Biomedical subjects
A Rolland
Publications and source records attributed to A Rolland.
A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.
Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).
Target specific optimization of cationic lipid-based systems for pulmonary gene therapy.
PURPOSE: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. METHODS: The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. RESULTS: Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. CONCLUSIONS: Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.
Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration.
PURPOSE: This study relates to the development of gene therapies for the treatment of lung diseases. It describes for the first time the use of ultrasonic nebulization for administration of plasmid/lipid complexes to the lungs to transfect lung epithelial cells. METHODS: Plasmid complexed to cationic liposomes at a specific stoichiometric ratio was nebulized using an ultrasonic nebulizer. We assessed: (i) the stability of plasmid and plasmid/lipid complexes to ultrasonic nebulization, (ii) the in vitro activity of plasmid in previously nebulized plasmid/lipid complex, (iii) the in vivo transgene expression in lungs following intratracheal instillation of nebulized plasmid/lipid formulations compared to un-nebulized complexes, (iv) the emitted dose from an ultrasonic nebulizer using plasmid/lipid complexes of different size, and (v) the transgene expression in lungs following oral inhalation of aerosolized plasmid/lipid complex generated using an ultrasonic nebulizer. RESULTS: Integrity of plasmid formulated with cationic lipids, and colloidal stability of the plasmid/lipid complex were maintained during nebulization. In contrast, plasmid alone formulated in 10% lactose was fragmented during nebulization. The efficiency of transfection of the complex before and after nebulization was comparable. Nebulization produced respirable aerosol particles. Oral exposure of rodents for 10 minutes to aerosol produced from the ultrasonic nebulizer resulted in transgene expression in lungs in vivo. CONCLUSIONS: The performance characteristics of the ultrasonic nebulizer with our optimized plasmid/lipid formulations suggests that this device can potentially be used for administering gene medicines to the airways in clinical settings for the treatment of respiratory disorders.
Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy.
Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk- HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43(+)-tk+ and Cx43(+)-tk- while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, i.e. Cx43(+)-tk+/Cx43(-)-tk-. When GCV was given after the appearance of tumors, the size of the tumors from Cx43- cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.
Expression of biologically active human insulin-like growth factor-I following intramuscular injection of a formulated plasmid in rats.
Recent evidence has shown that insulin-like growth factor-I (IGF-I) plays an important role in the development, maintenance, and regeneration of peripheral nerves and skeletal muscle. IGF-I offers the potential to treat neuromuscular diseases in humans. We have developed a nonviral gene therapy method to express and produce localized and sustained therapeutic levels of IGF-I within target muscles by intramuscular injection of formulated plasmids. The purpose of the present study was to demonstrate that intramuscular injection of a plasmid encoding human IGF-I (hIGF-I) and engineered to restrict expression to skeletal muscle produces sustained local concentrations of biologically active hIGF-I. Normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinylpyrrolidone (PVP). Results show that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study (28 days), whereas the hIGF-I protein was not detected in blood. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP-43, an indicator of motor neuron sprouting. Placement of human growth hormone (hGH) 3' untranslated region enhanced GAP-43 staining, probably due to improved secretion of hIGF-I. Enhanced immunoreactivity of GAP-43 was observed in muscles injected with the formulated hIGF-I plasmid when compared to controls. These results demonstrate that intramuscular injection of hIGF-I plasmid formulated as a complex with PVP produces a localized and sustained level of biologically active hIGF-I.
Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA.
p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45-46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45-46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.
Cationic lipid-based gene delivery systems: pharmaceutical perspectives.
Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.
[The Blue House. Ethical principles and therapeutic choices].
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[The Blue House. Beyond loneliness].
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[The Blue House. Acting up and therapeutic actions].
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[The Blue House. The body and sexuality].
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[The Blue House. Jean or "my father's garden..."].
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Determination of the surface tension of block copolymer micelles by phagocytosis.
PURPOSE: This work was carried out to determine the surface tension of block copolymer micelles of 14C labelled ABA poly (oxyethylene-bi-isoprene-b-oxyethylene) which have a long circulating half life in animals. METHODS: The method used was that of phagocytosis. The percentage of micelles phagocytosed by human mononuclear cells was determined in solutions of different surface tension. RESULTS: The values obtained were 72 mN/m which may be predicted for a particle with a long circulating half life in animals. The method also gave an estimate of the surface tension for the mononuclear cells. CONCLUSIONS: This technique has the advantage of determining the surface tension of highly hydrated small particles including stable micelles in an environment similar to that in which they normally exist.
Site-specific drug delivery to pilosebaceous structures using polymeric microspheres.
In order to improve the therapeutic index of adapalene, a new drug under development for the treatment of acne, site-specific delivery to the hair follicles using 50:50 poly(DL-lactic-co-glycolic acid) microspheres as particulate carriers was investigated in vitro and in vivo. The percutaneous penetration pathway of the microspheres was shown to be dependent on their mean diameter. Thus, after topical application onto hairless rat or human skin, adapalene-loaded microspheres (5-microns diameter) were specifically targeted to the follicular ducts and did not penetrate via the stratum corneum. The in vitro release of adapalene from the microspheres into artificial sebum at 37 degrees C was controlled and faster than the in vivo sebum excretion in humans. Aiming to reduce either the applied dose of drug or the frequency of administration, different formulations of adapalene-loaded microspheres were evaluated in vivo in the rhino mouse model. A dose-related comedolytic activity of topical formulations of adapalene-loaded microspheres was observed in this model. Furthermore, by applying a site-specific drug delivery system (0.1% adapalene) every other day or by administering a 10-fold less concentrated targeted formulation (0.01%) every day, a pharmacological activity equivalent to a daily application of an aqueous gel containing drug crystals (0.1% adapalene) was observed. Since an aqueous gel containing 10% adapalene-loaded microspheres was not irritating in a rabbit skin irritancy test, this formulation was applied onto forearms of human volunteers. Site-specific drug delivery was further evidenced by follicular biopsy. Since an aqueous gel containing 10% adapalene-loaded microspheres was not irritating in a rabbit skin irritancy test, this formulation was applied onto forearms of human volunteers. Site-specific drug delivery was further evidenced by follicular biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of formulation, receptor fluid, and occlusion, on in vitro drug release from topical dosage forms, using an automated flow-through diffusion cell.
An automated flow-through diffusion cell apparatus was used for comparing the release rates of a naphthoic acid derivative, CD 271, from different topical formulations. The influence of the following parameters on CD 271 release from the formulations was investigated: receptor fluid composition, occlusion, weight of tested formulation, and dosage form type. The amount of tested formulation was shown to have no significant effect on the apparent release constant and lag time for an anionic oil-in-water emulsion and an aqueous gel. Occlusion affected drug release from the different dosage forms. Thus, occlusion increased CD 271 pharmaceutical availability for a lotion and a hydroalcoholic gel containing 0.1% of solubilized drug. The release profile of CD 271 from the formulations was highly dependent on the receptor fluid composition. Drug release was dramatically enhanced with n-octanol as compared to an aqueous solution of surfactant. Using occlusive or nonocclusive procedures, CD 271 apparent release constant and lag time were found to be highly dependent on the type of tested formulation. The flow-through diffusion cell proposed in the present study allows an accurate comparison of drug release characteristics from prototype topical formulations and therefore represents a valuable tool for formulation research or quality control process.
Behavioural effects of the American traditional plant Eschscholzia californica: sedative and anxiolytic properties.
Eschsholzia californica Cham. is a traditional medicinal plant of the Indians used by the rural population of California for its analgesic and sedative properties. Our study on the aqueous extract shows that this plant reduced the behavioural parameters measured in a familiar environment test in mice (novelty preference, locomotion and rearings in two compartments test) at doses above 100 mg/kg and in non-familiar environment tests (staircase test) at doses above 200 mg/kg. This finding validates its traditional sedative properties confirmed by the sleeping induction at doses above 100 mg/kg. Furthermore, when administered at a dose a of 25 mg/kg, E. californica appeared to also have an anxiolytic action since it produced an increase of the number of steps climbed by mice in the staircase test (anticonflict effect) and that of the time spent by animals in the lit box when they were confronted with the light/dark choice situation. Before evaluation of the behavioural effects, it was verified that our aqueous extract did not induce any toxic effect when administered i.p. and p.o.