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1.
Curr Protoc Stem Cell Biol ; 55(1): e122, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32956578

RESUMO

Translating basic biological knowledge into applications remains a key issue for effectively tackling neurodegenerative, neuroinflammatory, or neuroendocrine disorders. Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) still poses a demanding challenge for drug development targeting central nervous system diseases. Validated in vitro models of the BBB could facilitate effective testing of drug candidates targeting the brain early in the drug discovery process during lead generation. We here review the potential of mono- or (isogenic) co-culture BBB models based on brain capillary endothelial cells (BCECs) derived from human-induced pluripotent stem cells (hiPSCs), and compare them to several available BBB in vitro models from primary human or non-human cells and to rodent in vivo models, as well as to classical and widely used barrier models [Caco-2, parallel artificial membrane permeability assay (PAMPA)]. In particular, we are discussing the features and predictivity of these models and how hiPSC-derived BBB models could impact future discovery and development of novel CNS-targeting therapeutics. © 2020 The Authors.

2.
Chemistry ; 26(32): 7299-7308, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32358806

RESUMO

Alzheimer's disease (AD) is a neurological disorder with still no preventive or curative treatment. Flavonoids are phytochemicals with potential therapeutic value. Previous studies described the flavanone sterubin isolated from the Californian plant Eriodictyon californicum as a potent neuroprotectant in several in vitro assays. Herein, the resolution of synthetic racemic sterubin (1) into its two enantiomers, (R)-1 and (S)-1, is described, which has been performed on a chiral chromatographic phase, and their stereochemical assignment online by HPLC-ECD coupling. (R)-1 and (S)-1 showed comparable neuroprotection in vitro with no significant differences. While the pure stereoisomers were configurationally stable in methanol, fast racemization was observed in the presence of culture medium. We also established the occurrence of extracted sterubin as its pure (S)-enantiomer. Moreover, the activity of sterubin (1) was investigated for the first time in vivo, in an AD mouse model. Sterubin (1) showed a significant positive impact on short- and long-term memory at low dosages.


Assuntos
Eriodictyon/química , Flavanonas/química , Flavonoides/química , Luteolina/química , Fármacos Neuroprotetores/química , Animais , Cromatografia Líquida de Alta Pressão , Camundongos , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Estereoisomerismo
3.
Mol Pharm ; 17(6): 1835-1847, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32315193

RESUMO

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.

4.
Sci Rep ; 9(1): 12297, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444389

RESUMO

The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.

5.
Front Microbiol ; 10: 1181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191497

RESUMO

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.

6.
Macromol Biosci ; 18(11): e1800155, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30256527

RESUMO

A known limitation of polymer micelles for the formulation of hydrophobic drugs is their low loading capacity (LC), which rarely exceeds 20 wt%. One general strategy to overcome this limitation is to increase the amphiphilic contrast, that is, to make the hydrophobic core of the micelles more hydrophobic. However, in the case of poly(2-oxazoline) (POx)-based amphiphilic triblock copolymers, a minimal amphiphilic contrast was reported to be beneficial. Here, this subject is revisited in more detail using long hydrophobic side chains that are either linear (nonyl) or branched (3-ethylheptyl). Two different backbones within the hydrophobic block are investigated, in particular POx and poly(2-oxazine) (POzi), for the solubilization and co-solubilization of the two highly water insoluble compounds, curcumin and paclitaxel. Even though high loading capacities can be achieved for curcumin using POzi-based triblock copolymers, the solubilization capacity of all investigated polymers with longer side chains is significantly lower compared to POx and poly(2-oxazine)s with shorter side chains. Although the even lower LC for paclitaxel can be somehow improved by co-formulating curcumin, this study corroborates that in the case of POx and POzi-based polymer micelles, an increased amphiphilic contrast leads to less drug solubilization.


Assuntos
Curcumina , Fibroblastos/metabolismo , Oxazóis/química , Paclitaxel , Células Cultivadas , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacologia , Fibroblastos/citologia , Humanos , Paclitaxel/química , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Solubilidade
7.
Curr Protoc Stem Cell Biol ; 47(1): e62, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30261129

RESUMO

Human blood-brain barrier (BBB) in vitro models pose a promising tool in drug development and understanding of mechanistic regulations during health and disease. Human-induced pluripotent stem cells (hiPS cells) represent an unlimited cell source to generate functional cells of the neurovascular unit (NVU), independent of variations or limitations during isolation and in vitro cultivation. This unit describes the standardized 2-D differentiation of adherent hiPS cells into BBB endothelial cells and neuronal stem cells (NSCs). Both cell types are combined with primary astrocytes and pericytes to develop complex, physiological BBB in vitro models. The endothelial cells in the apical compartment of the transwell models are separated from the basolateral seeded co-culture mixture by a synthetic membrane, simplifying analyses. The barrier integrity and functionality of the endothelium is improved by the specific mixture of NVU niche cells, determined here by decrease in the paracellular permeability of sodium-fluorescein and transendothelial electrical resistance (TEER) measurement. © 2018 by John Wiley & Sons, Inc.


Assuntos
Barreira Hematoencefálica , Técnicas de Cocultura , Células-Tronco Pluripotentes Induzidas/citologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Modelos Biológicos , Neurônios/citologia
8.
Biomacromolecules ; 19(7): 3119-3128, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746117

RESUMO

Many natural compounds with interesting biomedical properties share one physicochemical property, namely, low water solubility. Polymer micelles are, among others, a popular means to solubilize hydrophobic compounds. The specific molecular interactions between the polymers and the hydrophobic drugs are diverse, and recently it has been discussed that macromolecular engineering can be used to optimize drug-loaded micelles. Specifically, π-π stacking between small molecules and polymers has been discussed as an important interaction that can be employed to increase drug loading and formulation stability. Here, we test this hypothesis using four different polymer amphiphiles with varying aromatic content and various natural products that also contain different relative amounts of aromatic moieties. In the case of paclitaxel, having the lowest relative content of aromatic moieties, the drug loading decreases with increasing relative aromatic amount in the polymer, whereas the drug loading of curcumin, having a much higher relative aromatic content, is increased. Interestingly, the loading using schizandrin A, a dibenzo[ a, c]cyclooctadiene lignan with intermediate relative aromatic content is not influenced significantly by the aromatic content of the polymers employed. The very high drug loading, long-term stability, ability to form stable highly loaded binary coformulations in different drug combinations, small-sized formulations, and amorphous structures in all cases corroborate earlier reports that poly(2-oxazoline)-based micelles exhibit an extraordinarily high drug loading and are promising candidates for further biomedical applications. The presented results underline that the interaction between the polymers and the incorporated small molecules may be more complex and are significantly influenced by both sides, the used carrier and drug, and must be investigated in each specific case.


Assuntos
Antineoplásicos Fitogênicos/química , Produtos Biológicos/química , Hidrocarbonetos Aromáticos/análise , Antineoplásicos Fitogênicos/toxicidade , Produtos Biológicos/toxicidade , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Oxazóis/química , Tensoativos/química
9.
Stem Cell Reports ; 8(4): 894-906, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28344002

RESUMO

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.


Assuntos
Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Barreira Hematoencefálica/ultraestrutura , Permeabilidade Capilar , Células Cultivadas , Feto/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Acoplamento Neurovascular , Farmacocinética , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura
10.
J Vis Exp ; (96)2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25741763

RESUMO

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.


Assuntos
Fibroblastos/citologia , Queratinócitos/citologia , Pele/lesões , Animais , Movimento Celular/fisiologia , Células Cultivadas , Colágeno Tipo I , Células Epidérmicas , Humanos , Hidrogéis , Ratos , Pele/anatomia & histologia , Pele/citologia , Fenômenos Fisiológicos da Pele , Cicatrização/fisiologia
11.
Beilstein J Org Chem ; 10: 3087-96, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25670977

RESUMO

The mono-6-deoxy-6-azides of 2,6-di-O-methyl-ß-cyclodextrin (DIMEB) and randomly methylated-ß-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu(+)-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 °C (DIMEB-HES) and 84.5 °C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.

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