydrated by evaporation, and the physicochemical parameters and transfection efficiency (TE) of reconstituted LPD100 were compared to that of fresh LPD100. LPD100 previously dehydrated in the presence of 20% saccharose, displayed comparable size and surface charge as freshly prepared LPD100 but gave a better TE. CryoTEM experiments showed that the reconstituted LPD100 exhibited a shape similar to fresh ones. Moreover, when LPD100 were prepared with dehydrated pDNA/polymer complexes and fresh liposomes, TE was as efficient as with fresh LPD100 while a small increase of their size were observed. These results demonstrate that evaporation of LPD100 in the presence of saccharose is a powerful method to store them for a long period of time.

Lipid-Polycation-DNA complexes (LPD) is a promising non-viral system for nucleic acids delivery. Usually, LPD are prepared just before their use. In the present work, we have examined whether dehydration of a new type of LPD (named LPD100) might be a storage option. LPD100 comprises PEGylated histidylated polylysine/pDNA polyplexes and a liposomal formulation made with lipophosphoramidates containing N-methylimidazolium and histamine polar heads. LPD100 were dehydrated by evaporation, and the physicochemical parameters and transfection efficiency (TE) of reconstituted LPD100 were compared to that of fresh LPD100. LPD100 previously dehydrated in the presence of 20% saccharose, displayed comparable size and surface charge as freshly prepared LPD100 but gave a better TE. CryoTEM experiments showed that the reconstituted LPD100 exhibited a shape similar to fresh ones. Moreover, when LPD100 were prepared with dehydrated pDNA/polymer complexes and fresh liposomes, TE was as efficient as with fresh LPD100 while a small increase of their size were observed. These results demonstrate that evaporation of LPD100 in the presence of saccharose is a powerful method to store them for a long period of time.


2011   Références trouvées : 7

Delalande, A. ; Bouakaz, A. ; Renault, G. ; Tabareau, F. ; Kotopoulis, S. ; Midoux, P. ; Arbeille, B. ; Uzbekov, R. ; S., C. ; Postema, M. and Pichon, C.  (2011)

Ultrasound and microbubble-assisted gene delivery in Achilles tendons : long lasting gene expression and restoration of fibromodulin KO phenotype

Journal of Controlled Release 2011, 156, (2), 223-230
The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Bertrand, E. ; Goncalves, C. ; Billiet, L. ; Gomez, J. P. ; Pichon, C. ; Cheradame, H. ; Midoux, P. and Guégan, P  (2011)

Histidinylated linear PEI : a new efficient non-toxic polymer for gene transfer

Chem. Commun. 47 (46) 12547-12549
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.

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.

Berchel, M. ; Haelters, J. P. ; Couthon-Gourves, H. ; Deschamps, L. ; Midoux, P. ; Lehn, P. ; Jaffres, P. A.  (2011)

Modular Construction of Fluorescent Lipophosphoramidates by Click Chemistry

European Journal of Organic Chemistry 2011 (31) 6294-6303
The synthesis of fluorescent lipophosphoramidates is reported. Their modular construction allows several variations of the molecular structure including the separation of the fluorescent probe from the lipophosphoramidate moiety by a short methylene or by a tetraethylene glycol spacer. The last step of the convergent synthesis is a Huisgen click reaction, which assembles the fluorescent probe with the lipophosphoramidate part. Five fluorescent probes have been considered in this study including coumarin, NBD, fluorescein, naphthalimide and cyanine.

The synthesis of fluorescent lipophosphoramidates is reported. Their modular construction allows several variations of the molecular structure including the separation of the fluorescent probe from the lipophosphoramidate moiety by a short methylene or by a tetraethylene glycol spacer. The last step of the convergent synthesis is a Huisgen click reaction, which assembles the fluorescent probe with the lipophosphoramidate part. Five fluorescent probes have been considered in this study including coumarin, NBD, fluorescein, naphthalimide and cyanine.

Bennevault-Celton, V. ; Urbach, A. ; Martin, O. ; Pichon C. ; Guégan, P. and Midoux, P.  (2011)

Supramolecular Assemblies of Histidinylated α-Cyclodextrin in the Presence of DNA Scaffold during CDplexes Formation

Bioconjugate Chemistry 22 (12) 2404-2414
α-Cyclodextrin was transformed in a cationic unit after per substitution with histidine (His-α-CD) and lysine (Lys-α-CD) molecules on the primary face. His-α-CD and Lys-α-CD were used to form electrostatic complexes (CDplexes) with a plasmid DNA encoding luciferase gene, and the ability of CDplexes to transfect mammalian cells was examined using HEK293-T7 cells. The luciferase activity in cells transfected with His-α-CDplexes was 8-fold higher than that obtained Lys-α-CDplexes. When the transfection was carried out in the presence of chloroquine, the luciferase activity with His-α-CDplexes and Lys-α-CDplexes increased 6 and 25 times, respectively. The lower enhancement with His-α-CDplexes confirmed that histidine induced a proton sponge effect inside endosomes upon imidazole protonation, favoring DNA delivery in the cytosol. At the same time, we found that the condensation of DNA with His-α-CD was unexpectedly stronger than that obtained with the lysyl-α-CD counterpart. Moreover, it was as strong as that observed with high molecular weight polylysine. NMR (ROESY and DOSY) investigations in the absence of DNA showed that an inclusion complex is formed between the imidazole ring of histidine and the hydrophobic cavity of CD but no His-α-CD polymers can be formed by intermolecular interactions. These results suggest that intermolecular interactions between imidazole and His-α-CD cavity could be involved to form supramolecular assemblies in the presence of a DNA scaffold leading to DNA condensation into low diameter particles.

α-Cyclodextrin was transformed in a cationic unit after per substitution with histidine (His-α-CD) and lysine (Lys-α-CD) molecules on the primary face. His-α-CD and Lys-α-CD were used to form electrostatic complexes (CDplexes) with a plasmid DNA encoding luciferase gene, and the ability of CDplexes to transfect mammalian cells was examined using HEK293-T7 cells. The luciferase activity in cells transfected with His-α-CDplexes was 8-fold higher than that obtained Lys-α-CDplexes. When the transfection was carried out in the presence of chloroquine, the luciferase activity with His-α-CDplexes and Lys-α-CDplexes increased 6 and 25 times, respectively. The lower enhancement with His-α-CDplexes confirmed that histidine induced a proton sponge effect inside endosomes upon imidazole protonation, favoring DNA delivery in the cytosol. At the same time, we found that the condensation of DNA with His-α-CD was unexpectedly stronger than that obtained with the lysyl-α-CD counterpart. Moreover, it was as strong as that observed with high molecular weight polylysine. NMR (ROESY and DOSY) investigations in the absence of DNA showed that an inclusion complex is formed between the imidazole ring of histidine and the hydrophobic cavity of CD but no His-α-CD polymers can be formed by intermolecular interactions. These results suggest that intermolecular interactions between imidazole and His-α-CD cavity could be involved to form supramolecular assemblies in the presence of a DNA scaffold leading to DNA condensation into low diameter particles.

Perche, F., Benvegnu, T., Berchel, M., Lebègue, L., Pichon, C., Jaffrès, P.-A. & Midoux, P.,  (2011)

Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA

Nanomedicine : Nanotechnology, Biology and Medicine 7 (4) 445-453

Jaffres, P. A., Fraix, A., Lorilleux, C., Berchel, M., Couthon-Gourves, H., Haelters, J. P., Yaouanc, J. J., Burel, L., Giamarchi, P., Midoux, P., Montier, T. & Lehn, P.  (2011)

New lipo-phospharamidates for gene delivery

Phosphorus Sulfur and Silicon and the Related Elements 186 (4) 918-920

Perche, F., Gosset, D., Mével, M., Miramon, M.-L., Yaouanc, J.J., Pichon, C., Benvegnu, T., Jaffrès, P.A. & Midoux, P.  (2011)

Selective delivery in dendritic cells with Mannosylated and Histidylated Lipopolyplexes.

J. Drug Target. 19 (5) 315-325


2010   Références trouvées : 6

Kaddur, K., Lebegue, L., Tranquart, F., Midoux, P., Pichon, C. and Bouakaz, A.  (2010)

Transient transmembrane release of green fluorescent proteins with sonoporation.

IEEE Trans Ultrason Ferroelectr. Freq. Control. 57, 1558-1567.

Pichon, C., Billiet, L. and Midoux, P.  (2010)

Chemical vectors for gene delivery : uptake and intracellular trafficking.

Curr. Opin. 21 (5) 640-645

Suwalski, A., Dabboue, H., Delalande, A., Bensamoun, S., Canon, F., Midoux, P., Saillant, G., Klatzmann, D., Salvetat, J. & Pichon, C.  (2010)

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles.

Biomaterials 31, 5237-5245.

Fisichella, M., Dabboue, H., Bhattacharyya, S., Lelong, G., Warmont, F., Midoux, P., Pichon, C., Guérin, M., Hevor, T. and Salvetat, J.-P.  (2010)

Uptake of Functionalized Mesoporous Silica Nanoparticles by Human Cancer Cells.

J. Nanosci. Nanotechnol. 10 (4) : 2314-2324

Delalande, A., Bureau, M.F., Midoux, P., Bouakaz, A. & Pichon, C.  (2010)

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy.

Ultrasonics 50, 269-272.

Breton, M., Leblond, J., Seguin, J., Midoux, P., Schermani, D., Herscovici, J., Pichon, C. & Mignet, N.  (2010)

Comparative gene transfer between cationic and thiourea lipoplexes.

J. Gene Med. 12, 45-54.


2009   Références trouvées : 4

Bure, C., Pichon, C. & Midoux, P.  (2009)

Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

(2009) J Mass Spectrom 44, 1163-1170.

Midoux, P., Pichon, C., Yaouanc, J.J. & Jaffres, P.A.   (2009)

Chemical vectors for gene delivery : a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers.

Br. J. Pharmacol. 157, 166-178.

Goncalves, C., Ardourel, M.Y., Decoville, M., Breuzard, G., Midoux, P., Hartmann, B. & Pichon, C.  (2009)

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

J. Gene Med. 11, 401-411.

Bure, C., Maget, R., Delmas, A.F., Pichon, C. & Midoux, P.  (2009)

Histidine-rich peptide : evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH.

J. Mass Spectrom. 44, 81-89.


2008   Références trouvées : 5

Pichon, C., Kaddur, K., Midoux, P., Tranquart, F. & Bouakaz, A.  (2008)

Recent advances in gene delivery with ultrasound and microbubbles.

J. Exp. Nanosci. 3, 17-40.

Midoux, P., Breuzard, G., Gomez, J.P. & Pichon, C.  (2008)

Polymer-Based Gene Delivery : A Current Review on the Uptake and Intracellular Trafficking of Polyplexes.

Curr. Gene Ther. 8, 335-352.

Mevel, M., Neveu, C., Goncalves, C., Yaouanc, J.J., Pichon, C., Jaffres, P.A. & Midoux, P.  (2008)

Novel neutral imidazole-lipophosphoramides for transfection assays.

Chem. Commun. 27, 3124-3126 (hot article le 08 juillet 2008).

Mevel, M., Breuzard, G., Yaouanc, J.J., Clement, J.C., Lehn, P., Pichon, C., Jaffres, P.A. & Midoux, P.  (2008)

Synthesis and transfection activity of new cationic phosphoramidate lipids : High efficiency of an imidazolium derivative.

ChemBioChem 9, 1462-1471.

Breuzard, G., Tertil, M., Goncalves, C., Cheradame, H., Geguan, P., Pichon, C. & Midoux, P.  (2008)

Nuclear delivery of N kappa B-assisted DNA/polymer complexes : plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging.

Nucleic Acids Res. 36, 12, e71.


2007   Références trouvées : 2

Mockey, M ; Bourseau, E ; Chandrashekhar, V ; Chaudhuri, A ; Lafosse, S ; Le Cam, E ; Quesniaux, VFJ ; Ryffel, B ; Pichon, C ; Midoux, P  (2007)

mRNA-based cancer vaccine : prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes

Cancer Gene Therapy 14 (9) 802-814
Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route.

Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route.

Breuzard, G ; Tertil, M ; Pichon, C ; Midoux, P  (2007)

Intracellular trafficking and nuclear import of polyplexes by FRET coupled with cellular imaging

Human Gene Therapy 18 1048-1048


2006   Références trouvées : 2

Mockey, M ; Goncalves, C ; Dupuy, FP ; Lemoine, FM ; Pichon, C ; Midoux, P  (2006)

mRNA transfection of dendritic cells : Synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level

Biochemical and Biophysical Research Communications 340 (4) 1062-1068
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).

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).

Mennesson, E ; Erbacher, P ; Kuzak, M ; Kieda, C ; Midoux, P ; Pichon, C  (2006)

DNA/cationic polymer complex attachment on a human vascular endothelial cell monolayer exposed to a steady laminar flow

Journal of Controlled Release 114 (3) 389-397
This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.

This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.


2005   Références trouvées : 2

Mennesson, E ; Erbacher, P ; Piller, W ; Kieda, C ; Midoux, P ; Pichon C  (2005)

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells : a comparative study in non-polarized and polarized cells

Journal of Gene Medicine 7 (6) 729-738
Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.

Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.

Mennesson, E ; Erbacher, P ; Piller, V ; Kieda, C ; Midoux, P ; Pichon, C  (2005)

Transfection efficiency and uptake process of polyplexes in human lung endothetial cells : A comparative study in non-polarized and polarized cells

Journal of Gene Medicine 7 (6) 729-738
Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.

Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.


2004   Références trouvées : 6

Kamal, A., Kumar, B.A., Arifuddin, M. and Midoux, P.   (2004)

An efficient and facile nitration of phenols with nitric acid/zinc chloride under ultrasonic conditions

Ultrasonics Sonochemistry 11, 455-457.
An efficient and facile nitration of phenols using nitric acid/zinc chloride under ultrasonic condition has exhibited significant chemo as well as regioselectivity. This study has been extended to other aromatic compounds like naphthalene and anthracene.

An efficient and facile nitration of phenols using nitric acid/zinc chloride under ultrasonic condition has exhibited significant chemo as well as regioselectivity. This study has been extended to other aromatic compounds like naphthalene and anthracene.

Elmadbouh, I ; Rossignol, P ; Meilhac, O ; Vranckx, R ; Pichon, C ; Pouzet, B ; Midoux, P ; Michel, JB  (2004)

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications

Journal of Gene Medicine 6 (10) 1112-1124
Background : Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.

Background : Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.

Singh, RS ; Goncalves, C ; Sandrin, P ; Pichon, C ; Midoux, P ; Chaudhuri, A  (2004)

On the gene delivery efficacies of pH-sensitive cationic lipids via endosomal protonation : a chemical biology investigation

Chemistry & Biology 11 (6) 883-883

Montier, T ; Delepine, P ; Pichon, C ; Ferec, C ; Porteous, DJ ; Midoux, P  (2004)

Non-viral vectors in cystic fibrosis gene therapy : progress and challenges

Trends in Biotechnology 22 (11) 586-592
Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Kumar, VV ; Singh, RS ; Goncalves, C ; Sandrin, P ; Pichon, C ; Midoux, P ; Chaudhuri, A  (2004)

New histidylated cationic lipids for DNA- and mRNA-based lipofection

Molecular Therapy 9 S258-S259 Art No. 682 Suppl. 1

Goncalves, C ; Mennesson, E ; Fuchs, R ; Gorvel, JP ; Midoux, P ; Pichon, C  (2004)

Macropinocytosis of polyplexes and recycling of plasmid from clathrin-dependent pathway impair the transfection efficiency into human hepatocarcinoma cells

Molecular Therapy 9 S317-S317 Art No. 835 Suppl. 1


2003   Références trouvées : 3

Kumar, VV ; Pichon, C ; Refregiers, M ; Guerin, B ; Midoux, P ; Chaudhuri, A  (2003)

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids : evidence for histidine-mediated membrane fusion at acidic pH

Gene Therapy 10 (15) 1206-1215
Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles.

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles.

Midoux, P ; LeCam, E ; Coulaud, D ; Delain, E ; Pichon, C - Editor(s) : Luo, D ; Saltzman, WM ; Luo, D ; Saltzman, WM  (2003)

Histidine containing peptides and polypeptides as nucleic acid vectors

Synthetic Dna Delivery Systems 7 23-44

Madbouh, IEL ; Rossignol, P ; Midoux, P ; Michel, JB  (2003)

Optimization of primary cultured vascular cell transfection with non-viral vectors

Journal of Hypertension 21 (8) A6-A6


2002   Références trouvées : 3

Pichon, C ; Guerin, B ; Refregiers, M ; Goncalves, C ; Vigny, P ; Midoux, P  (2002)

Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Journal of Gene Medicine 4 (5) 548-559
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 : 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.

Pichon, C ; LeCam, E ; Guerin, B ; Coulaud, D ; Delain, E ; Midoux, P  (2002)

Poly[Lys-(AEDTP)] : A cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection

Bioconjugate Chemistry 13 (1) 76-82
Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly [Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio-)propionyl residues in order to have each amino group of poly [Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond.

Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly [Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio-)propionyl residues in order to have each amino group of poly [Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond.

Goncalves, C ; Pichon, C ; Guerin, B ; Midoux, P  (2002)

Intracellular processing and stability of DNA complexed with histidylated polylysine conjugates

Journal of Gene Medicine 4 (3) 271-281
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.

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.


2001   Références trouvées : 3

Pichon, C ; Goncalves, C ; Midoux, P  (2001)

Histidine-rich peptides and polymers for nucleic acids delivery

Advanced Drug Delivery Reviews 53 (1) 75-94
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.

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.

Ohan, J ; Gilbert, MA ; Leseche, G ; Panis, Y ; Midoux, P ; Drouet, L  (2001)

Nonviral gene transfer into primary cultures of human and porcine mesothelial cells

In Vitro Cellular & Developmental Biology-Animal 37 (7) 402-407
Due to their abundance and accessibility, mesothelial cells may be suitable toots for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter.

Due to their abundance and accessibility, mesothelial cells may be suitable toots for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter.

Bello Roufai, M. and Midoux, P.  (2001)

Histidylated polylysine as DNA vector : Elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes

Bioconjugate Chemistry 12 (1) 92-99
We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the a -amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free a -amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV.

We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the a -amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free a -amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV.


2000   Références trouvées : 4

Fajac, I ; Allo, JC ; Souil, E ; Merten, M ; Pichon, C ; Figarella, C ; Monsigny, M ; Briand, P ; Midoux, P  (2000)

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells

Journal of Gene Medicine 2 (5) 368-378
Background : We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.

Background : We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.

Pichon, C ; Roufai, MB ; Monsigny, M ; Midoux, P  (2000)

Histidylated oligolysines increase the transmembrane passage and the biological activity of antisense oligonucleotides

Nucleic Acids Research 28 (2) 504-512
We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN), Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cytosolic delivery of ODN from endosomes and increase their nuclear accumulation.

We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN), Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cytosolic delivery of ODN from endosomes and increase their nuclear accumulation.

Freulon, I ; Monsigny, M ; Midoux, P ; Mayer, R  (2000)

Spacer length dependence on the efficiency of dimeric anionic peptides in gene transfer by glycosylated polylysine/plasmid complexes

Bioscience Reports 20 (5) 383-398
Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol, It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-ß Ala-ß Ala residues. but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the calls to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol, It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-ß Ala-ß Ala residues. but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the calls to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Allo, JC ; Midoux, P ; Merten, M ; Souil, E ; Lipecka, J ; Figarella, C ; Monsigny, M ; Briand, P ; Fajac, I  (2000)

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors

American Journal of Respiratory Cell and Molecular Biology 22 (2) 166-175
Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of re porter gene transfer into immortalized normal (MM-39) and CF (CF-KM I) human airway epithelial gland serous cells using various synthetic vectors : glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using a-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF trans membrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using a-glycosylated poly lysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of re porter gene transfer into immortalized normal (MM-39) and CF (CF-KM I) human airway epithelial gland serous cells using various synthetic vectors : glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using a-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF trans membrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using a-glycosylated poly lysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane.


1999   Références trouvées : 6

Monsigny, M ; Midoux, P ; Mayer, R ; Roche, AC  (1999)

Glycotargeting : Influence of the sugar moiety on both the uptake and the intracellular trafficking of nucleic acid carried by glycosylated polymers

Bioscience Reports 19 (2) 125-132
Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.

Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.

Fajac, I ; Briand, P ; Monsigny, M ; Midoux, P  (1999)

Sugar-mediated uptake of glycosylated polylysines and gene transfer into normal and cystic fibrosis airway epithelial cells

Human Gene Therapy 10 (3) 395-406
We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins ; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes) ; and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors, The most efficient up-take of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing a-D-Man residues.

We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins ; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes) ; and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors, The most efficient up-take of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing a-D-Man residues.

Allo, JC ; Midoux, P ; Merten, M ; Monsigny, M ; Briand, P ; Figarella, C ; Fajac, I  (1999)

Histidylated polylysine/DNA complexes as efficient vectors for gene transfer into cystic fibrosis airway glandular serous cells.

American Journal of Respiratory and Critical Care Medicine 159 (3) A679-A679 Suppl. S

Kichler, A ; Freulon, I ; Boutin, V ; Mayer, R ; Monsigny, M ; Midoux, P  (1999)

Glycofection (TM) in the presence of anionic fusogenic peptides : A study of the parameters affecting the peptide-mediated enhancement of the transfection efficiency

Journal of Gene Medicine 1 (2) 134-143
Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line.

Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line.

Boutin, V ; Legrand, A ; Mayer, R ; Nachtigal, M ; Monsigny, M ; Midoux, P  (1999)

Glycofection : The ubiquitous roles of sugar bound on glycoplexes

Drug Delivery 6 (1) 45-50
Glycofection (transfection by using sugar-substituted polylysine) was assessed in order to provide an alternative to viral vectors for the transfer of genes into vascular smooth muscle cells, A rabbit vascular smooth muscle cell line (Rb-l cells) was selectively transfected by using glycoplexes (glycosylated polylysine/pSV2LUC complexes) in the presence of 10 mu M of the fusogenic peptide GALA, A sugar-specific transfection was obtained when the glycofection was conducted for 1 h with glycoplexes containing either a-Gal, a-Glc, a-GalNAc, ß-GlcNAc, or ß-GalNAc residues.

Glycofection (transfection by using sugar-substituted polylysine) was assessed in order to provide an alternative to viral vectors for the transfer of genes into vascular smooth muscle cells, A rabbit vascular smooth muscle cell line (Rb-l cells) was selectively transfected by using glycoplexes (glycosylated polylysine/pSV2LUC complexes) in the presence of 10 mu M of the fusogenic peptide GALA, A sugar-specific transfection was obtained when the glycofection was conducted for 1 h with glycoplexes containing either a-Gal, a-Glc, a-GalNAc, ß-GlcNAc, or ß-GalNAc residues.

Midoux, P ; Monsigny, M  (1999)

Efficient gene transfer by histidylated polylysine pDNA complexes

Bioconjugate Chemistry 10 (3) 406-411
Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides.


1998   Références trouvées : 4

Quetard, C ; Bourgerie, S ; Normand-Sdiqui, N ; Mayer, R ; Strecker, G ; Midoux, P ; Roche, AC ; Monsigny, M  (1998)

Novel glycosynthons for glycoconjugate preparation : Oligosaccharylpyroglutamylanilide derivatives

Bioconjugate Chemistry 9 (2) 268-276
The reducing sugar of an oligosaccharide reacting with the a-amino group of an amino acid is converted to an N-oligosaccharylamino acid which can then be stabilized by N-acylation. Oligosaccharides in solution in N,N-dimethylformamide reacted with a-glutamyl-p-nitroanilide at 50 degrees C for a few hours, leading to an N-oligosaccharylglutamyl-p-nitroanilide. Then, the gamma-carboxylic group of the glutamyl moiety, activated by adding (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), reacted with the substituted a-amino group of the glutamyl residue, leading to an N-oligosaccharylpyroglutamyl-p-nitroanilide within 0.5 h.

The reducing sugar of an oligosaccharide reacting with the a-amino group of an amino acid is converted to an N-oligosaccharylamino acid which can then be stabilized by N-acylation. Oligosaccharides in solution in N,N-dimethylformamide reacted with a-glutamyl-p-nitroanilide at 50 degrees C for a few hours, leading to an N-oligosaccharylglutamyl-p-nitroanilide. Then, the gamma-carboxylic group of the glutamyl moiety, activated by adding (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), reacted with the substituted a-amino group of the glutamyl residue, leading to an N-oligosaccharylpyroglutamyl-p-nitroanilide within 0.5 h.

Monsigny, M ; Quetard, C ; Bourgerie, S ; Delay, D ; Pichon, C ; Midoux, P ; Mayer, R ; Roche, AC  (1998)

Glycotargeting : The preparation of glyco-amino acids and derivatives from unprotected reducing sugars

Biochimie 80 (2) 99-108
Lectins are present on the surface of many cells. Many lectins actively recycle from membrane to endosomes and efficiently take up glycoconjugates in a sugar-dependent manner. On this basis, glycoconjugates, specially those obtained by chemical means, are good candidates as carriers of drugs, oligonucleotides or genes. In this paper, we present a panel of methods suitable to transform unprotected reducing oligosaccharides into glycosynthons designed to be easily linked to therapeutic agents. All the glycosynthons presented here are glycosylamines or derivatives, mainly glyco-amino acids or glycopeptides. Glycosylamines are easy to obtain, but they are very labile in slightly acidic or neutral medium ; they must be stabilized, by acylation for instance. The coupling efficiency of a reducing sugar with ammonia as well as an alkylamine or an arylamine is higher at high temperature, however, because of the Amadori rearrangement, special conditions have to be selected to prepare the expected glycosylamine derivative with a high yield.

Lectins are present on the surface of many cells. Many lectins actively recycle from membrane to endosomes and efficiently take up glycoconjugates in a sugar-dependent manner. On this basis, glycoconjugates, specially those obtained by chemical means, are good candidates as carriers of drugs, oligonucleotides or genes. In this paper, we present a panel of methods suitable to transform unprotected reducing oligosaccharides into glycosynthons designed to be easily linked to therapeutic agents. All the glycosynthons presented here are glycosylamines or derivatives, mainly glyco-amino acids or glycopeptides. Glycosylamines are easy to obtain, but they are very labile in slightly acidic or neutral medium ; they must be stabilized, by acylation for instance. The coupling efficiency of a reducing sugar with ammonia as well as an alkylamine or an arylamine is higher at high temperature, however, because of the Amadori rearrangement, special conditions have to be selected to prepare the expected glycosylamine derivative with a high yield.

Midoux, P ; Kichler, A ; Boutin, V ; Maurizot, JC ; Monsigny, M  (1998)

Membrane permeabilization and efficient gene transfer by a peptide containing several histidines

Bioconjugate Chemistry 9 (2) 260-267
We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH ; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum.

We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH ; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum.

Bartholeyns, J ; Romet-Lemonne, JL ; Chokri, M ; Buyse, M ; Velu, T ; Bruyns, C ; Van de Winkel, JJ ; Heeney, J ; Koopman, G ; Malmsten, M ; De Groote, D ; Monsigny, M ; Midoux, P ; Alarcon, B  (1998)

Cellular vaccines

Research in Immunology 149 (7-8) 647-649
This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient's own tumour. (1) Autologous cellular vaccines are prepared by processing circulating brood mononuclear cells outside of the patient's body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation.

This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient’s own tumour. (1) Autologous cellular vaccines are prepared by processing circulating brood mononuclear cells outside of the patient’s body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation.


1997   Références trouvées : 6

Stewart, AJ ; Pichon, C ; Midoux, P ; Mayer, R ; Monsigny, M ; Roche, AC ; Aubertin, AM  (1997)

Fluorescent labelling of unmodified phosphorothioate oligodeoxynucleotides : Synthesis and characterization

New Journal of Chemistry 21 (1) 87-98
In this paper we describe the preparation and characterization of phosphorothioate oligodeoxynucleotides (pt-odn) substituted with a fluorescein molecule linked to the non-bridging sulfur of the internucleotidic linkage. This technique is original in that the starting material is a perphosphorothioate oligonucleotide. The phosphorothioate oligonucleotides, after reaction at pH 8.0 and 50 degrees C with iodoacetamido-fluorescein, yielded a fluorescent derivative, termed F-pt-odn. The two F-pt-odn used in this report contained 1.6 and 3.4 fluorescein per oligonucleotide and were found to be respectively 1.5 and 2.2 times more fluorescent than an alkylamidothiocarbamyl-fluoresceinyl-pt-odn (F-NH-pt-odn) containing a single reporter group.

In this paper we describe the preparation and characterization of phosphorothioate oligodeoxynucleotides (pt-odn) substituted with a fluorescein molecule linked to the non-bridging sulfur of the internucleotidic linkage. This technique is original in that the starting material is a perphosphorothioate oligonucleotide. The phosphorothioate oligonucleotides, after reaction at pH 8.0 and 50 degrees C with iodoacetamido-fluorescein, yielded a fluorescent derivative, termed F-pt-odn. The two F-pt-odn used in this report contained 1.6 and 3.4 fluorescein per oligonucleotide and were found to be respectively 1.5 and 2.2 times more fluorescent than an alkylamidothiocarbamyl-fluoresceinyl-pt-odn (F-NH-pt-odn) containing a single reporter group.

Pichon, C ; Freulon, I ; Midoux, P ; Mayer, R ; Monsigny, M ; Roche, AC  (1997)

Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide

Antisense & Nucleic Acid Drug Development 7 (4) 335-343
Antisense oligonucleotides (ODN) were easily introduced into the cytosol of mammalian cells on permeabilization of the plasma membrane by an amphiphilic anionic peptide. The E5CA peptide (GLFEAIAEFIEGG-WEGLIEGCA) is an E5 peptide analog derived from the N-terminal segment of the HA2 subunit of influenza virus hemagglutinin.

Antisense oligonucleotides (ODN) were easily introduced into the cytosol of mammalian cells on permeabilization of the plasma membrane by an amphiphilic anionic peptide. The E5CA peptide (GLFEAIAEFIEGG-WEGLIEGCA) is an E5 peptide analog derived from the N-terminal segment of the HA2 subunit of influenza virus hemagglutinin.

Erbacher, P ; Roche, AC ; Monsigny, M ; Midoux, P  (1997)

The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes

Biochimica et Biophysica Acta-Biomembranes 1324 (1) 27-36
A polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of polylysine were blocked with gluconoyl residues.

A polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of polylysine were blocked with gluconoyl residues.

Erbacher, P ; Roche, AC ; Monsigny, M ; Midoux, P  (1997)

Specific gene transfer based on biotinylated and gluconoylated polylysine/plasmid complexes

Drug Delivery 4 (3) 173-179
Complexes formed between plasmids and polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated polylysine/DNA complexes were made between a plasmid and biotinylated polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin.

Complexes formed between plasmids and polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated polylysine/DNA complexes were made between a plasmid and biotinylated polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin.

Kollen, W ; Erbacher, P ; Midoux, P ; Roche, AC ; Monsigny, M ; Glick, MC ; Scanlin, TF  (1997)

Glycosylated polylysines - Nonviral vectors for gene transfer into cystic fibrosis airway epithelial cells

Chest 111 (6) S95-S96 Suppl. S

Stewart, AJ ; Pichon, C ; Meunier, L ; Midoux, P ; Monsigny, M ; Roche, AC  (1997)

Enhanced biological activity of antisense oligonucleotides complexed with glycosylated poly-L-lysine

Molecular Pharmacology 50 (6) 1487-1494
We sought to exploit glycosylated poly-L-lysine (pLK) to increase the uptake and biological antisense activity of a phosphorothioate oligonucleotide (pt-odn) [pt-odn complementary to the 3' noncoding region of intercellular adhesion molecule-1 ICAM-1)(odn(ICAM-1))] complementary to the 3'-noncoding region of ICAM-1 in A549 cells. Dose-dependent inhibition of ICAM-1 expression was obtained (IC50 = 500 nM) through treatment of cells with odn(ICAM-1) complexed with pLK carrying fucose residues in the presence of 100 mu M chloroquine. Alteration in the charge ratio between fucosylated pLK and pt-odn had a significant effect on the efficacy of inhibition (optimal conditions, charge ratio = 1.1).

We sought to exploit glycosylated poly-L-lysine (pLK) to increase the uptake and biological antisense activity of a phosphorothioate oligonucleotide (pt-odn) [pt-odn complementary to the 3’ noncoding region of intercellular adhesion molecule-1 ICAM-1)(odn(ICAM-1))] complementary to the 3’-noncoding region of ICAM-1 in A549 cells. Dose-dependent inhibition of ICAM-1 expression was obtained (IC50 = 500 nM) through treatment of cells with odn(ICAM-1) complexed with pLK carrying fucose residues in the presence of 100 mu M chloroquine. Alteration in the charge ratio between fucosylated pLK and pt-odn had a significant effect on the efficacy of inhibition (optimal conditions, charge ratio = 1.1).


1996   Références trouvées : 5

Erbacher, P ; Roche, AC ; Monsigny, M ; Midoux, P  (1996)

Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA lactosylated polylysine complexes

Experimental Cell Research 225 (1) 186-194
Chloroquine improves drastically the transfection of cells upon exposure to plasmid DNA/glycosylated polylysine complexes. So far the mechanism of action of chloroquine is not well understood. In this paper, the effect of chloroquine was investigated by measuring the transfection efficiency of a human hepatocarcinoma (HepG2 cells) by pSV2LUC/lactosylated polylysine complexes involving their internalization via the galactose-specific membrane lectin of these cells. The luciferase activity in the transfected cells was maximal when the transfection was performed for 3 or 4 h in the presence of 100 mu M chloroquine. The luciferase activity was also enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does. Chloroquine enters cells and accumulates in vesicular compartments ; the overall intracellular concentration increases to 9 mM, which means that in the vesicular compartment, the chloroquine concentration is still higher. At such high concentrations, chloroquine induces the dissociation of plasmid DNA/lactosylated polylysine complexes, as shown in acellular experiments. (C) 1996 Academic Press, Inc.

Chloroquine improves drastically the transfection of cells upon exposure to plasmid DNA/glycosylated polylysine complexes. So far the mechanism of action of chloroquine is not well understood. In this paper, the effect of chloroquine was investigated by measuring the transfection efficiency of a human hepatocarcinoma (HepG2 cells) by pSV2LUC/lactosylated polylysine complexes involving their internalization via the galactose-specific membrane lectin of these cells. The luciferase activity in the transfected cells was maximal when the transfection was performed for 3 or 4 h in the presence of 100 mu M chloroquine. The luciferase activity was also enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does. Chloroquine enters cells and accumulates in vesicular compartments ; the overall intracellular concentration increases to 9 mM, which means that in the vesicular compartment, the chloroquine concentration is still higher. At such high concentrations, chloroquine induces the dissociation of plasmid DNA/lactosylated polylysine complexes, as shown in acellular experiments. (C) 1996 Academic Press, Inc.

Kollen, WJW ; Midoux, P ; Erbacher, P ; Yip, A ; Roche, AC ; Monsigny, M ; Glick, MC ; Scanlin, TF  (1996)

Gluconoylated and glycosylated polylysines as vectors for gene transfer into cystic fibrosis airway epithelial cells

Human Gene Therapy 7 (13) 1577-1586
To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed, Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups, In addition, polylysine was substituted with sugar residues on a specified number of amino groups, Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 mu M chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection.

To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed, Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups, In addition, polylysine was substituted with sugar residues on a specified number of amino groups, Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 mu M chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection.

Erbacher, P ; Bousser, MT ; Raimond, J ; Monsigny, M ; Midoux, P ; Roche, AC  (1996)

Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages

Human Gene Therapy 7 (6) 721-729
Macrophages are putative target cells for expressing an exogenous gene with therapeutical effects. Knowing that macrophages express membrane lectins mediating endocytosis of their ligands, DNA/glycosylated polylysine complexes were used to transfect human blood monocyte-derived macrophages. Monocytes from human peripheral blood were matured in culture for 7 days to differentiate into macrophage-like cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adherent cells, which displayed characteristic macrophage markers, CD 14, CD 11b, HLA-DR, and HLA-ABC antigens and mannose receptor, were transfected by DNA/glycosylated polylysine complexes in the presence of chloroquine.

Macrophages are putative target cells for expressing an exogenous gene with therapeutical effects. Knowing that macrophages express membrane lectins mediating endocytosis of their ligands, DNA/glycosylated polylysine complexes were used to transfect human blood monocyte-derived macrophages. Monocytes from human peripheral blood were matured in culture for 7 days to differentiate into macrophage-like cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adherent cells, which displayed characteristic macrophage markers, CD 14, CD 11b, HLA-DR, and HLA-ABC antigens and mannose receptor, were transfected by DNA/glycosylated polylysine complexes in the presence of chloroquine.

Avrameas, A ; McIlroy, D ; Hosmalin, A ; Autran, B ; Debre, P ; Monsigny, M ; Roche, AC ; Midoux, P  (1996)

Expression of a mannose/fucose membrane lectin on human dendritic cells

European Journal of Immunology 26 (2) 394-400
Dendritic cells (DC) are the most efficient antigen presenting cells for T lymphocytes. CD1a CD14(-) DC with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al.. J. Exp. Med. 1994, 1795, 1109). Human macrophages express a membrane lectin. or sugar-specific receptor. which specifically mediates the binding and endocytosis of mannose- and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens.

Dendritic cells (DC) are the most efficient antigen presenting cells for T lymphocytes. CD1a CD14(-) DC with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al.. J. Exp. Med. 1994, 1795, 1109). Human macrophages express a membrane lectin. or sugar-specific receptor. which specifically mediates the binding and endocytosis of mannose- and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens.

Kollen, WJW ; Erbacher, P ; Midoux, P ; Roche, AC ; Monsigny, M ; Glick, MC ; Scanlin, TF  (1996)

Nonviral vectors for gene transfer into cystic fibrosis (CF) airway epithelial cells

Pediatric Research 39 (4) 2312-2312 Part 2


1995   Références trouvées : 3

Sdiqui, N., Arar, K., Midoux, P., Monsigny, M. & Ropche, A.C.  (1995)

Inhibition of human mammary cell line proliferation by membrane lectin-mediated uptake of Ha-ras antisense oligonucleotide

Drug Delivery 2, 63-72

Erbacher, P ; Roche, AC ; Monsigny, M ; Midoux, P  (1995)

Glycosylated polylysine DNA complexes - gene-transfer efficiency in relation with the size and the sugar substitution level of glycosylated polylysines and with the plasmid size

Bioconjugate Chemistry 6 (4) 401-410
A DNA delivery system based on the use of polylysine substituted with small recognition signals, such as carbohydrate moieties specifically recognized by membrane lectins present in a given cell line, has been developed [Midoux et al. (1993) Nucleic Acids Res. 21, 871-878]. Human hepatoma (HepG2) cells which express a galactose-specific membrane lectin are efficiently transfected in the presence of chloroquine with pSV2Luc plasmid complexed with a lactosylated polylysine.

A DNA delivery system based on the use of polylysine substituted with small recognition signals, such as carbohydrate moieties specifically recognized by membrane lectins present in a given cell line, has been developed [Midoux et al. (1993) Nucleic Acids Res. 21, 871-878]. Human hepatoma (HepG2) cells which express a galactose-specific membrane lectin are efficiently transfected in the presence of chloroquine with pSV2Luc plasmid complexed with a lactosylated polylysine.

Midoux, P ; Mayer, R ; Monsigny, M  (1995)

Membrane permeabilization by a-helical peptides - a flow-cytometry study

Biochimica et Biophysica Acta-Biomembranes 1239 (2) 249-256
The permeabilization by a-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by a-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (

The permeabilization by a-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by a-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (


1994   Références trouvées : 1

Monsigny, M ; Roche, AC ; Midoux, P ; Mayer, R  (1994)

Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes

Advanced Drug Delivery Reviews 14 (1) 1-24
Cell surface receptors are good candidates to selectively target drugs, oligonucleotides or even genes by making use of their specific ligands. A large number of mammalian cells express cell surface sugar-binding proteins, also called ''membrane lectins''. Therefore, sugars may be used as specific recognition signals to specifically deliver biological active components. Tens of membrane lectins with different sugar specificities have been characterized ; some of them actively carry their ligands to intracellular compartments, including endosomes, lysosomes and, in some cases, Golgi apparatus.

Cell surface receptors are good candidates to selectively target drugs, oligonucleotides or even genes by making use of their specific ligands. A large number of mammalian cells express cell surface sugar-binding proteins, also called ’’membrane lectins’’. Therefore, sugars may be used as specific recognition signals to specifically deliver biological active components. Tens of membrane lectins with different sugar specificities have been characterized ; some of them actively carry their ligands to intracellular compartments, including endosomes, lysosomes and, in some cases, Golgi apparatus.


1993   Références trouvées : 3

Midoux, P ; Mendes, C ; Legrand, A ; Raimond, J ; Mayer, R ; Monsigny, M ; Roche, AC  (1993)

Specific gene-transfer mediated by lactosylated poly-l-lysine into hepatoma-cells

Nucleic Acids Research 21 (4) 871-878
Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner : i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner : i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes.

Monsigny, M ; Midoux, P ; Roche, AC  (1993)

Targeted plasmid-polylysine complex as putative tools to selectively transfer genes ex vivo and in vivo

M S-Medecine Sciences 9 (4) 441-449
Gene therapy will be an ideal method when it is possible to transfer DNA in cells with a great efficacy and an absolute safety in vivo. Along with viral carriers which are efficient but may not be strictly safe, non viral carriers could be advantageously used. Polylysine, a polycation giving stable DNA complexes, is proposed as the basis of such a carrier system. Indeed, polylysine substituted with either a protein easily taken up by cells due to specific cell surface receptors, or carbohydrate moieties which selectively interact with lectins expressed at the surface of specific cells, allows a cell specific delivery of genes. In addition, when such a targeted carrier is used together with components that protect the plasmid-targeted polylysine complex from the hydrolytic activities of lysosomes and/or help the complex to cross the cell membranes to reach the cytosol, the specificity and the efficacy of the transfection are conspicuously higher.

Gene therapy will be an ideal method when it is possible to transfer DNA in cells with a great efficacy and an absolute safety in vivo. Along with viral carriers which are efficient but may not be strictly safe, non viral carriers could be advantageously used. Polylysine, a polycation giving stable DNA complexes, is proposed as the basis of such a carrier system. Indeed, polylysine substituted with either a protein easily taken up by cells due to specific cell surface receptors, or carbohydrate moieties which selectively interact with lectins expressed at the surface of specific cells, allows a cell specific delivery of genes. In addition, when such a targeted carrier is used together with components that protect the plasmid-targeted polylysine complex from the hydrolytic activities of lysosomes and/or help the complex to cross the cell membranes to reach the cytosol, the specificity and the efficacy of the transfection are conspicuously higher.

Carpentier, V ; Midoux, P ; Monsigny, M ; Roche, AC  (1993)

Endocytosis of a(1)-acid glycoprotein variants by human monocytic lineage cells

Biology of The Cell 77 (2) 187-193
Human a1-acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte-derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.

Human a1-acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte-derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.


1992   Références trouvées : 4

Bonfils, E ; Mendes, C ; Roche, AC ; Monsigny, M ; Midoux, P  (1992)

Uptake by macrophages of a biotinylated oligo- ?-deoxythymidylate by using mannosylated streptavidin

Bioconjugate Chemistry 3 (4) 277-284
Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(a-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis(a-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.

Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(a-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3’-biotinylated and 5’-fluoresceinylated dodecakis(a-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.

Bonfils, E ; Depierreux, C ; Midoux, P ; Thuong, NT ; Monsigny, M ; Roche, AC  (1992)

Drug targeting - synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates

Nucleic Acids Research 20 (17) 4621-4629
Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides-substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,-were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides.

Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides-substituted on their 5’-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3’-end, on the other hand,-were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides.

Midoux, P ; Martin, A ; Collet, B ; Monsigny, M ; Roche, AC ; Toujas, L  (1992)

Activation of mouse macrophages by muramyl dipeptide coupled with an antimacrophage monoclonal-antibody

Bioconjugate Chemistry 3 (2) 194-199
A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer ; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues was prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer ; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules.

A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer ; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues was prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer ; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules.

Bonfils, E ; Mendes, C ; Roche, AC ; Monsigny, M ; Midoux, P  (1992)

Uptake by macrophages of a biotinylated oligo-a-deoxythymidylate by using mannosylated streptavidin

Bioconjugate Chemistry 3 (4) 277-284
Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(a-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis(a-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin.

Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(a-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3’-biotinylated and 5’-fluoresceinylated dodecakis(a-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin.


1991   Références trouvées : 2

Breton, P ; Midoux, P ; Petit, C ; Bousser, MT ; Roche, AC ; Mayer, R ; Monsigny, M  (1991)

Production of macrophage-derived cytotoxic factor by n-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins

Bioconjugate Chemistry 2 (1) 16-18
6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-](carbamoylmethyl)thio]propionyl residues are potent macrophage activators.

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-](carbamoylmethyl)thio]propionyl residues are potent macrophage activators.

Pimpaneau, V ; Midoux, P ; Monsigny, M ; Roche, AC  (1991)

Characterization and isolation of an intracellular d-mannose-specific receptor from human promyelocytic-hl-60 cells

Carbohydrate Research 213 95-108
Most mammalian macrophages express D-mannose-specific receptor (membrane lectin, M(r) 175000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl a-D-Mannopyranoside-substituted Affi-gel as a 60 000-M(r) membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175 000-M(r) D-Mannose-specific receptor but not the 60 000-M(r) receptor.

Most mammalian macrophages express D-mannose-specific receptor (membrane lectin, M(r) 175000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl a-D-Mannopyranoside-substituted Affi-gel as a 60 000-M(r) membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175 000-M(r) D-Mannose-specific receptor but not the 60 000-M(r) receptor.


1990   Références trouvées : 3

Roche, AC ; Midoux, P ; Pimpaneau, V ; Negre, E ; Mayer, R ; Monsigny, M  (1990)

Endocytosis mediated by monocyte and macrophage membrane lectins - application to antiviral drug targeting

Research in Virology 141 (2) 243-249

Midoux, P ; Negre, E ; Roche, AC ; Mayer, R ; Monsigny, M ; Balzarini, J ; Declercq, E ; Mayer, E ; Ghaffar, A ; Gangemi, JD  (1990)

Drug targeting - anti-hsv-1 activity of mannosylated polymer-bound 9-(2-phosphonylmethoxyethyl)adenine

Biochemical and Biophysical Research Communications 167 (3) 1044-1049

Monsigny, M ; Midoux, P ; Depierreux, C ; Bebear, C ; Lebris, MT ; Valeur, B  (1990)

Benzoxazinone kanamycin-a conjugate - a new fluorescent-probe suitable to detect mycoplasmas in cell-culture

Biology of The Cell 70 (3) 101-105
The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[ß-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]-phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[ß-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]-phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.


1989   Références trouvées : 4

Derrien, D ; Midoux, P ; Petit, C ; Negre, E ; Mayer, R ; Monsigny, M ; Roche, AC  (1989)

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Glycoconjugate Journal 6 (2) 241-255

Midoux, P ; Petit, C ; Pellen, P ; Toujas, L ; Monsigny, M ; Roche, AC  (1989)

Macrophage antigens associated with adhesion - identification by a monoclonal-antibody specific for lewis lung-carcinoma cells

Experimental Cell Research 183 (1) 168-178

Monsigny, M ; Midoux, P ; Lebris, MT ; Roche, AC ; Valeur, B  (1989)

Benzoxazinone derivatives - new fluorescent-probes for 2-color flow-cytometry analysis using ole excitation wavelength

Biology of The Cell 67 (2) 193-200

Pimpaneau, V ; Midoux, P ; Durand, G ; Debaetselier, P ; Monsigny, M ; Roche, AC  (1989)

Endocytosis of a-1-acid glycoprotein variants and of neoglycoproteins containing mannose derivatives by a mouse hybridoma cell-line (2C11-12) - comparison with mouse peritoneal-macrophages

Glycoconjugate Journal 6 (4) 561-574


1988   Références trouvées : 2

Monsigny, M ; Roche, AC ; Kieda, C ; Midoux, P ; Obrenovitch, A  (1988)

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Biochimie 70 (11) 1633-1649

Monsigny, M ; Roche, AC ; Midoux, P  (1988)

Endogenous lectins and drug trageting

Annals of The New York Academy of Sciences 551 399-414


1987   Références trouvées : 2

Midoux, P ; Roche, AC ; Monsigny, M  (1987)

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow-cytometry

Cytometry 8 (3) 327-334

Gabius, HJ ; Engelhardt, R ; Hellmann, T ; Midoux, P ; Monsigny, M ; Nagel, GA ; Vehmeyer, K  (1987)

Characterization of membrane lectins in human-colon carcinoma-cells by flow cytofluorometry, drug targeting and affinity-chromatography

Anticancer Research 7 (1) 109-112


1986   Références trouvées : 3

Seve, A.P., Hubert, J., Bouvier, D., Bourgeois, C., Midoux, P., Roche, A.C. & Monsigny, M.  (1986)

Analysis of sugar-binding sites in mammalian cell nuclei by quantitative flow microfluorometry

Proc. Natl. Acad. Sci. USA 83, 5997-6001

Roche, AC ; Midoux, P ; Petit, C ; Mayer, R ; Monsigny, M ; Patchen, ML  (1986)

Immunostimulating drugs targeted by neoglycoproteins - effects on hematopoiesis and therapeutic properties

Bulletin Du Cancer 73 (4) 424-424

Midoux, P ; Roche, AC ; Monsigny, M  (1986)

Estimation of the degradation of endocytosed material by flow cytofluorometry using 2 neoglycoproteins containing different numbers of fluorescein molecules

Biology of The Cell 58 (3) 221-225


1985   Références trouvées : 3

Monsigny, M ; Midoux, P ; Roche, AC  (1985)

Use of flow-cytometry in the study of endocytosis

Biology of The Cell 55 (1-2) A23-A23

Roche, AC ; Midoux, P ; Bouchard, P ; Monsigny, M  (1985)

Membrane lectins on human-monocytes - maturation-dependent modulation of 6-phosphomannose and mannose receptors

Febs Letters 193 (1) 63-68

Roche, AC ; Mayer, R ; Midoux, P ; Monsigny, M  (1985)

Immunostimulant drugs targeted by monoclonal-antibodies and neoglycoproteins

Journal of Leukocyte Biology 38 (1) 138-138


1984   Références trouvées : 9

Monsigny, M ; Roche, AC ; Midoux, P  (1984)

Uptake of neoglycoproteins via membrane lectin(s) of l1210 cells evidenced by quantitative flow cytofluorometry and drug targeting

Biology of The Cell 51 (2) 187-196

Midoux, P ; Maillet, T ; Therain, F ; Monsigny, M ; Roche, AC  (1984)

Tumor-localization of lewis lung-carcinoma with radiolabeled monoclonal-antibodies

Cancer Immunology Immunotherapy 18 (1) 19-23

Roche, AC ; Bailly, P ; Midoux, P ; Monsigny, M  (1984)

Selective macrophage activation by muramyldipeptide bound to monoclonal-antibodies specific for mouse-tumor cells

Cancer Immunology Immunotherapy 18 (3) 155-159

Monsigny, M., Roche, A.C. and Midoux, P.  (1984)

Monoclonal-antibodies (igm) against a murine carcinoma, lewis lung-carcinoma (3ll) - production, purification and in vitro selectivity

Immunology Letters 8 (3) 131-136

Midoux, P ; Roche, AC ; Monsigny, M  (1984)

Flow cytofluorometry analysis of neoglycoproteins endocytosis mediated by membrane lectin of lewis lung-carcinoma cells

Biology of The Cell 52 (2) A76-A76

Roche, AC ; Midoux, P ; Lescanne, PJ ; Marteau, C ; Delmotte, F ; Monsigny, M  (1984)

Drug targeting via membrane lectins of mouse leukemic and carcinoma-cells

Biology of The Cell 51 (2) A59-A59

Midoux, P ; Grivet, JP ; Delmotte, F ; Monsigny, M  (1984)

The binding of monosaccharides to wheat-germ-agglutinin - fluorescence and NMR investigations

Biochemical and Biophysical Research Communications 119 (2) 603-611

Philippe, M ; Midoux, P ; Hommel, M ; Monsigny, M ; Schrevel, J  (1984)

Preliminary-study of glycosidases during the endoerythrocytary phase of plasmodium

Journal of Protozoology 31 (4) A75-A75

Midoux, P ; Wahl, P ; Auchet, JC ; Monsigny, M  (1984)

Fluorescence quenching of tryptophan by trifluoroacetamide

Biochimica et Biophysica Acta 801 (1) 16-25


1983   Références trouvées : 4

Roche, AC ; Barzilay, M ; Midoux, P ; Junqua, S ; Sharon, N ; Monsigny, M  (1983)

Sugar-specific endocytosis of glycoproteins by lewis lung-carcinoma cells

Journal of Cellular Biochemistry 22 (3) 131-140

Midoux, P ; Roche, AC ; Monsigny, M  (1983)

Preparation of monoclonal-antibodies directed against tumoral cells of a murine carcinoma, pulmonary lewis carcinoma

Biology of The Cell 49 (1) A33-A33

Maillet, T ; Therain, F ; Midoux, P ; Bailly, P ; Roche, AC ; Monsigny, M  (1983)

In vivo study of the localization of tumoral anti-cellular monoclonal-antibodies in mice

Biology of The Cell 49 (1) A32-A32

Midoux, P ; Delmotte, F ; GRIVET, JP ; Monsigny, M  (1983)

Protein sugar interactions - environmental-effect on the fluorescence of ortho-(4-methylumbelliferyl)-glycosides

Biochemical and Biophysical Research Communications 110 (3) 926-933


1980   Références trouvées : 1

Midoux, P ; Grivet, JP ; Monsigny, M  (1980)

Lectin-sugar interactions - the binding of 1-0-methyl-di-N-trifluoroacetyl-ß-chitobioside to wheat-germ-agglutinin

Febs Letters 120 (1) 29-32


Mots-clés

Directeur de recherche , Responsable de groupe thématique , Thérapies innovantes et nanomédecine