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1.
J Infect Dis ; 230(3): 614-623, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-38537273

RESUMO

BACKGROUND: Bone infections with Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. METHODS: CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. RESULTS: CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9 of 12 and full eradication in 5 of 12 pigs. CONCLUSIONS: Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.


Assuntos
Antibacterianos , Clindamicina , Osteomielite , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Suínos , Clindamicina/administração & dosagem , Clindamicina/uso terapêutico , Ratos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Levofloxacino/administração & dosagem , Preparações de Ação Retardada , Feminino
2.
Microcirculation ; 27(7): e12643, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32542908

RESUMO

OBJECTIVE: The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. METHODS: In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high-pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high-pressure frozen and by transmission soft x-rays, imaged under cryogenic conditions. RESULTS: The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high-pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x-rays. In non-contrasted vessels, the glycocalyx was not resolved. LaDy-contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. CONCLUSIONS: Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x-ray tomography based solely on amplitude contrast seems unsuitable.


Assuntos
Criopreservação/métodos , Células Endoteliais/química , Células Endoteliais/ultraestrutura , Glicocálix/química , Glicocálix/ultraestrutura , Fixação de Tecidos/métodos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Células Cultivadas , Feminino , Substituição ao Congelamento/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Microvasos/citologia , Tomografia por Raios X
3.
Anal Bioanal Chem ; 412(24): 6371-6380, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32451643

RESUMO

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)-sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. Graphical abstract.


Assuntos
Antineoplásicos/administração & dosagem , Endocitose , Lipossomos , Oxaliplatina/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Espectroscopia Dielétrica , Humanos , Oxaliplatina/farmacologia
4.
Exp Cell Res ; 379(1): 73-82, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30922921

RESUMO

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.


Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular/fisiologia , Glioblastoma/patologia , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Estudos de Avaliação como Assunto , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Nestina/metabolismo , Vimentina/metabolismo
5.
Cell Mol Life Sci ; 74(13): 2467-2485, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28293718

RESUMO

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.


Assuntos
Encéfalo/citologia , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Eritropoetina/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/metabolismo , Transfecção/métodos , Animais , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células HeLa , Humanos , Imuno-Histoquímica , Mitose , Modelos Biológicos , Ratos Sprague-Dawley
6.
Nanomedicine ; 14(1): 27-34, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28882674

RESUMO

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.


Assuntos
Neoplasias Colorretais/metabolismo , Raios gama/uso terapêutico , Neoplasias de Cabeça e Pescoço/metabolismo , Lipossomos/farmacocinética , Animais , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/farmacocinética , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Hipóxia/metabolismo , Lipossomos/administração & dosagem , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Doses de Radiação , Distribuição Tecidual , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Biol Chem ; 291(38): 20220-31, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27502277

RESUMO

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium animalis/crescimento & desenvolvimento , Oligossacarídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Bifidobacterium animalis/genética , Humanos
8.
Eur J Nucl Med Mol Imaging ; 43(5): 941-952, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26646780

RESUMO

PURPOSE: The objective of this study was to evaluate the potential of PEGylated (64)Cu-liposomes in clinical diagnostic positron emission tomography (PET) imaging and PEGylated (177)Lu-liposomes in internal tumor radiotherapy through in vivo characterization and dosimetric analysis in a human xenograft mouse model. METHODS: Liposomes with 5 and 10 mol% PEG were characterized with respect to size, charge, and (64)Cu- and (177)Lu-loading efficiency. The tumor imaging potential of (64)Cu-loaded liposomes was evaluated in terms of in vivo biodistribution, tumor accumulation and tumor-to-muscle (T/M) ratios, using PET imaging. The potential of PEGylated liposomes for diagnostic and therapeutic applications was further evaluated through dosimetry analysis using OLINDA/EXM software. The (64)Cu-liposomes were used as biological surrogates to estimate the organ and tumor kinetics of (177)Lu-liposomes. RESULTS: High remote loading efficiency (>95 %) was obtained for both (64)Cu and (177)Lu radionuclides with PEGylated liposomes, and essentially no leakage of the encapsulated radionuclide was observed upon storage and after serum incubation for 24 h at 37 °C. The 10 mol% PEG liposomes showed higher tumor accumulation (6.2 ± 0.2 %ID/g) than the 5 mol% PEG liposomes, as evaluated by PET imaging. The dosimetry analysis of the (64)Cu-liposomes estimated an acceptable total effective dose of 3.3·10(-2) mSv/MBq for diagnostic imaging in patients. A high absorbed tumor dose (114 mGy/MBq) was estimated for the potential radiotherapeutic (177)Lu-liposomes. CONCLUSION: The overall preclinical profile of PEGylated (64)Cu-liposomes showed high potential as a new PET theranostic tracer for imaging in humans. Dosimetry results predicted that initial administered activity of 200 MBq of (64)Cu-liposomes should be acceptable in patients. Work is in progress to validate the utility of PEGylated (64)Cu-liposomes in a clinical research programme. The high absorbed tumor dose (114 mGy/MBq) estimated for (177)Lu-liposomes and the preliminary dosimetric studies justify further therapeutic and dosimetry investigation of (177)Lu-liposomes in animals before potential testing in man.


Assuntos
Radioisótopos de Cobre/farmacocinética , Lipossomos/farmacocinética , Tumores Neuroendócrinos/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/farmacocinética , Animais , Linhagem Celular Tumoral , Radioisótopos de Cobre/administração & dosagem , Humanos , Lipossomos/química , Lutécio/administração & dosagem , Lutécio/farmacocinética , Lutécio/uso terapêutico , Camundongos , Camundongos Nus , Tumores Neuroendócrinos/radioterapia , Polietilenoglicóis/química , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/uso terapêutico , Distribuição Tecidual
9.
J Biomol NMR ; 61(3-4): 347-59, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25556885

RESUMO

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors' function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static (15)N NMR spectra and quantitative determination of (1)H-(13)C order parameters through measurement of the (1)H-(13)C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) obtained in directly polarized (13)C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.


Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Receptores de Neuropeptídeo Y/metabolismo , Membrana Celular/metabolismo , Ácidos Graxos Monoinsaturados , Humanos , Modelos Moleculares , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfolipídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Neuropeptídeo Y/genética
10.
Angew Chem Int Ed Engl ; 54(25): 7446-9, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25924821

RESUMO

Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.


Assuntos
Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Neuropeptídeo Y/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Receptores de Neuropeptídeo Y/química
11.
Biochim Biophys Acta ; 1828(8): 1822-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23583923

RESUMO

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.


Assuntos
Colesterol/análogos & derivados , Ciclodextrinas/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Células CHO , Colesterol/metabolismo , Cricetinae , Ciclodextrinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Fluorescência , Esteróis/química
12.
Chemistry ; 20(17): 4986-92, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24623336

RESUMO

In spite of the recent success in crystallizing several G-protein-coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid-state NMR spectroscopy. Static (15) N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional (1) H,(13) C order parameter of the CH, CH2 , and CH3 groups. In directly polarized (13) C NMR experiments, these order parameters showed astonishingly low values of SCH =0.55, S CH 2=0.33, and S CH 3=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50-60° in the side chain. At physiological temperature, (2) H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α-helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid-crystalline membrane at physiological temperature.


Assuntos
Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Receptores de Neuropeptídeo Y/química , Humanos , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
13.
Phys Chem Chem Phys ; 16(16): 7461-71, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24626742

RESUMO

A series of peptide mutants was studied to understand the influence of local physical interactions on the fibril formation mechanism of amyloid ß (Aß)(1-40). In the peptide variants, the well-known hydrophobic contact between residues phenylalanine 19 and leucine 34 was rationally modified. In single site mutations, residue phenylalanine 19 was replaced by amino acids that introduce higher structural flexibility by a glycine mutation or restrict the backbone flexibility by introduction of proline. Next, the aromatic phenylalanine was replaced by tyrosine or tryptophan, respectively, to probe the influence of additional hydrogen bond forming capacity in the fibril interior. Furthermore, negatively charged glutamate or positively charged lysine was introduced to probe the influence of electrostatics. In double mutants, the hydrophobic contact was replaced by a putative salt bridge (glutamate and lysine) or two electrostatically repelling lysine residues. The influence of these mutations on the fibrillation kinetics and morphology, cross-ß structure as well as the local structure and dynamics was probed using fluorescence, transmission electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. While the fibrillation kinetics and the local structure and dynamics of the peptide variants were influenced by the introduction of these local fields, the overall morphology and cross-ß structure of the fibrils remained very robust against all the probed interactions. Overall, 7 out of the 8 mutated peptides formed fibrils of very similar morphology compared to the wildtype. However, characteristic local structural and dynamical changes indicate that amyloid fibrils show an astonishing ability to respond to local perturbations but overall show a very homogenous mesoscopic organization.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/síntese química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Microscopia Eletrônica de Transmissão , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/síntese química , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Difração de Raios X
14.
Int J Pharm ; 654: 123957, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38430950

RESUMO

The oral bioavailability of therapeutic peptides is generally low. To increase peptide transport across the gastrointestinal barrier, permeation enhancers are often used. Despite their widespread use, mechanistic knowledge of permeation enhancers is limited. To address this, we here investigate the interactions of six commonly used permeation enhancers with lipid membranes in simulated intestinal environments. Specifically, we study the interactions of the permeation enhancers sodium caprate, dodecyl maltoside, sodium cholate, sodium dodecyl sulfate, melittin, and penetratin with epithelial cell-like model membranes. To mimic the molecular composition of the real intestinal environment, the experiments are performed with two peptide drugs, salmon calcitonin and desB30 insulin, in fasted-state simulated intestinal fluid. Besides providing a comparison of the membrane interactions of the studied permeation enhancers, our results demonstrate that peptide drugs as well as intestinal-fluid components may substantially change the membrane activity of permeation enhancers. This highlights the importance of testing permeation enhancement in realistic physiological environments and carefully choosing a permeation enhancer for each individual peptide drug.


Assuntos
Absorção Intestinal , Mucosa Intestinal , Humanos , Mucosa Intestinal/metabolismo , Células CACO-2 , Absorção Intestinal/fisiologia , Transporte Biológico , Lipídeos , Permeabilidade
15.
J Control Release ; 371: 288-297, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38705519

RESUMO

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.


Assuntos
Antineoplásicos , Lipossomos , Neoplasias Ovarianas , Oxaliplatina , Polietilenoglicóis , Receptor ErbB-2 , Trastuzumab , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Animais , Humanos , Receptor ErbB-2/metabolismo , Receptor ErbB-2/imunologia , Oxaliplatina/administração & dosagem , Linhagem Celular Tumoral , Polietilenoglicóis/química , Polietilenoglicóis/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/química , Trastuzumab/administração & dosagem , Trastuzumab/química , Camundongos Nus , Sistemas de Liberação de Medicamentos , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
16.
Biochim Biophys Acta ; 1818(9): 2302-13, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22521809

RESUMO

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31-32 amino acid repeat regions exhibits both helical and ß-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively (13)C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a ß-structure. In addition, motionally averaged (1)H-(13)C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain (2)H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.


Assuntos
Bicamadas Lipídicas/metabolismo , Fosfolipídeos/química , Proteínas tau/química , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Benzotiazóis , Biofísica/métodos , Dicroísmo Circular , Humanos , Lipídeos/química , Lipossomos/química , Lisina/química , Espectroscopia de Ressonância Magnética/métodos , Metilação , Dados de Sequência Molecular , Fosfatidilcolinas/química , Fosfatidilserinas/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Tiazóis/química , Lipossomas Unilamelares/química
17.
Glycobiology ; 23(11): 1260-9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23982278

RESUMO

Although the interaction between interleukin-8 (IL-8) and glycosaminoglycans (GAGs) is crucial for the mediation of inflammatory effects, little is known about the site specificity of this interaction. Therefore, we studied complexes of IL-8 and heparin (HEP) as well as other GAGs in a multidisciplinary approach, involving site-directed mutagenesis, mass spectrometry, fluorescence and solution NMR spectroscopy as well as computer modeling. The interaction between GAG and IL-8 is largely driven by the amine groups of the lysine and the guanidinium groups of arginine side chains. However, due to fast exchange with the solvent, it is typically not possible to detect NMR signals of those groups. Here, we applied reductive (13)C-methylation of the lysine side chains providing sensitive NMR probes for monitoring directly the sites of GAG interaction in (1)H-(13)C correlation experiments. We focused on the lysine side chains K25, K28, K59, K69 and K72 of IL-8 (1-77), which were reported to be involved in the binding to GAGs. The NMR signals of these residues were assigned in (1)H-(13)C HSQC spectra through the help of site-directed mutagenesis. NMR and fluorescence titration experiments in combination with molecular docking and molecular dynamics simulations were applied to investigate the involvement of each lysine in the binding with HEP and various GAG hexasaccharides. We identified K25, K69 and K72 to be the most relevant binding anchors of IL-8(1-77) for the analyzed GAGs.


Assuntos
Heparina/química , Interleucina-8/química , Lisina/química , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Glicosaminoglicanos/química , Interleucina-8/genética , Interleucina-8/fisiologia , Espectroscopia de Ressonância Magnética , Metilação , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Transdução de Sinais , Soluções
18.
Biol Chem ; 394(8): 1045-56, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23732681

RESUMO

In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2. We identified the free cysteines in the receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cysteine residues were mutated to alanine or serine without any significant loss of functionality of the receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cysteines was irreversibly formed by applying oxidative in vitro folding. Applying this strategy, the stability of the functionally folded Y2 receptor could be increased to 20 days at a concentration of 15 µm in a micelle environment consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine and n-dodecyl-ß-D-maltoside.


Assuntos
Cisteína/química , Dobramento de Proteína , Receptores de Neuropeptídeo Y/química , Clonagem Molecular , Cisteína/genética , Escherichia coli/genética , Humanos , Modelos Moleculares , Oxirredução , Mutação Puntual , Estabilidade Proteica , Receptores de Neuropeptídeo Y/genética
19.
Angew Chem Int Ed Engl ; 52(49): 12848-51, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24382636

RESUMO

The influence of cholesterol's alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterol's side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Membrana Celular/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Relação Estrutura-Atividade
20.
Int J Biol Macromol ; 235: 123658, 2023 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-36822285

RESUMO

The growing interest in biopharmaceuticals combined with the challenges regarding formulation and delivery continues to encourage the development of new and improved formulations of this class of therapeutics. Nanoclusters (NCs) represent a type of formulation strategy where the biopharmaceutical is clustered in a reversible manner to function as both the therapeutic and the vehicle. In this study, insulin NCs (INCs) were formulated by a new methodology of first crosslinking proteins followed by desolvation. Crosslinking of the protein with the reducible DTSSP crosslinker improved control of the INC synthesis process to give INCs with a mean size of 198 ± 7 nm and a mean zeta potential of -39 ± 1 mV. Crosslinking and clustering of insulin did not induce cytotoxicity or major differences in the biological activity compared to the free unmodified protein. The potency of the crosslinked insulin and the INCs appeared slightly lower than that of the unmodified protein, and significantly higher doses of the INCs compared to the free protein were applied to achieve similar blood sugar lowering effects in vivo. Interestingly, the INCs allowed for high doses to be subcutaneously delivered with prolonged efficacy without being lethal in rats.


Assuntos
Insulina , Proteínas , Ratos , Animais , Preparações de Ação Retardada/farmacologia , Excipientes
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