RESUMO
The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.
Assuntos
Formação de Anticorpos/imunologia , Quitosana/administração & dosagem , DNA/administração & dosagem , Antígenos de Superfície da Hepatite B/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Nanopartículas/administração & dosagem , Mucosa Nasal/imunologia , Administração Intranasal , Animais , Quitosana/química , DNA/química , Portadores de Fármacos , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Mucosa Nasal/efeitos dos fármacos , Transfecção , VacinasRESUMO
MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation of gene expression at the posttranscriptional level. Due to their ability to simultaneously modulate the fate of different genes, these molecules are particularly well suited to act as key regulators during immune cell differentiation and activation, and their dysfunction can contribute to pathological conditions associated with neuroinflammation. Recent studies have addressed the role of miRNAs in the differentiation of progenitor cells into microglia and in the activation process, aiming at clarifying the origin of adult microglia cells and the contribution of the central nervous system (CNS) environment to microglia phenotype, in health and disease. Altered expression of several miRNAs has been associated with Alzheimer's disease, multiple sclerosis, and ischemic injury, hence strongly advocating the use of these small molecules as disease markers and new therapeutic targets. This review summarizes the recent advances in the field of miRNA-mediated regulation of microglia development and activation. We discuss the role of specific miRNAs in the maintenance and switching of microglia activation states and illustrate the potential of this class of nucleic acids both as biomarkers of inflammation and new therapeutic tools for the modulation of microglia behavior in the CNS.
Assuntos
Sistema Nervoso Central/imunologia , MicroRNAs/imunologia , Microglia/imunologia , Doenças Neurodegenerativas/imunologia , Biomarcadores/metabolismo , Diferenciação Celular , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Microglia/efeitos dos fármacos , Microglia/patologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Oligonucleotídeos Antissenso/farmacologiaRESUMO
Since clinical application of conventional cancer therapies is usually limited by drug resistance and toxic side effects, combination of chemotherapeutic agents with gene therapy appears as an attractive therapeutic strategy to overcome these issues. Being selectively expressed in tumor tissues, survivin is a promising target for the development of anticancer strategies aimed at eliminating tumor cells while sparing normal tissues. In this work, we achieved substantial protein knockdown in a number of human cell lines, namely, A549, HeLa and MCF-7 cells which overexpress survivin, after treatment with anti-survivin siRNAs, which was associated with a significant reduction of cell viability, when compared to treatment with control siRNAs. Interestingly, when the survivin-silencing approach was combined with a chemotherapeutic agent, an enhancement of the therapeutic effect was achieved. Treatment with anti-survivin siRNAs resulted in high levels of caspase 3/7 activation, and an enhancement of this effect was observed when survivin silencing was combined with vinblastine. In addition, we showed that for A549 and HeLa cells survivin silencing contributes to the reversion of cell resistance to doxorubicin. Overall, we demonstrate that the combination of a survivin-directed silencing strategy with chemotherapeutic agents constitutes a valuable approach for cancer treatment.
Assuntos
Doxorrubicina/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citometria de Fluxo , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Vimblastina/farmacologiaRESUMO
In the present work, we used a novel albumin-associated lipoplex formulation, containing the cationic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) and cholesterol (Chol), to evaluate the antitumoral efficacy of two gene therapy strategies: immuno-gene therapy, mediated by IL-12 gene expression, and "suicide" gene therapy, mediated by HSV-tk gene expression followed by ganciclovir (GCV) treatment. Our data show that, in an animal model bearing a subcutaneous TSA (mouse mammary adenocarcinoma) tumor, intratumoral administration of the albumin-associated complexes containing the plasmid encoding IL-12 results in a strong antitumoral effect, as demonstrated by the smaller tumor size, the higher T-lymphocyte tumor infiltration and the more extensive tumor necrotic and hemorrhagic areas, as compared to that observed in animals treated with control complexes. On the other hand, the application of the "suicide" gene therapy strategy results in a significant antitumoral activity, which is similar to that achieved with the immuno-gene therapy strategy, although involving different antineoplastic mechanisms. For the tested model, albumin-associated complexes were shown to efficiently mediate intratumoral delivery of therapeutic genes, thus leading to a significant antitumoral effect. This finding is particularly relevant since TSA tumors are characterized for being poorly immunogenic, aggressive and exhibiting high proliferation capacity.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Interleucina-12/administração & dosagem , Interleucina-12/genética , Neoplasias/genética , Neoplasias/terapia , Simplexvirus/enzimologia , Timidina Quinase/administração & dosagem , Timidina Quinase/genética , Animais , Antineoplásicos , Sobrevivência Celular , DNA Viral/genética , Relação Dose-Resposta a Droga , Feminino , Ganciclovir/uso terapêutico , Interleucina-12/metabolismo , Lipossomos , Luciferases/genética , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Linfócitos T/imunologia , Timidina Quinase/metabolismoRESUMO
New molecular biology techniques have uncovered the hidden role of genes in cancer. Identification of activated oncogenes, as fundamental genetic differences relative to normal cells, has made it possible to consider such genes as targets for antitumor therapy, namely by applying gene silencing strategies. In this regard, antisense oligonucleotides or small interfering RNAs, constitute promising therapeutic tools. The widespread clinical application of such molecules as modulators of gene expression, is still dependent on several aspects that limit their bioavailability, including: enhanced biological stability, favourable pharmacokinetics, enhanced tumor cell uptake and, consequently, efficient targeted delivery. One of the most promising strategies to overcome the barriers faced by gene silencing molecules, upon systemic administration, involves the use of lipid-based nanoparticles. The first part of this review aims at providing the reader with the molecular mechanism of action of the most important gene silencing molecules used in anticancer therapy. The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. Therefore, an overview of different lipid-based strategies for nucleic acid delivery will be presented on the second part. As we learn more about the pharmacokinetics and pharmacodynamics of the carrier and/or of the gene silencing molecules, it will be possible to further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer systemic gene silencing.
Assuntos
Inativação Gênica , Lipídeos/química , Nanopartículas , Animais , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/terapia , RNA Interferente PequenoRESUMO
The use of tailored particle-based adjuvants constitutes a promising way to enhance antigen-specific humoral and cellular immune responses. However, a thorough understanding of the mechanisms underlying their adjuvanticity is crucial to generate more effective vaccines. We studied the ability of chitosan-aluminum nanoparticles (CH-Al NPs), which combine the immunostimulatory effects of chitosan and aluminum salts, to promote dendritic cell activation, assess their impact on innate and adaptive immune responses, and compare the results to those reported for conventional chitosan particles (CH-Na NPs). All tested CH-NP formulations were capable of modulating cytokine secretion by dendritic cells. CH-Al NPs promoted NLRP3 inflammasome activation, enhancing the release of IL-1ß without significantly inhibiting Th1 and Th17 cell-polarizing cytokines, IL-12p70 or IL-23, and induced DC maturation, but did not promote pro-inflammatory cytokine production on their own. In vivo results showed that mice injected with CH-Al NPs generated a local inflammatory response comparable to that elicited by the vaccine adjuvant alum. Importantly, after subcutaneous immunization with CH-Al NPs combined with the hepatitis B surface antigen (HBsAg), mice developed antigen-specific IgG titers in serum, nasal and vaginal washes. Overall, our results established CH-Al NPs as a potential adjuvant to enhance both innate and adaptive immune responses.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Alumínio/administração & dosagem , Quitosana/administração & dosagem , Antígenos de Superfície da Hepatite B/administração & dosagem , Nanopartículas/administração & dosagem , Animais , Citocinas/imunologia , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.
Assuntos
Antimetabólitos/uso terapêutico , Citosina Desaminase/genética , Flucitosina/uso terapêutico , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Transgênicos Suicidas , Terapia Genética , Neoplasias Hepáticas Experimentais/terapia , Animais , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Terapia Combinada , Citocinas/genética , Células Matadoras Naturais/imunologia , Lipossomos , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/secundário , Masculino , Plasmídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Transfecção , Células Tumorais CultivadasRESUMO
Central nervous system (CNS) diseases constitute a set of challenging pathological conditions concerning diagnosis and therapeutics. For most of these disorders, there is a lack of early diagnosis, biomarkers to allow proper follow-up of disease progression and effective therapeutic strategies to allow a persistent cure. The poor prognosis of most CNS diseases is, therefore, a global concern, especially regarding chronic age-related neurodegenerative disorders, which are already considered problems of public health due to the increasing average of life expectancy. The difficulties associated with the treatment of CNS diseases are owed, at least in part, to very specific characteristics of the brain and spinal cord, when compared to peripheral organs. In this regard, the CNS is physically and chemically protected by the blood-brain barrier (BBB), which, while maintaining essential brain homeostasis, significantly restricts the delivery of most therapeutic agents to the brain parenchyma. On the other hand, regenerative properties of the tissue are lacking, meaning that a CNS insult resulting in neuronal death is a permanent phenomenon. Approaches for transposing the BBB aiming to treat CNS diseases, relying on specific properties of nanosystems, have been reported for therapeutic delivery to CNS without interfering with the normal function of the brain. In this chapter, we address the latest advances concerning the principles of such approaches, employing lipid-based nanoparticles and cell-produced exosomes as drug and nucleic acid delivery systems, and summarize recent example of applications in the context of neurological diseases. Major achievements obtained in preclinical studies and the trends identified by these studies are emphasized to provide new prospects for further developments in this area, thus enabling us to move from the research realm to the clinical arena.
Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Exossomos , Nanopartículas/uso terapêutico , Nanotecnologia/tendências , Animais , Sistemas de Liberação de Medicamentos , Humanos , Lipídeos/administração & dosagem , Nanopartículas/administração & dosagemRESUMO
We investigated the molecular mechanisms by which pH-sensitive liposomes surpass the cytoplasmic and endosomal membranes to deliver their aqueous contents into the cytoplasm. Various liposome formulations were evaluated for their efficacy to mediate intracellular delivery of encapsulated material, including a novel sterically stabilized pH-sensitive formulation ((DOPE:CHEMS:DSPE-PEG(2000) (6:4:0.3)) that was previously developed in our laboratories. In an attempt to fully characterize the nature of liposome-cell interactions different approaches based on a dual-labeling fluorescence assay were used. Our results indicate that the efficacy of interaction of pH-sensitive liposomes, both plain and sterically stabilized, with cells is strongly determined by the inclusion of DOPE in their composition, independently of the type of the amphiphilic stabilizer used. In fact, DOPE-containing liposomes shown to be non-pH sensitive by biophysical assays, mediated cytoplasmic delivery of their contents as efficiently as well known pH-sensitive formulations (e.g. DOPE:CHEMS). However, among the different formulations studied, DOPE:CHEMS liposomes were those exhibiting the highest extent of cell association. Moreover, our results with cells pretreated with metabolic inhibitors or lysosomotropic agents clearly indicate that DOPE-containing liposomes are internalized essentially by endocytosis and that acidification of the endosomes is not the only mechanism involved in the destabilization of the liposomes inside the cell.
Assuntos
Endocitose , Lipossomos/química , Macrolídeos , Fosfatidiletanolaminas/química , Antibacterianos/farmacologia , Antimicina A/farmacologia , Linhagem Celular , Química Farmacêutica , Cloroquina/farmacologia , Citoplasma/química , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Endossomos/química , Fluoresceínas , Corantes Fluorescentes , Glicerofosfolipídeos/química , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/química , Microscopia de Fluorescência , Rodaminas , Azida Sódica/farmacologia , Fluoreto de Sódio/farmacologia , Espectrometria de FluorescênciaRESUMO
African swine fever virus (ASFV) enters cells by receptor mediated endocytosis and requires a fusion event between the viral envelope and the limiting membrane of the endosome at low pH. In order to investigate the role of cholesterol in the early stages of ASFV infection, we have studied the effect of the removal of cell and viral membrane cholesterol by cholesterol oxidase treatment of cells and virions, as well as the effect of some inhibitors of cholesterol synthesis on the infectious pathway. In addition, we have investigated viral infection in cholesterol-depleted Vero cells. Both cholesterol-depleted and cholesterol oxidase-treated Vero cells were unaltered in their ability to bind or internalize the virus, but were blocked in ASFV fusion and subsequent virus replication. Our results indicate that ASFV infection is affected by cholesterol in the target membrane.
Assuntos
Vírus da Febre Suína Africana/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Febre Suína Africana/metabolismo , Animais , Membrana Celular/virologia , Cerulenina/farmacologia , Chlorocebus aethiops , Colesterol Oxidase/farmacologia , Miconazol/farmacologia , Sulfonamidas/farmacologia , Células VeroRESUMO
The kinetics of fusion of Sendai virus (Z strain) with the human promyelocytic leukemia cell line HL-60, and the human T lymphocytic leukemia cell line CEM was investigated. Fusion was monitored by fluorescence dequenching of octadecylrhodamine (R-18) incorporated in the viral membrane. For one virus isolate (Z/G), the overall rate of fusion (at 37 degrees C) increased as the pH was lowered, reaching a maximum at about pH 5, the lowest pH tested. For another isolate (Z/SF) the rate and extent of fusion were lower at pH 5 than at neutral pH. Lowering the pH from neutral to 5 after several minutes of incubation of either isolate with HL-60 cells resulted in an enhanced rate of fluorescence dequenching. Nevertheless, experiments utilizing NH4Cl indicated that fusion of the virus with cells was not enhanced by the mildly acidic pH of the endosome lumen. Analysis of the kinetics of fusion by means of a mass action model resulted in good simulation and predictions for the time-course of fusion. For the isolate which showed maximal fusogenic activity at pH 5, the rate constant of fusion (approx. 0.1 s-1) at neutral pH was in the range found previously for virus-liposome fusion, whereas the rate constant of adhesion was close to the upper limit for diffusion-controlled processes (1.4.10(10) M-1 s-1). However, for the other isolate (Z/SF) the rate constant of fusion at neutral pH was very small (less than 0.01 s-1), whereas the rate constant of adhesion was larger (greater than or equal to 2.10(10) M-1 s-1). Lowering the temperature decreased the fusion rate. Experiments involving competition with excess unlabeled virions indicated that not all binding sites for Sendai virus on HL-60 cells are fusion sites. The virus fusion activity towards HL-60 cells at neutral pH was not altered significantly by pre-incubation of the virus at pH 5 or 9, in contrast to earlier observations with liposomes and erythrocyte ghosts, or results based on erythrocyte hemolysis or cell-cell fusion.
Assuntos
Fusão de Membrana , Vírus da Parainfluenza 1 Humana/fisiologia , Cloreto de Amônio/farmacologia , Linhagem Celular , Membrana Eritrocítica/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Leucemia Promielocítica Aguda , Leucemia de Células T , Lipossomos , Fusão de Membrana/efeitos dos fármacos , Espectrometria de Fluorescência , TemperaturaRESUMO
The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10-40 degrees C. The fusogenic activity of the cations decreases in the sequence Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+ for cholesterol concentrations in the range 20-40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca2+ and Ba2+ at 25 degrees C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25 degrees C, Mg2+ is ineffective in causing fusion at all cholesterol concentrations. However, at 30 degrees C, Mg2+-induced fusion is observed with vesicles containing cholesterol. At 40 degrees C, Mg2+ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca2+, Ba2+ and Sr2+. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na+ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (Tm) in Na+ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the Tm upward, with a sequence of effectiveness Ba2+ greater than Sr2+ greater than Mg2+. The Tm of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na+, Sr2+ or Mg2+, the addition of Ba2+ reveals endotherms with Tm progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the Tm, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg2+ concentrations. With Ca2+, saturation is not observed for cholesterol-containing vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cátions Bivalentes/farmacologia , Colesterol/farmacologia , Fusão de Membrana/efeitos dos fármacos , Lipídeos de Membrana , Fosfolipídeos , Animais , Varredura Diferencial de Calorimetria , Bovinos , Fenômenos Químicos , Físico-Química , Técnicas In Vitro , Cinética , Fosfatidiletanolaminas , Fosfatidilserinas , TemperaturaRESUMO
Cationic liposome-DNA complexes ('lipoplexes') are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1, 2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.
Assuntos
Albumina Sérica/farmacologia , Transfecção/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linfócitos B , Sangue , Células COS , Linhagem Celular , Portadores de Fármacos , Ácidos Graxos Monoinsaturados , Células HeLa , Humanos , Lipossomos , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Peptídeos , Fosfatidiletanolaminas , Plasmídeos , Compostos de Amônio Quaternário , Transfecção/métodosRESUMO
Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.
Assuntos
Adesão Celular , DNA/química , Lipossomos/química , Ligação Competitiva , Portadores de Fármacos , Endocitose/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/química , Humanos , Leucemia Monocítica Aguda , Compostos de Amônio Quaternário/química , Temperatura , Transfecção , Células Tumorais Cultivadas/químicaRESUMO
The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non-dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH-sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected lack of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo.
Assuntos
DNA/administração & dosagem , Endossomos/metabolismo , Luciferases/genética , Macrófagos , Transfecção/métodos , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Genes Reporter , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/farmacologia , Transfecção/efeitos dos fármacos , Transfecção/genética , Transferrina/metabolismo , Transferrina/farmacologiaRESUMO
Great expectations from the application of gene therapy approaches to human disease have been impaired by the unsatisfactory clinical progress observed. Among others, the use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of this promising therapeutic strategy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. This review focuses on the various parameters that are considered to be crucial to optimize the use of cationic lipid-DNA complexes for gene therapy purposes. Particular emphasis is devoted to the analysis of the different stages involved in the transfection process, from the biophysical aspects underlying the formation of the complexes to the different biological barriers that need to be surpassed for gene expression to occur.
Assuntos
DNA/química , Terapia Genética/métodos , Lipídeos/química , Lipossomos/química , Transfecção/métodos , Animais , Fenômenos Biofísicos , Biofísica , Cátions , Membrana Celular/metabolismo , DNA/administração & dosagem , Humanos , Lipídeos/administração & dosagem , Lipossomos/administração & dosagem , Membrana Nuclear/metabolismoRESUMO
The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome/DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Lipossomos/administração & dosagem , Lipossomos/química , Animais , Fenômenos Biofísicos , Biofísica , Cátions , Humanos , Transfecção/métodosRESUMO
The development of new low molecular weight drugs against human immunodeficiency virus Type 1 (HIV-1) targets other than reverse transcriptase (RT) and protease, such as the integrase and the envelope glycoprotein, is likely to take many years. Macromolecular drugs, including antisense oligonucleotides, ribozymes, RNA decoys and transdominant mutant proteins, may be able to interfere with a relatively large number of viral targets, thereby decreasing the likelihood of the emergence of drug-resistant strains. It may also be relatively easy to alter the sequence of some of the macromolecular drugs to counter emerging drug-resistant viruses. The delivery of antisense oligonucleotides and ribozymes to HIV-1 infected or potentially infectable cells by antibody-targeted liposomes, certain cationic lipid formulations and pH-sensitive liposomes results in significant anti-HIV-1 activity. These carriers not only facilitate cytoplasmic delivery but also protect the drugs from nuclease digestion. Delivery of therapeutic genes (another form of macromolecular drug) to target cells is an important challenge of gene therapy. Following delivery by a viral vector, sufficient levels of gene expression must be maintained over an extended period of time to have therapeutic activity. Robust expression of therapeutically useful ribozymes, antisense, decoys and aptamers can be achieved by the use of Pol III expression systems. Moloney murine leukaemia virus- (MoMuLV), adeno-associated virus (AAV)-, or HIV-derived vectors expressing a variety of therapeutic genes have been used successfully to inhibit HIV-1 replication in cultured cells.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Vírus de DNA/genética , Vetores Genéticos , HIV-1 , Oligonucleotídeos Antissenso/administração & dosagem , Vírus de RNA/genética , RNA Catalítico/administração & dosagemRESUMO
Liposomes can be used as carriers of drugs in the treatment of viral, bacterial and protozoal infections. The potential for liposome-mediated therapy of Mycobacterium avium-intracellulare complex infections, one of the most common opportunistic infections in AIDS, is currently under study. Here, we have investigated the effect of the lipid-soluble antimycobacterial drugs ansamycin, clofazimine and CGP7040 on the thermotropic behavior of liposomes composed of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) using differential scanning calorimetry (DSC). In the presence of ansamycin (rifabutine), the peak gel-liquid crystalline phase transition temperature (Tm) of DPPG was reduced, as was the sub-transition temperature (Ts), whereas the Tm of DPPC was reduced only slightly. The temperature of the pre-transition (Tp) of DPPC was lowered, while the pre-transition of DPPG was abolished. Ansamycin also caused the broadening of the transition endotherm of both lipids. Equilibration of the drug/lipid complex for 1 or 5 days produced different thermotropic behavior. In the presence of clofazimine, the cooperativity of the phase transition of DPPG decreased. Above 10 mol% clofazimine formed two complexes with DPPG, as indicated by two distinguishable peaks in DSC thermograms. The Tm of both peaks were lowered as the mole fraction increased. Clofazimine had minimal interaction with DPPC. In contrast, CGP7040 interacted more effectively with DPPC than with DPPG, causing a reduction of the size of the cooperative unit of DPPC even at 2 mol%. The main transition of DPPC split into 3 peaks at 5 mol% drug. The pre-transition was abolished at all drug concentrations and the sub-transition disappeared at 10 mol% CGP7040. These studies suggest that maximal encapsulation of clofazimine in liposomes would require a highly negatively charged membrane, while that of CGP7040 would necessitate a zwitterionic membrane. We have also investigated the interaction of the water-soluble antibiotic pentamidine, which has been used against Pneumocystis carinii, the most lethal of AIDS-related opportunistic pathogens. Aerosol administration of this drug leads to long-term sequestration of the drug in the lungs. The DPPG/pentamidine complex exhibited a pre-transition at 3.5 degrees C, an endothermic peak at 42 degrees C, and an exothermic peak at 44.5 degrees C, followed by another endothermic peak at 55 degrees C. The exotherm depended on the history of the sample, requiring pre-incubation for several minutes below the 42 degrees C transition. These observations suggest that upon melting of the DPPG chains at 42 degrees C, the DPPG crystallizes as a DPPG/pentamidine complex that melts at 55 degrees C.
Assuntos
Antibacterianos/metabolismo , Antifúngicos/metabolismo , Lipídeos de Membrana/metabolismo , Complexo Mycobacterium avium/efeitos dos fármacos , Fosfolipídeos/metabolismo , Pneumocystis/efeitos dos fármacos , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Antituberculosos/metabolismo , Calorimetria/métodos , Clofazimina/metabolismo , Temperatura Alta , Humanos , Lactamas Macrocíclicas , Pentamidina/metabolismo , Fosfatidilgliceróis/metabolismo , Rifabutina , Rifamicinas/metabolismoRESUMO
We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R 18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.