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Biomedical subjects

Maria C Pedroso de Lima

Publications and source records attributed to Maria C Pedroso de Lima.

13 recordsLinked to original sources
PubMed2006

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy.

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.

Antibiotics, Antineoplastic↗
PubMed2006

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions.

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.

Amino Acid Sequence↗
PubMed2006

Nuclear export of African swine fever virus p37 protein occurs through two distinct pathways and is mediated by three independent signals.

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.

Active Transport, Cell Nucleus↗
PubMed2005

On the mechanisms of the internalization of S4(13)-PV cell-penetrating peptide.

Cell-penetrating peptides have been shown to translocate across eukaryotic cell membranes through a temperature-insensitive and energy-independent mechanism that does not involve membrane receptors or transporters. Although cell-penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest both in vitro and in vivo, the mechanisms by which cellular uptake occurs remain unclear. In the face of recent reports demonstrating that uptake of cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of fixation artifacts, we conducted a critical re-evaluation of the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. We report that the S4(13)-PV peptide is able to accumulate inside live cells very efficiently through a rapid, dose-dependent and non-toxic process, providing clear evidence that the cellular uptake of this peptide cannot be attributed to fixation artifacts. Comparative analysis of peptide uptake into mutant cells lacking heparan sulphate proteoglycans demonstrates that their presence at the cell surface facilitates the cellular uptake of the S4(13)-PV peptide, particularly at low peptide concentrations. Most importantly, our results clearly demonstrate that, in addition to endocytosis, which is only evident at low peptide concentrations, the efficient cellular uptake of the S4(13)-PV cell-penetrating peptide occurs mainly through an alternative, non-endocytic mechanism, most likely involving direct penetration across cell membranes.

Amino Acid Sequence↗
PubMed2005

Targeting of sterically stabilised pH-sensitive liposomes to human T-leukaemia cells.

The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)-phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.

Cell Line, Tumor↗
PubMed2005

Delivery of antiviral agents in liposomes.

The intracellular activity of certain antiviral agents, including antisense oligonucleotides, acyclic nucleoside phosphonates, and protease inhibitors, is enhanced when they are delivered in liposome-encapsulated form. In this chapter we describe the preparation of pH-sensitive liposomes encapsulating antisense oligonucleotides, ribozymes, and acyclic nucleoside phosphonate analogues and their effects on HIV replication in macrophages. We outline the use of liposomal HIV protease inhibitors in infected macrophages. We present two methods for the covalent coupling of soluble CD4 to liposomes and show the association of these liposomes with HIV-infected cells. We also describe the synthesis of a novel antiviral agent based on cyclodextrin and its incorporation into liposomes.

Antiviral Agents↗
PubMed2005

Liposomal and viral vectors for gene therapy of the central nervous system.

Due to the presence of the blood-brain barrier, the central nervous system (CNS) is not easily accessible to systemically delivered macromolecules with therapeutic activity such as growth factors, cytokines or enzymes. Therefore, the expression of exogenously administered genes in the brain has been proposed for a wide variety of inherited and acquired diseases of the CNS, for which classical pharmacotherapy is unavailable or not easily applicable. Gene therapy to the CNS has been the target of a great number of studies aiming at finding a viable therapeutic strategy for the treatment of neurological disorders. This approach has already been used as a promising tool for brain protection and repair from neuronal insults and degeneration in several animal models, and is currently being applied in clinical trials. The choice of an appropriate vector system for transferring the desired gene into the affected brain area is an important issue for developing a safe and efficient gene therapy approach for the CNS. In this review, we focus on the various types of vectors that have been used for gene delivery into the CNS. Particular emphasis is given to their mode of preparation, biological activity, safety and in vivo behavior. Examples illustrating the potential of both viral and non-viral vectors in therapeutic applications to brain disorders are provided. In addition, the use of lentiviral vectors for in vivo modeling of genetic disorders of the CNS is discussed.

Animals↗
PubMed2004

On the formulation of pH-sensitive liposomes with long circulation times.

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo.

Animals↗
PubMed2004

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons.

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.

Animals↗
PubMed2004

Caveolae as an additional route for influenza virus endocytosis in MDCK cells.

Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded.

Animals↗
PubMed2003

Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides.

Cationic liposome-DNA complexes, also called "lipoplexes", constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations to emulate in vivo conditions.

Animals↗
PubMed2002

Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability.

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.

Erythrocyte Membrane↗