Lipoxin A4 impairment of apoptotic signaling in macrophages: Implication of the PI3K/Akt and the ERK/Nrf-2 defense pathways

El pdf del artículo es la versión pre-print.Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires transcellular metabolic traffic for its synthesis. The targets of LXA4 on neutrophils are well described, contributing to attenuation of inflammation. However, effects of lipoxins on macrophage are less known, particularly the action of LXA4 on the regulation of apoptosis of these cells. Our data show that pretreatment of human or murine macrophages with LXA4 at the concentrations prevailing in the course of resolution of inflammation (nanomolar range) significantly inhibits the apoptosis induced by staurosporine, etoposide and S-nitrosoglutathione or by more pathophysiological stimuli, such as LPS/IFNγ challenge. The release of mitochondrial mediators of apoptosis and the activation of caspases was abrogated in the presence of LXA4. In addition to this, the synthesis of reactive oxygen species induced by staurosporine was attenuated and antiapoptotic proteins of the Bcl-2 family accumulated in the presence of lipoxin. Analysis of the targets of LXA4 identified an early activation of the PI3K/Akt and ERK/Nrf-2 pathways, which was required for the observation of the antiapoptotic effects of LXA4. These data suggest that the LXA 4, released after the recruitment of neutrophils to sites of inflammation, exerts a protective effect on macrophage viability that might contribute to a better resolution of inflammation. © 2010 Macmillan Publishers Limited All rights reserved.PP was supported by a fellowship from Ministerio de Ciencia e Innovación (MICINN; Spain). This work was supported by grant BFU2008-02161 from MICINN, S-BIO-0283/2006 from Comunidad de Madrid and FIS-RECAVA RD06/0014/0025. RECAVA and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Lipid-polymer membranes as carriers for L-tryptophan : Molecular and metabolic properties

Polymerized liposomes encapsulating L-tryptophan were studied with the aim to characterize them as drug delivery systems for the treatment of several metabolic diseases that need an increased systemic L-tryptophan concentration, polymerized liposomes were obtained by UV irradiation of vesicles containing 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a 1:1 molar ratio, in the presence of 10 and 50 mol% of L-tryptophan (respect to total lipid concentration). Polymerization efficiency was studied spectrophotometrically. Also, bilayer packing at the polar head region was followed with the Merocyanine 540 (MC540) and specific interactions in the lipopolymers were studied by FTIR. High L-tryptophan concentrations (50 mol% respect to total lipid concentration) induced a higher amount of six- and nine-unit polymers. This phenomenon was induced because the L-tryptophan located outside the lipid membrane was included in it during the polymerization process and was thus responsible for the better accommodate of the polar head region. This was not possible with the lower amount of L-tryptophan (10 mol%). The stability of lipopolymers with different amounts of L-tryptophan was studied through release profiles. Polymerized liposomes with 50 mol% of L-tryptophan were able to retain around 80% of the amino acid after 24 hours, whereas those with 10 mol % of the amino acid were able to retain 20%. The metabolic activity of the Caco-2 cell line was also studied. Cytotoxic effects were low in the presence of polymerized liposomes, rendering a maximum percentage of cell death of 30%. In summary, this work stresses the relevance of nonspecific drug-polymerized membrane binding on L-tryptophan pharmacological interaction with possible pharmaceutical applications in liposomal drug delivery. Moreover, the absence of significant cytotoxic effects allows the system proposed to be applied in human health.Instituto Multidisciplinario de Biología Celula

Lipid-polymer membranes as carriers for L-tryptophan : Molecular and metabolic properties

Polymerized liposomes encapsulating L-tryptophan were studied with the aim to characterize them as drug delivery systems for the treatment of several metabolic diseases that need an increased systemic L-tryptophan concentration, polymerized liposomes were obtained by UV irradiation of vesicles containing 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a 1:1 molar ratio, in the presence of 10 and 50 mol% of L-tryptophan (respect to total lipid concentration). Polymerization efficiency was studied spectrophotometrically. Also, bilayer packing at the polar head region was followed with the Merocyanine 540 (MC540) and specific interactions in the lipopolymers were studied by FTIR. High L-tryptophan concentrations (50 mol% respect to total lipid concentration) induced a higher amount of six- and nine-unit polymers. This phenomenon was induced because the L-tryptophan located outside the lipid membrane was included in it during the polymerization process and was thus responsible for the better accommodate of the polar head region. This was not possible with the lower amount of L-tryptophan (10 mol%). The stability of lipopolymers with different amounts of L-tryptophan was studied through release profiles. Polymerized liposomes with 50 mol% of L-tryptophan were able to retain around 80% of the amino acid after 24 hours, whereas those with 10 mol % of the amino acid were able to retain 20%. The metabolic activity of the Caco-2 cell line was also studied. Cytotoxic effects were low in the presence of polymerized liposomes, rendering a maximum percentage of cell death of 30%. In summary, this work stresses the relevance of nonspecific drug-polymerized membrane binding on L-tryptophan pharmacological interaction with possible pharmaceutical applications in liposomal drug delivery. Moreover, the absence of significant cytotoxic effects allows the system proposed to be applied in human health.Instituto Multidisciplinario de Biología Celula

Effect of Risperidone and Fluoxetine on the Movement and Neurochemical Changes of Zebrafish

Brain developmental disorders in humans, including Autism Spectrum Disorders (ASD) and Down’s syndrome, have been linked to increased serotonin levels. This work was designed to study changes in serotonin levels in the early stages of development with two classes of antipsychotic drugs: Risperidone, a drug that blocks serotonin and dopamine receptors, and fluoxetine, a serotonin reuptake inhibitor. The use of antipsychotic drugs is a solid choice to study the decrease and increase of these neurotransmitters and their influence on development. The study of these parameters will give an idea of the effects of serotonin in early developmental stages. To this end, we examined the effects of risperidone and fluoxetine on the locomotor activity, heart rate and brain development of zebrafish larvae. Our results showed that in larvae exposed to fluoxetine alone, swimming was significantly increased at 9 dpf (days post-fertilization). Erratic and abnormal movements were observed suggesting a toxic effect of fluoxetine. No erratic swimming was observed in larvae treated with fluoxetine plus risperidone. Both drugs presented morphological changes in dopaminergic neurons and mononeurons. Exposure to fluoxetine plus risperidone indicated possible reversal effects. Studies in zebrafish allow obtaining new insights into the side effects of these drugs as well as into the brain control of locomotor activity. Testing several drug-induced changes in behavior and serotonin levels is one of the experimental approaches for screening a new therapeutically relevant compound, and thus, merits further research.Instituto Multidisciplinario de Biología Celula

Effect of Risperidone and Fluoxetine on the Movement and Neurochemical Changes of Zebrafish

Brain developmental disorders in humans, including Autism Spectrum Disorders (ASD) and Down’s syndrome, have been linked to increased serotonin levels. This work was designed to study changes in serotonin levels in the early stages of development with two classes of antipsychotic drugs: Risperidone, a drug that blocks serotonin and dopamine receptors, and fluoxetine, a serotonin reuptake inhibitor. The use of antipsychotic drugs is a solid choice to study the decrease and increase of these neurotransmitters and their influence on development. The study of these parameters will give an idea of the effects of serotonin in early developmental stages. To this end, we examined the effects of risperidone and fluoxetine on the locomotor activity, heart rate and brain development of zebrafish larvae. Our results showed that in larvae exposed to fluoxetine alone, swimming was significantly increased at 9 dpf (days post-fertilization). Erratic and abnormal movements were observed suggesting a toxic effect of fluoxetine. No erratic swimming was observed in larvae treated with fluoxetine plus risperidone. Both drugs presented morphological changes in dopaminergic neurons and mononeurons. Exposure to fluoxetine plus risperidone indicated possible reversal effects. Studies in zebrafish allow obtaining new insights into the side effects of these drugs as well as into the brain control of locomotor activity. Testing several drug-induced changes in behavior and serotonin levels is one of the experimental approaches for screening a new therapeutically relevant compound, and thus, merits further research.Instituto Multidisciplinario de Biología Celula

PAMAM dendrimers as a carbamazepine delivery system for neurodegenerative diseases : A biophysical and nanotoxicological characterization

Carbamazepine (CBZ) is an antiepileptic drug, which also could be used in the treatment of neurodegenerative diseases, such as the Alzheimer's disease. However, its use has been limited due to its low solubility, inefficient pharmacokinetic profiles, and multiple side effects. PAMAM dendrimers, ethylenediamine core, generation 4.0 (amine terminal groups) and 4.5 (carboxylate terminal groups) (DG4.0 and DG4.5 respectively) are polymers that can increase drug solubility through complexation. Thus, the aim of this work was to obtain and characterize complexes between CBZ and dendrimers. Both DG4.0 and DG4.5 allowed the incorporation of ∼20 molecules of CBZ per dendrimer, into their hydrophobic pockets. DG4.0-CBZ and DG4.5-CBZ complexes were found to be stable for 90 days at 37 °C and resistant to a lyophilization process, presenting controlled drug release. Also, the complexes nanotoxicity was tested ex vivo (human red blood cells), in vitro (N2a cell line), and in vivo (zebrafish). No hemolytic effect was observed in the ex vivo model. As regards in vitro toxicity, the DG4.5-CBZ complexes significantly reduced the toxicity caused by the free drug. Moreover, the DG4.5-CBZ did not cause neurotoxicity or cardiotoxicity in zebrafish larvae. In conclusion, a stable and biocompatible drug delivery system based on the DG4.5 capable of complex the CBZ has been developed. This achievement highlights the advantages of using negatively charged dendrimers for nanomedicine.Instituto Multidisciplinario de Biología Celula

PAMAM dendrimers as a carbamazepine delivery system for neurodegenerative diseases : A biophysical and nanotoxicological characterization

Carbamazepine (CBZ) is an antiepileptic drug, which also could be used in the treatment of neurodegenerative diseases, such as the Alzheimer's disease. However, its use has been limited due to its low solubility, inefficient pharmacokinetic profiles, and multiple side effects. PAMAM dendrimers, ethylenediamine core, generation 4.0 (amine terminal groups) and 4.5 (carboxylate terminal groups) (DG4.0 and DG4.5 respectively) are polymers that can increase drug solubility through complexation. Thus, the aim of this work was to obtain and characterize complexes between CBZ and dendrimers. Both DG4.0 and DG4.5 allowed the incorporation of ∼20 molecules of CBZ per dendrimer, into their hydrophobic pockets. DG4.0-CBZ and DG4.5-CBZ complexes were found to be stable for 90 days at 37 °C and resistant to a lyophilization process, presenting controlled drug release. Also, the complexes nanotoxicity was tested ex vivo (human red blood cells), in vitro (N2a cell line), and in vivo (zebrafish). No hemolytic effect was observed in the ex vivo model. As regards in vitro toxicity, the DG4.5-CBZ complexes significantly reduced the toxicity caused by the free drug. Moreover, the DG4.5-CBZ did not cause neurotoxicity or cardiotoxicity in zebrafish larvae. In conclusion, a stable and biocompatible drug delivery system based on the DG4.5 capable of complex the CBZ has been developed. This achievement highlights the advantages of using negatively charged dendrimers for nanomedicine.Instituto Multidisciplinario de Biología Celula

Diacetylenic lipids in the design of stable lipopolymers able to complex and protect plasmid DNA

Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel nonviral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.Instituto Multidisciplinario de Biología Celula

Diacetylenic lipids in the design of stable lipopolymers able to complex and protect plasmid DNA

Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel nonviral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.Instituto Multidisciplinario de Biología Celula

Combined Therapy for Alzheimer’s Disease : Tacrine and PAMAM Dendrimers Co-Administration Reduces the Side Effects of the Drug without Modifying its Activity

Alzheimer’s disease has become a public health priority, so an investigation of new therapies is required. Tacrine (TAC) was licensed for treatments; however, its oral administration caused hepatotoxicity, so it is essential to reduce the side effects. PAMAM dendrimer generation 4.0 and 4.5 (DG4.0 and DG4.5) can be used as drug delivery systems and as nanodrugs per se. Our work aims to propose a combined therapy based on TAC and PAMAM dendrimer co-administration. TAC and dendrimer interactions were studied by in vitro drug release, drug stability, and FTIR. The toxicity profile of co-administration was evaluated in human red blood cells, in Neuro-2a cell culture, and in zebrafish larvae. Also, the anti-acetylcholinesterase activity was studied in cell culture. It was possible to obtain DG4.0-TAC and DG4.5-TAC suspensions, without reducing the drug solubility and stability. FTIR and in vitro release studies confirmed that interaction between TAC and DG4.5 was of the electrostatic type. No toxicity effects on human red blood cells were observed, whereas the co-administration with DG4.5 reduced cytotoxicity of TAC on the Neuro-2a cell line. Moreover, in vivo co-administration of both DG4.0-TAC and DG4.5-TAC reduced the morphological and hepatotoxic effects of TAC in zebrafish larvae. The reduction of TAC toxicity was not accompanied by a reduction in its activity since the anti-acetylcholinesterase activity remains when it is co-administrated with dendrimers. In conclusion, the co-administration of TAC with both DG4.0 and DG4.5 is a novel therapy since it was less-toxic, was more biocompatible, and has the same effectiveness than the free drug.Instituto Multidisciplinario de Biología Celula