RESUMO
Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.
Assuntos
Barreira Hematoencefálica , Modelos Animais de Doenças , AVC Isquêmico , Lipossomos , Nanopartículas , Molécula 1 de Adesão de Célula Vascular , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Animais , Camundongos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Nanopartículas/química , AVC Isquêmico/metabolismo , AVC Isquêmico/tratamento farmacológico , Lipídeos/química , Sistemas de Liberação de Medicamentos/métodos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , HumanosRESUMO
Two camps have emerged for targeting nanoparticles to specific organs and cell types: affinity moiety targeting and physicochemical tropism. Here we directly compare and combine both using intravenous (IV) lipid nanoparticles (LNPs) designed to target the lungs. We utilized PECAM antibodies as affinity moieties and cationic lipids for physicochemical tropism. These methods yield nearly identical lung uptake, but aPECAM LNPs show higher endothelial specificity. LNPs combining these targeting methods had >2-fold higher lung uptake than either method alone and markedly enhanced epithelial uptake. To determine if lung uptake is because the lungs are the first organ downstream of IV injection, we compared IV vs intra-arterial (IA) injection into the carotid artery, finding that IA combined-targeting LNPs achieve 35% of the injected dose per gram (%ID/g) in the first-pass organ, the brain, among the highest reported. Thus, combining the affinity moiety and physicochemical strategies provides benefits that neither targeting method achieves alone.
RESUMO
Metal-oxide nanoparticles (MO-NPs), such as the highly bioreactive copper-based nanoparticles (CuO-NPs), are widely used in manufacturing of hundreds of commercial products. Epidemiological studies correlated levels of nanoparticles in ambient air with a significant increase in lung disease. CuO-NPs, specifically, were among the most potent in a set of metal-oxides and carbons studied in parallel regarding DNA damage and cytotoxicity. Despite advances in nanotoxicology research and the characterization of their toxicity, the exact mechanism(s) of toxicity are yet to be defined. We identified chlorination toxicity as a damaging consequence of inflammation and myeloperoxidase (MPO) activation, resulting in macromolecular damage and cell damage/death. We hypothesized that the inhalation of CuO-NPs elicits an inflammatory response resulting in chlorination damage in cells and lung tissues. We further tested the protective action of LGM2605, a synthetic small molecule with known scavenging properties for reactive oxygen species (ROS), but most importantly, for active chlorine species (ACS) and an inhibitor of MPO. CuO-NPs (15 µg/bolus) were instilled intranasally in mice and the kinetics of the inflammatory response in lungs was evaluated 1, 3, and 7 days later. Evaluation of the protective action of LGM2605 was performed at 24 h post-challenge, which was selected as the peak acute inflammatory response to CuO-NP. LGM2605 was given daily via gavage to mice starting 2 days prior to the time of the insult (100 mg/kg). CuO-NPs induced a significant inflammatory influx, inflammasome-relevant cytokine release, and chlorination damage in mouse lungs, which was mitigated by the action of LGM2605. Preventive action of LGM2605 ameliorated the adverse effects of CuO-NP in lung.
Assuntos
Butileno Glicóis/farmacologia , Glucosídeos/farmacologia , Inflamação/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar/citologia , Butileno Glicóis/metabolismo , Cloro/metabolismo , Cobre/metabolismo , Cobre/toxicidade , Dano ao DNA/efeitos dos fármacos , Feminino , Glucosídeos/metabolismo , Inflamassomos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Óxidos/farmacologia , Peroxidase/farmacologia , Espécies Reativas de Oxigênio/farmacologiaRESUMO
Malignant mesothelioma (MM), linked to asbestos exposure, is a highly lethal form of thoracic cancer with a long latency period, high mortality and poor treatment options. Chronic inflammation and oxidative tissue damage caused by asbestos fibers are linked to MM development. Flaxseed lignans, enriched in secoisolariciresinol diglucoside (SDG), have antioxidant, anti-inflammatory and cancer chemopreventive properties. As a prelude to chronic chemoprevention studies for MM development, we tested the ability of flaxseed lignan component (FLC) to prevent acute asbestos-induced inflammation in MM-prone Nf2(+/mu) mice. Mice (n = 16-17 per group) were placed on control (CTL) or FLC-supplemented diets initiated 7 days prior to a single intraperitoneal bolus of 400 µg of crocidolite asbestos. Three days post asbestos exposure, mice were evaluated for abdominal inflammation, proinflammatory/profibrogenic cytokine release, WBC gene expression changes and oxidative and nitrosative stress in peritoneal lavage fluid (PLF). Asbestos-exposed mice fed CTL diet developed acute inflammation, with significant (P < 0.0001) elevations in WBCs and proinflammatory/profibrogenic cytokines (IL-1ß, IL-6, TNFα, HMGB1 and active TGFß1) relative to baseline (BL) levels. Alternatively, asbestos-exposed FLC-fed mice had a significant (P < 0.0001) decrease in PLF WBCs and proinflammatory/profibrogenic cytokine levels relative to CTL-fed mice. Importantly, PLF WBC gene expression of cytokines (IL-1ß, IL-6, TNFα, HMGB1 and TGFß1) and cytokine receptors (TNFαR1 and TGFßR1) were also downregulated by FLC. FLC also significantly (P < 0.0001) blunted asbestos-induced nitrosative and oxidative stress. FLC reduces acute asbestos-induced peritoneal inflammation, nitrosative and oxidative stress and may thus prove to be a promising agent in the chemoprevention of MM.
Assuntos
Asbesto Crocidolita/toxicidade , Butileno Glicóis/administração & dosagem , Glucosídeos/administração & dosagem , Inflamação/patologia , Lignanas/administração & dosagem , Peritônio/patologia , Animais , Antioxidantes/farmacologia , Cromatografia Líquida , Dieta , Suplementos Nutricionais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Linho , Mesotelioma/patologia , Camundongos , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Lavagem Peritoneal , Peritônio/efeitos dos fármacos , Lesões Pré-Cancerosas/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and â¼4-5 Ab (or â¼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.
Assuntos
Endotélio Vascular/metabolismo , Ferritinas/química , Nanopartículas , Animais , Microscopia Eletrônica de TransmissãoRESUMO
Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.
Assuntos
Dano ao DNA , Hiperóxia , Modelos Biológicos , Estresse Oxidativo , Radiação Ionizante , Voo Espacial , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/efeitos da radiação , Animais , Antioxidantes/metabolismo , Apoptose/genética , Apoptose/efeitos da radiação , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Regulação Enzimológica da Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions.
Assuntos
Radiação Cósmica/efeitos adversos , Lesão Pulmonar/etiologia , Animais , Apoptose , Autofagia , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar , Proliferação de Células , Modelos Animais de Doenças , Hipóxia/sangue , Hipóxia/complicações , Hipóxia/patologia , Lesão Pulmonar/sangue , Lesão Pulmonar/patologia , Masculino , Camundongos Endogâmicos C3H , Estresse Oxidativo , Oxigênio/sangue , Pneumonia/sangue , Pneumonia/complicações , Pneumonia/patologia , Transdução de SinaisRESUMO
Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed's protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.
Assuntos
Antioxidantes/farmacologia , Butileno Glicóis/farmacologia , Dano ao DNA , Células Endoteliais/efeitos dos fármacos , Linho/química , Raios gama , Glucosídeos/farmacologia , Pulmão/citologia , Extratos Vegetais/farmacologia , Animais , Morte Celular , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismoRESUMO
Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has yet to achieve this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs' numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils' uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels.
Assuntos
Pulmão , Nanopartículas , Neutrófilos , Animais , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Nanopartículas/química , Sistema Fagocitário Mononuclear/metabolismo , Endotélio/metabolismo , Camundongos Endogâmicos C57BL , NanomedicinaRESUMO
BACKGROUND: Wholegrain flaxseed (FS), and its lignan component (FLC) consisting mainly of secoisolariciresinol diglucoside (SDG), have potent lung radioprotective properties while not abrogating the efficacy of radiotherapy. However, while the whole grain was recently shown to also have potent mitigating properties in a thoracic radiation pneumonopathy model, the bioactive component in the grain responsible for the mitigation of lung damage was never identified. Lungs may be exposed to radiation therapeutically for thoracic malignancies or incidentally following detonation of a radiological dispersion device. This could potentially lead to pulmonary inflammation, oxidative tissue injury, and fibrosis. This study aimed to evaluate the radiation mitigating effects of FLC in a mouse model of radiation pneumonopathy. METHODS: We evaluated FLC-supplemented diets containing SDG lignan levels comparable to those in 10% and 20% whole grain diets. 10% or 20% FLC diets as compared to an isocaloric control diet (0% FLC) were given to mice (C57/BL6) (n=15-30 mice/group) at 24, 48, or 72-hours after single-dose (13.5 Gy) thoracic x-ray treatment (XRT). Mice were evaluated 4 months post-XRT for blood oxygenation, lung inflammation, fibrosis, cytokine and oxidative damage levels, and survival. RESULTS: FLC significantly mitigated radiation-related animal death. Specifically, mice fed 0% FLC demonstrated 36.7% survival 4 months post-XRT compared to 60-73.3% survival in mice fed 10%-20% FLC initiated 24-72 hours post-XRT. FLC also mitigated radiation-induced lung fibrosis whereby 10% FLC initiated 24-hours post-XRT significantly decreased fibrosis as compared to mice fed control diet while the corresponding TGF-beta1 levels detected immunohistochemically were also decreased. Additionally, 10-20% FLC initiated at any time point post radiation exposure, mitigated radiation-induced lung injury evidenced by decreased bronchoalveolar lavage (BAL) protein and inflammatory cytokine/chemokine release at 16 weeks post-XRT. Importantly, neutrophilic and overall inflammatory cell infiltrate in airways and levels of nitrotyrosine and malondialdehyde (protein and lipid oxidation, respectively) were also mitigated by the lignan diet. CONCLUSIONS: Dietary FLC given early post-XRT mitigated radiation effects by decreasing inflammation, lung injury and eventual fibrosis while improving survival. FLC may be a useful agent, mitigating adverse effects of radiation in individuals exposed to incidental radiation, inhaled radioisotopes or even after the initiation of radiation therapy to treat malignancy.
Assuntos
Butileno Glicóis/administração & dosagem , Citocinas/metabolismo , Linho , Glucosídeos/administração & dosagem , Lesão Pulmonar/prevenção & controle , Fitoterapia , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Sementes , Ração Animal , Animais , Líquido da Lavagem Broncoalveolar , Feminino , Fibrose/etiologia , Fibrose/prevenção & controle , Estimativa de Kaplan-Meier , Lignanas/administração & dosagem , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos da radiação , Lesão Pulmonar/complicações , Lesão Pulmonar/metabolismo , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Oxigênio/sangue , Lesões Experimentais por Radiação/complicações , Lesões Experimentais por Radiação/metabolismo , Pneumonite por Radiação/etiologia , Pneumonite por Radiação/patologia , Taxa de Sobrevida , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS. Graphical abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.
RESUMO
BACKGROUND: Flaxseed (FS), a nutritional supplement consisting mainly of omega-3 fatty acids and lignan phenolics has potent anti-inflammatory, anti-fibrotic and antioxidant properties. The usefulness of flaxseed as an alternative and complimentary treatment option has been known since ancient times. We have shown that dietary FS supplementation ameliorates oxidative stress and inflammation in experimental models of acute and chronic lung injury in mice resulting from diverse toxicants. The development of lung tissue damage in response to direct or indirect oxidant stress is a complex process, associated with changes in expression levels of a number of genes. We therefore postulated that flaxseed might modulate gene expression of vital signaling pathways, thus interfering with the development of tissue injury. METHODS: We evaluated gene expression in lungs of flaxseed-fed (10%FS) mice under unchallenged, control conditions. We reasoned that array technology would provide a powerful tool for studying the mechanisms behind this response and aid the evaluation of dietary flaxseed intervention with a focus on toxicologically relevant molecular gene targets. Gene expression levels in lung tissues were analyzed using a large-scale array whereby 28,800 genes were evaluated. RESULTS: 3,713 genes (12.8%) were significantly (p < 0.05) differentially expressed, of which 2,088 had a >1.5-fold change. Genes affected by FS include those in protective pathways such as Phase I and Phase II. CONCLUSIONS: The array studies have provided information on how FS modulates gene expression in lung and how they might be related to protective mechanisms. In addition, our study has confirmed that flaxseed is a nutritional supplement with potentially useful therapeutic applications in complementary and alternative (CAM) medicine especially in relation to treatment of lung disease.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Linho/química , Pneumopatias , Pulmão/efeitos dos fármacos , Preparações de Plantas/farmacologia , Transcriptoma , Animais , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/uso terapêutico , Perfilação da Expressão Gênica/métodos , Lignanas/farmacologia , Lignanas/uso terapêutico , Pulmão/metabolismo , Pneumopatias/tratamento farmacológico , Pneumopatias/genética , Pneumopatias/metabolismo , Camundongos , Fenóis/farmacologia , Fenóis/uso terapêutico , Fitoterapia , Preparações de Plantas/uso terapêutico , Sementes/química , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Flaxseed (FS) is a dietary supplement known for its antioxidant and anti-inflammatory properties. Radiation exposure of lung tissues occurs either when given therapeutically to treat intrathoracic malignancies or incidentally, such as in the case of exposure from inhaled radioisotopes released after the detonation of a radiological dispersion devise (RDD). Such exposure is associated with pulmonary inflammation, oxidative tissue damage and irreversible lung fibrosis. We previously reported that dietary FS prevents pneumonopathy in a rodent model of thoracic X-ray radiation therapy (XRT). However, flaxseed's therapeutic usefulness in mitigating radiation effects post-exposure has never been evaluated. METHODS: We evaluated the effects of a 10%FS or isocaloric control diet given to mice (C57/BL6) in 2 separate experiments (n = 15-25 mice/group) on 0, 2, 4, 6 weeks post a single dose 13.5 Gy thoracic XRT and compared it to an established radiation-protective diet given preventively, starting at 3 weeks prior to XRT. Lungs were evaluated four months post-XRT for blood oxygenation levels, inflammation and fibrosis. RESULTS: Irradiated mice fed a 0%FS diet had a 4-month survival rate of 40% as compared to 70-88% survival in irradiated FS-fed mouse groups. Additionally, all irradiated FS-fed mice had decreased fibrosis compared to those fed 0%FS. Lung OH-Proline content ranged from 96.5 ± 7.1 to 110.2 ± 7.7 µg/ml (Mean ± SEM) in all irradiated FS-fed mouse groups, as compared to 138 ± 10.8 µg/ml for mice on 0%FS. Concomitantly, bronchoalveolar lavage (BAL) protein and weight loss associated with radiation cachexia was significantly decreased in all FS-fed groups. Inflammatory cell influx to lungs also decreased significantly except when FS diet was delayed by 4 and 6 weeks post XRT. All FS-fed mice (irradiated or not), maintained a higher blood oxygenation level as compared to mice on 0%FS. Similarly, multiplex cytokine analysis in the BAL fluid revealed a significant decrease of specific inflammatory cytokines in FS-fed mice. CONCLUSIONS: Dietary FS given post-XRT mitigates radiation effects by decreasing pulmonary fibrosis, inflammation, cytokine secretion and lung damage while enhancing mouse survival. Dietary supplementation of FS may be a useful adjuvant treatment mitigating adverse effects of radiation in individuals exposed to inhaled radioisotopes or incidental radiation.
Assuntos
Linho , Pulmão/efeitos da radiação , Fitoterapia , Preparações de Plantas/uso terapêutico , Pneumonite por Radiação/dietoterapia , Protetores contra Radiação/uso terapêutico , Radioterapia/efeitos adversos , Sementes , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Antioxidantes/administração & dosagem , Antioxidantes/uso terapêutico , Líquido da Lavagem Broncoalveolar/química , Caquexia/dietoterapia , Caquexia/etiologia , Caquexia/prevenção & controle , Citocinas/análise , Dieta , Avaliação Pré-Clínica de Medicamentos , Feminino , Lignanas/sangue , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Oxigênio/sangue , Preparações de Plantas/administração & dosagem , Fibrose Pulmonar/dietoterapia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/prevenção & controle , Pneumonite por Radiação/etiologia , Pneumonite por Radiação/prevenção & controle , Protetores contra Radiação/administração & dosagem , Distribuição Aleatória , Redução de PesoRESUMO
Background: Thoracic radiotherapy is complicated by acute radiation-induced adverse events such as radiation pneumonitis (RP) and radiation esophagitis (RE). Based on preclinical work and a randomized pilot trial from our laboratory, this single-arm phase II trial investigated administering flaxseed as a radioprotector in patients receiving definitive chemoradiation for nonsmall cell lung cancer (NSCLC). Methods: Between June 2015 and February 2018, 33 patients with locally advanced or metastatic NSCLC with planned definitive chemoradiation were enrolled. Finely-ground Linum usitatissimum L. (Linaceae; flaxseed or linseed) in 40-g packets were provided for daily consumption in any patient-desired formulation 1 week before radiotherapy and throughout radiotherapy as tolerated. The primary outcomes were overall adverse events, with particular focus on Grade ≥3 RP, and flaxseed tolerability. Adverse events were graded according to CTCAE v4.0. Results: Of the 33 patients enrolled, 5 patients (15%) did not receive chemoradiation, 4 (12%) withdrew promptly after enrollment, 4 (12%) did not return a flaxseed consumption log, and 1 patient had irritable bowel syndrome (3%). The remaining 19 patients (57%) had chemoradiation and flaxseed ingestion with a mean completion and standard deviation of the intended flaxseed course of 62% ± 8.3%. Nine (50%) of these 19 patients reported difficulties with flaxseed consumption, citing nausea, constipation, odynophagia, or poor taste or texture. One patient (5%), with unverifiable flaxseed consumption, developed Grade 3 RP. There were no cases of Grade 2 RP. Six patients (32%) developed Grade 2 RE, but no patients developed Grade ≥3 RE. Median overall and progression-free survival were 31 and 12 months, respectively. Conclusions: Despite the low incidence of acute radiation-induced complications reported, significant treatment-related gastrointestinal toxicities and subsequently low flaxseed tolerability inhibit accurate determination of flaxseed effect in patients receiving concurrent thoracic chemoradiation. Thus, further investigations should focus on optimizing flaxseed formulation for improved tolerability and evaluation. ClinicalTrials.gov ID: NCT02475330.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Linho , Neoplasias Pulmonares , Lesões por Radiação , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quimiorradioterapia , Terapia Combinada , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapiaRESUMO
The therapeutic efficacy of adoptive transfer of T cells transduced with chimeric antigen receptors (CARs) has been limited in the treatment of solid cancers, partly due to tumor antigen heterogeneity. Overcoming lack of universal tumor antigen expression would be achieved if CAR T cells could induce bystander effects. To study this process, we developed a system where CAR T cells targeting mesothelin could cure tumors containing 100% antigen-positive cells in immunocompetent mice. Using this model, we found that the CAR T cells were unable to cure tumors, even when only 10% of the tumor cells were mesothelin negative. A bystander effect was not induced by co-administration of anti-PD-1, anti-CTLA-4, or anti-TGF-ß (transforming growth factor ß) antibodies; agonistic CD40 antibodies; or an IDO (indoleamine 2,3-dioxygenase) inhibitor. However, pretreatment with a non-lymphodepleting dose of cyclophosphamide (CTX) prior to CAR T cells resulted in cures of tumors with up to 25% mesothelin-negative cells. The mechanism was dependent on endogenous CD8 T cells but not on basic leucine zipper transcription factor ATF-like 3 (BATF3)-dependent dendritic cells. These data suggest that CAR T cell therapy of solid tumors, in which the targeted antigen is not expressed by the vast majority of tumor cells, will not likely be successful unless combination strategies to enhance bystander effects are used.
RESUMO
Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H(2)O(2), respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H(2)O(2) produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H(2)O(2) in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.
Assuntos
Anticorpos/farmacologia , Antioxidantes/farmacologia , Catalase/farmacologia , Endotélio Vascular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Superóxido Dismutase/farmacologia , Angiotensina II/farmacologia , Animais , Anticorpos/metabolismo , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Biopterinas/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Catalase/administração & dosagem , Reagentes de Ligações Cruzadas , Glucose Oxidase/farmacologia , Peróxido de Hidrogênio/metabolismo , Pneumopatias/induzido quimicamente , Pneumopatias/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Circulação Pulmonar/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Superóxido Dismutase/administração & dosagem , Superóxidos/metabolismoRESUMO
Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2-/-) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm²) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1ß (IL-1ß), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2-/- macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.
RESUMO
Inflammatory mediators binding to Toll-Like receptors (TLR) induce an influx of superoxide anion in the ensuing endosomes. In endothelial cells, endosomal surplus of superoxide causes pro-inflammatory activation and TLR4 agonists act preferentially via caveolae-derived endosomes. To test the hypothesis that SOD delivery to caveolae may specifically inhibit this pathological pathway, we conjugated SOD with antibodies (Ab/SOD, size ~10nm) to plasmalemmal vesicle-associated protein (Plvap) that is specifically localized to endothelial caveolae in vivo and compared its effects to non-caveolar target CD31/PECAM-1. Plvap Ab/SOD bound to endothelial cells in culture with much lower efficacy than CD31 Ab/SOD, yet blocked the effects of LPS signaling with higher efficiency than CD31 Ab/SOD. Disruption of cholesterol-rich membrane domains by filipin inhibits Plvap Ab/SOD endocytosis and LPS signaling, implicating the caveolae-dependent pathway(s) in both processes. Both Ab/SOD conjugates targeted to Plvap and CD31 accumulated in the lungs after IV injection in mice, but the former more profoundly inhibited LPS-induced pulmonary inflammation and elevation of plasma level of interferon-beta and -gamma and interleukin-27. Taken together, these results indicate that targeted delivery of SOD to specific cellular compartments may offer effective, mechanistically precise interception of pro-inflammatory signaling mediated by reactive oxygen species.
Assuntos
Anti-Inflamatórios/administração & dosagem , Anticorpos/administração & dosagem , Proteínas de Transporte/imunologia , Proteínas de Membrana/imunologia , Superóxido Dismutase/administração & dosagem , Animais , Cavéolas/metabolismo , Células Cultivadas , Citocinas/sangue , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BLRESUMO
One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50â¯nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20â¯nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics.
Assuntos
Cavéolas/metabolismo , Portadores de Fármacos/metabolismo , Ferritinas/metabolismo , Nanopartículas/metabolismo , Superóxido Dismutase/administração & dosagem , Animais , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Imunoconjugados/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Superóxido Dismutase/farmacocinéticaRESUMO
Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.