RESUMO
The synthetic compound dendritic polyglycerol sulfate (dPGS) is a pleiotropic acting molecule but shows a high binding affinity to immunological active molecules as L-/P-selectin or complement proteins leading to well described anti-inflammatory properties in various mouse models. In order to make a comprehensive evaluation of the direct effect on the innate immune system, macrophage polarization is analyzed in the presence of dPGS on a phenotypic but also metabolic level. dPGS administered macrophages show a significant increase of MCP1 production paralleled by a reduction of IL-10 secretion. Metabolic analysis reveals that dPGS could potently enhance the glycolysis and mitochondrial respiration in M0 macrophages as well as decrease the mitochondrial respiration of M2 macrophages. In summary the data indicate that dPGS polarizes macrophages into a pro-inflammatory phenotype in a metabolic pathway-dependent manner.
Assuntos
Dendrímeros/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Dendrímeros/síntese química , Regulação da Expressão Gênica/imunologia , Glicerol/química , Glicólise/genética , Imunidade Inata , Imunofenotipagem , Interleucina-10/genética , Interleucina-10/imunologia , Lectinas/genética , Lectinas/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Fenótipo , Polímeros/química , Cultura Primária de Células , Piridinas/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/imunologiaRESUMO
A thorough understanding of nanoparticle bio-distribution at the feto-maternal interface will be a prerequisite for their diagnostic or therapeutic application in women of childbearing age and for teratologic risk assessment. Therefore, the tissue interaction of biocompatible dendritic polyglycerol nanoparticles (dPG-NPs) with first- trimester human placental explants were analyzed and compared to less sophisticated trophoblast-cell based models. First-trimester human placental explants, BeWo cells and primary trophoblast cells from human term placenta were exposed to fluorescence labeled, â¼5 nm dPG-NPs, with differently charged surfaces, at concentrations of 1 µM and 10 nM, for 6 and 24 h. Accumulation of dPGs was visualized by fluorescence microscopy. To assess the impact of dPG-NP on trophoblast integrity and endocrine function, LDH, and hCG releases were measured. A dose- and charge-dependent accumulation of dPG-NPs was observed at the early placental barrier and in cell lines, with positive dPG-NP-surface causing deposits even in the mesenchymal core of the placental villi. No signs of plasma membrane damage could be detected. After 24 h we observed a significant reduction of hCG secretion in placental explants, without significant changes in trophoblast apoptosis, at low concentrations of charged dPG-NPs. In conclusion, dPG-NP's surface charge substantially influences their bio-distribution at the feto-maternal interface, with positive charge facilitating trans-trophoblast passage, and in contrast to more artificial models, the first-trimester placental explant culture model reveals potentially hazardous influences of charged dPG-NPs on early placental physiology.
Assuntos
Gonadotropina Coriônica/metabolismo , Células Dendríticas/metabolismo , Glicerol/farmacologia , Glicerol/farmacocinética , Nanopartículas/química , Placenta/metabolismo , Polímeros/farmacologia , Polímeros/farmacocinética , Apoptose , Disponibilidade Biológica , Células Cultivadas , Feminino , Glicerol/química , Humanos , Polímeros/química , Gravidez , Primeiro Trimestre da Gravidez , Propriedades de Superfície , Trofoblastos/metabolismoRESUMO
Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-ß (Aß42) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aß42 peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aß42, i.e., a direct interaction between dPGS and Aß42 species interfered with Aß fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.
Assuntos
Dendrímeros/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Glicerol/análogos & derivados , Glicerol/farmacologia , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Sinapses/efeitos dos fármacos , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Lipocalina-2/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neuroglia/metabolismo , Neuroglia/patologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Ressonância de Plasmônio de Superfície , Sinapses/metabolismo , Sinapses/patologia , Técnicas de Cultura de TecidosRESUMO
Dendritic polyglycerols (dPG) are water soluble, polyether-based nanomaterials which hold great potential in diagnostic as well as therapeutic applications. In order to translate them for in vivo applications, a systematic assessment regarding their cell and tissue interactions as well as their metabolic fate in vivo is a crucial step. Herein, we explore the structure-activity relationship of three different sizes (ca. 3, 5, and 10 nm) of neutral dendritic polyglycerol (dPG) and their corresponding negatively charged sulfate analogs (dPGS) on their in vitro and in vivo characteristics. Cellular metabolic activity was studied in A431 and HEK293 cells. Biomolecular corona formation was determined using an electrophoretic mobility shift assay, which showed an increased protein binding of the dPGS even with serum concentrations as low as 20%. An in situ technique, microscale thermophoresis, was employed to address the binding affinities of these nanomaterials with serum proteins such as serum albumin, apo-transferrin, and fibrinogen. In addition, nanoparticle-cell interactions were studied in differentiated THP-1 cells which showed a charge dependent scavenger receptor-mediated uptake. In line with this data, detailed biodistribution and small animal PET imaging studies in Wistar rats using 68Ga-labeled dPG-/dPGS-NOTA conjugates showed that the neutral dPG-NOTA conjugates were quantitatively excreted via the kidneys with a subsequent hepatobiliary excretion with an increase in their size, whereas the polysulfated analogs (dPGS-NOTA) were sequestered preferentially in the liver and kidneys irrespective of their size. Taken together, this systematic study accentuates that the pharmacokinetics of dPGs is critically dependent on the overall size and charge and can be, fine-tuned for the intended requirements in nano-theranostics.
RESUMO
The destruction of articular cartilage is a critical feature in joint diseases. An approach to selectively target the damaged tissue is promising for the development of diagnostic and therapeutic agents. We herein present the interaction of dendritic polyglycerol (dPG) anions with native and inflamed cartilage. Confocal laser scanning microscopy revealed the inert character of dPG and low functionalized dPG bisphosphonate (dPGBP7%) toward cartilage in vitro. An enhanced binding was observed for highly functionalized dPG bisphosphonate, sulfate, and phosphate, which additionally showed a higher affinity to IL-1ß treated tissue. The mixed anion containing sulfate and bisphosphonate groups exhibited an exceptionally high affinity to cartilage and strongly bound to collagen type II, as shown by a normalized fluorescence-based binding assay. All polyglycerol anions, except dPGBP7%, were taken up by chondrocytes within 24 h and no cytotoxicity was found up to 10-5 M. In a rheumatoid arthritis model, dPGBP7% accumulated in mineralized compartments of inflamed joints and showed an increasing affinity to cartilage with higher clinical scores, as evident from histological examinations. For dPGS no interaction with bone but a strong binding to cartilage, independent of the score, was demonstrated. These results make dPG anions promising candidates for the selective targeting of cartilage tissue.
RESUMO
A new class of fully synthetic shell cleavable multivalent polysulfates is prepared by introducing degradable linkers into a stable biocompatible dendritic polyglycerol scaffold and subsequent sulfation. The sulfated polymers show different degradation profiles, low anticoagulant and high anti-inflammatory properties, are able to efficiently bind to L-selectin and inhibit the complement activation at very low concentrations in vitro.
RESUMO
Targeting bone with anionic macromolecules is a potent approach for the development of novel diagnostics and therapeutics for bone related diseases. A highly efficient modular synthesis of dendritic polyglycerol (dPG) polyanion dye conjugates, namely, sulfates, sulfonates, carboxylates, phosphates, phosphonates, and bisphosphonates via click chemistry is presented. By investigating the microarchitecture of stained bone sections with confocal laser scanning microscopy, the bisphosphonate, phosphonate, and phosphate functionalized polymers are identified as strongly penetrating compounds, whereas sulfates, sulfonates, and carboxylates reveal a weaker binding to hydroxyapatite (HA) but a more pronounced affinity toward collagen. In a quantitative HA binding assay, the affinity of the dPG sulfonate, sulfate, and carboxylate toward collagen and the exceptional high HA affinity of the phosphorous containing polyelectrolytes are validated. This shows the potential of dendritic polyphosphates and phosphonates as alternatives to the commonly employed bisphosphonate modification. In cytotoxicity studies with murine fibroblasts, the conjugates have no significant effect on the cell viability at 10(-5) m. All polyanions are taken up into the cells within 24 h. The presented synthetic approach allows versatile extensions for preparing conjugates for selective bone imaging applications, tissue engineering, and drug delivery.