Biomedical subjects

J S Remy

Publications and source records attributed to J S Remy.

At least 19 recordsLinked to original sources

PubMed2001

Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in culture.

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.

3T3 Cells↗

PubMed2001

Polyethylenimine-mediated gene transfer into pancreatic tumor dissemination in the murine peritoneal cavity.

Although peritoneal dissemination of cancer cells often occurs at the advanced stages of pancreatic, gastric or ovarian cancers, no effective therapy has been established. Cationic lipid-mediated gene transfer into peritoneal dissemination may offer a prospect of safe therapies, but vector improvements are needed with regard to the efficiency and specificity of the gene transfer. In this study, the intraperitoneal injection of plasmid DNA:polyethylenimine (PEI) complexes into mice was evaluated as a gene delivery system for the peritoneal disseminations. The luciferase and beta-galactosidase genes were used as marker genes. PEI was more efficient than the cationic lipids examined in this study in vivo, and the transgene was preferentially expressed in the tumors. Although PCR analysis showed that the injected DNA was delivered to various organs, the distributed DNA became undetectable by 6 months after the gene transfer. Blood chemistry and histological analysis showed no significant toxicity in the injected mice. This study demonstrated that the intraperitoneal injection of DNA:PEI is a promising delivery method to transduce a gene into disseminated cancer nodules in the peritoneal cavity.

Animals↗

PubMed2001

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

Animals↗

PubMed2000

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells.

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.

Adenocarcinoma↗

PubMed2000

Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery.

Uncontrolled interactions of gene vectors and drug carriers in and with an in vivo environment pose serious limitations to their applicability. In order to reduce such interactions we have designed, synthesized and applied novel copolymers of poly(ethylene glycol) and reactive linkers which are derivatized with anionic peptides after copolymerization. The anionic copolymer derivatives are used to coat positively charged nonviral gene vectors by electrostatic interactions. The copolymer coat confers to polyelectrolyte colloids of DNA and polycations steric stabilization in their minimal size and prevents salt- and serum albumin-induced aggregation. Furthermore, complement activation and the interaction with serum proteins are drastically reduced or abolished in contrast to unprotected DNA complexes. The designed vectors are compatible with the intracellular steps of gene delivery and can even enhance transfection efficiency as demonstrated with various adherent and nonadherent cell lines in culture. The synthetic concept is amenable to the principles of combinatorial chemistry and the copolymeric products may be applicable beyond gene delivery in targeted drug delivery. Gene Therapy (2000) 7, 1183-1192.

Cells, Cultured↗

PubMed1999

Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway.

The interaction between cationic DNA-containing particles and cell surface anionic proteoglycans is an efficient means of entering cultured cells. Therapeutic in vivo gene delivery levels, however, require binding to less ubiquitous molecules. In an effort to follow adenovirus, thiol-derivatized polyethylenimine (PEI) was conjugated to the integrin-binding peptide CYGGRGDTP via a disulfide bridge. The most extensively conjugated derivative (5.5% of the PEI amine functions) showed physical properties of interest for systemic gene delivery. In the presence of excess PEI-RGD, plasmid DNA was condensed into a rather homogeneous population of 30-100 nm toroidal particles as revealed by electron microscopy images in 150 mM salt. Their surface charge was close to neutrality as a consequence of the shielding effect of the prominent zwitterionic peptide residues. Transfection efficiency of integrin-expressing epithelial (HeLa) and fibroblast (MRC5) cells was increased by 10- to 100-fold as compared with PEI, even in serum. This large enhancement factor was lost when aspartic acid was replaced by glutamic acid in the targeted peptide sequence (RGD/RGE), confirming the involvement of integrins in transfection. PEI-RGD/DNA complexes thus share with adenovirus constitutive properties such as size and a centrally protected DNA core, and 'early' properties, i.e. cell entry mediated by integrins and acid-triggered endosome escape.

Cations↗

PubMed1998

Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.

The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

Animals↗

PubMed1998

Monomolecular collapse of plasmid DNA into stable virus-like particles.

Cationic lipids are being widely used for cell transfection in vitro. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use in vivo. Cationic detergents, on the contrary, do not mediate cell transfection per se, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply "frozen" by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.

Animals↗

PubMed1998

Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability.

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. METHODS: Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. RESULTS: In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. CONCLUSIONS: Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.

Biopolymers↗

PubMed1998

Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system.

Currently in vivo gene delivery by synthetic vectors is hindered by the limited diffusibility of complexes in extra-cellular fluids and matrices. Here we show that certain formulations of plasmid DNA with linear polyethylenimine (22 kDa PEI, ExGene 500) can produce complexes that are sufficiently small and stable in physiological fluids so as to provide high diffusibility. When plasmid DNA was formulated with 22 kDa PEI in 5% glucose, it produced a homogeneous population of complexes with mean diameters ranging from 30 to 100 nm according to the amount of PEI used. In contrast, formulation in physiological saline produced complexes an order of magnitude greater (> or = 1 micron). Intraventricular injection of complexes formulated in glu-cose showed the complexes to be highly diffusible in the cerebrospinal fluid of newborn and adult mice, diffusing from a single site of injection throughout the entire brain ventricular spaces. Transfection efficiency was followed by histochemistry of beta-galactosidase activity and double immunocytochemistry was used to identify the cells transfected. Transgene expression was found in both neurons and glia adjacent to ventricular spaces. Thus, this method of formulation is promising for in vivo work and may well be adaptable to other vectors and physiological models.

Animals↗

PubMed1997

Lipospermine-mediated gene transfer technique into murine cultured cortical cells.

In order to transfer exogenous DNA into embryonic cortical cells, we have chosen a transfection technique using a synthetic lipospermine (dipalmitoylphosphatidylethanolamylspermine, DPPES) which complexes DNA molecules and allows their penetration into the intracellular compartment. The procedure was optimized after testing several parameters: DPPES/DNA ratio, incubation time, kinetics of transgene expression, and growth medium. The protocol was achieved by following the expression of the E. coli LacZ reporter gene under the control of the cytomegalovirus promoter. The lipopolyamine-mediated transfection is efficient for terminally differentiated cells, since we routinely obtained transfection efficiencies of 30% for neurons.

Animals↗

PubMed1995

Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids.

The presence of thiol-reactive phospholipid derivatives, such as N-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine (MPB-DPPE), in electrically neutral lipospermine/DNA particles results in more than a 100-fold increased transfection efficiency of human hepatoma HepG2 cells and murine 3T3 fibroblasts. These effects could be ascribed to the presence of thiol-reactive functions, such as maleimide, bromoacetamide and dithiopyridyl linkage, on the transfecting particles. We propose that such particles react with thiol groups present at the surface of the cells, leading to their covalent anchoring, a process that is probably followed by an endocytosis of the complex.

3T3 Cells↗

PubMed1995

Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses.

Optimal in vitro gene delivery with cationic lipids requires an excess of cationic charges with respect to DNA phosphates. In these conditions, in vivo delivery will be hampered by interference from cationic lipid-binding macromolecules either circulating or in the extracellular matrix. To overcome this problem, we are developing a modular transfection system based on lipid-coated DNA particles reminiscent of enveloped viruses. The particle core consists of the lipopolyamine-condensed nucleic acid in an electrically neutral ratio to which other synthetic lipids with key viral properties are hydrophobically adsorbed. As a first result, we have found that a good transfection level can be achieved simply with the neutral core particle, provided a zwitterionic lipid (dioleoyl phosphatidylethanolamine) is added to completely coat the DNA. Addition of lipids bearing a fusogenic or a nuclear localization peptide head group to the particles does not significantly improve an already efficient system, in contrast to polylysine-based gene transfer methods that rely on lysosomotropic or fusogenic agents to be effective. This emphasizes the distinctive properties of the lipopolyamines, including cell membrane destabilization, endosome buffering capacity, and possibly nuclear tropism. Most importantly, addition of lipids with a triantennary galactosyl residue drives the neutral nucleolipidic particles to the asialoglycoprotein receptor of human hepatoma HepG2 cells: Transfection increases approximately 1000-fold with 25% galactolipid. This receptor-mediated process is saturable and slightly less efficient than receptor-independent transfection obtained in vitro with a large excess of cationic lipid alone. Yet, electrically silent particles may provide an attractive solution for gene transfer in vivo where their external saccharide coat should allow them to diffuse within the organism and reach their target cells.

Animals↗

PubMed1994

High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes.

The ability to introduce DNA into mammalian cells has provided a powerful means to examine the regulation of gene expression and the function of gene products. However, the most commonly used techniques for DNA transfection are not always suitable for primary cells. Primary human keratinocytes are particularly stringent in their growth requirements and are also very refractory to transfection, rendering transient gene expression studies difficult. We have investigated the ability of several polycationic lipids to promote DNA uptake into human epidermal keratinocytes, as monitored with the bacterial beta-galactosidase reporter gene. We report that the cationic lipopolyamine dipalmitoyl phosphatidylethanolamine spermine as well as another procedure using Polybrene can achieve a 20% to 30% transfection efficiency, superior to any other agent tested on these cells. Gene transfer was accomplished by a 3-h exposure of monolayer cells to DNA complexes formed with either reagent by simple mixing in a serum-free medium, followed by a brief osmotic shock with glycerol. Neither DNA carrier showed any toxicity at the effective concentrations nor interfered with cell attachment, growth or differentiation. The use of a fully biodegradable lipopolyamine as DNA carrier should make it possible to extend this transfection method to gene transfer for in vivo therapeutic applications.

DNA↗

PubMedDate not supplied

Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI).

BACKGROUND: The ideal non-viral vector should be cell-type directed and form complexes with DNA that are physically stable, small and electrically neutral. METHODS: We have synthesized several PEI derivatives that coat the PEI/DNA complexes with water-soluble residues able to stabilize the particles, to mask their surface charge and eventually to direct them to a particular tissue. The morphologies and sizes of the complexes were observed by TEM and DLS techniques, and their apparent surface charge was quantitated by zeta potential measurements; in vitro transfection efficacies were determined in serum-containing cell culture medium. RESULTS: When compared to DNA complexes formed with the unmodified PEI, extensive grafting with maltose (15-25% of the amine functions) led to beneficial electrostatic shielding of the particle surface, but was unable to prevent aggregation in physiological salt concentration. More extended hydrophilic residues were therefore explored as a mean of physical repulsion between the particles. Low grafting (2.7%) with a linear dextran non-asaccharide led to small and stable toroids having no apparent surface charge, yet still reaching effective transfection levels. Electron microscopy of complexes with a higher extent of grafting showed worm-like structures unsuited for cell entry. Conjugation of PEI with as little as 0.5% of a terminally galactose-derivatized polyethyleneglycol (PEG)-3400 also gave neutral complexes of another worm-like structure that failed to transfect receptor-expressing hepatocytes. CONCLUSION: These results show that conjugation of large and flexible hydrophilic residues to PEI, while protecting the complexes from parasitic interactions also interfere with DNA condensation. PEG conjugation after PEI/DNA complex formation may avoid this problem, provided intracomplex reorganization is slow. Finally an anti-GD2 antibody (mAb) grafted with PEI was synthesized. The corresponding protein-coated DNA complexes were compact and small (50-60 nm), yet did not enhance transfection of GD2 ganglioside-expressing cells.

Animals↗

PubMedDate not supplied

Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse.

BACKGROUND: Several nonviral vectors including linear polyethylenimine (L-PEI) confer a pronounced lung tropism to plasmid DNA when injected into the mouse tail vein in a nonionic solution. METHODS: and results We have optimized this route by injecting 50 microg DNA with excess L-PEI (PEI nitrogen/DNA phosphate = 10) in a large volume of 5% glucose (0.4 ml). In these conditions, 1-5% of lung cells were transfected (corresponding to 2 ng luciferase/mg protein), the other organs remaining essentially refractory to transfection (1-10 pg luciferase/mg protein). beta-Galactosidase histochemistry confirmed alveolar cells, including pneumocytes, to be the main target, thus leading to the puzzling observation that the lung microvasculature must be permeable to cationic L-PEI/DNA particles of ca 60 nm. A smaller injected volume, premixing of the complexes with autologous mouse serum, as well as removal of excess free L-PEI, all severely decreased transgene expression in the lung. Arterial or portal vein delivery did not increase transgene expression in other organs. CONCLUSIONS: These observations suggest that effective lung transfection primarily depends on the injection conditions: the large nonionic glucose bolus prevents aggregation as well as mixing of the cationic complexes and excess free L-PEI with blood. This may favour vascular leakage in the region where the vasculature is dense and fragile, i.e. around the lung alveoli. Cationic particles can thus reach the epithelium from the basolateral side where their receptors (heparan sulphate proteoglycans) are abundant.

Animals↗

PubMedDate not supplied

Gene transfer with a series of lipophilic DNA-binding molecules.

Synthetic gene transfer vectors could be an attractive alternative to biological vehicles for gene therapy. In an effort to improve the previously developed lipopolyamine-mediated transfection technique, various amphiphilic DNA-binding molecules have been synthesized. Besides Transfectam, several lipospermines display very high gene delivery levels. The structure-activity relationship obtained points to the central role played by the polyamine headgroup in condensing the plasmid and binding it to the cell surface, provided the hydrophobic moiety is capable to generate nonmicellar mesomorphic structures. It also highlights other favorable (albeit more speculative) properties shared by protonable lipospermines as compared to quaternary ammonium-bearing lipids, such as their ability to act as a buffer and their strong affinity for chromatin. The former property may prevent the pH decrease along the degradative lysosomial pathway. The ability to bind to chromatin even in the presence of endogeneous polyamines should have two consequences: a nuclear tropism of the transfecting particles and plasmid uncoating in the nucleus by competitive dilution of the lipopolyamine into an ocean of DNA.

3T3 Cells↗

PubMedDate not supplied

Convenient polymer-supported synthetic route to heterobifunctional polyethylene glycols.

Conventional synthesis of heterobifunctional poly(ethylene glycol) derivatives, especially of medium size, is a rather tedious task. A straightforward solid-phase methodology has been developed that is illustrated here by the synthesis of alpha-pyridyldithio-omega-hydroxy-poly(ethylene glycol)600. This derivative was prepared from resin-bound PEG600 with a global yield of 65% for 6 individual steps, i.e., with an average yield of 93%/step. Intermediate purification steps simply consisted of resin washing. Progress of each reaction toward completion could conveniently be monitored by 13C NMR of the resin-bound PEG derivatives. This example highlights both the versatility and efficiency of combining polymer-supported synthesis with direct 13C-NMR characterization of the intermediate compounds.

Magnetic Resonance Spectroscopy↗