Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population

In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds

Combination antiretroviral drugs in PLGA nanoparticle for HIV-1

<p>Abstract</p> <p>Background</p> <p>Combination antiretroviral (AR) therapy continues to be the mainstay for HIV treatment. However, antiretroviral drug nonadherence can lead to the development of resistance and treatment failure. We have designed nanoparticles (NP) that contain three AR drugs and characterized the size, shape, and surface charge. Additionally, we investigated the <it>in vitro </it>release of the AR drugs from the NP using peripheral blood mononuclear cells (PBMCs).</p> <p>Methods</p> <p>Poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing ritonavir (RTV), lopinavir (LPV), and efavirenz (EFV) were fabricated using multiple emulsion-solvent evaporation procedure. The nanoparticles were characterized by electron microscopy and zeta potential for size, shape, and charge. The intracellular concentration of AR drugs was determined over 28 days from NPs incubated with PBMCs. Macrophages were imaged by fluorescent microscopy and flow cytometry after incubation with fluorescent NPs. Finally, macrophage cytotoxicity was determined by MTT assay.</p> <p>Results</p> <p>Nanoparticle size averaged 262 ± 83.9 nm and zeta potential -11.4 ± 2.4. AR loading averaged 4% (w/v). Antiretroviral drug levels were determined in PBMCs after 100 μg of NP in 75 μL PBS was added to media. Intracellular peak AR levels from NPs (day 4) were RTV 2.5 ± 1.1; LPV 4.1 ± 2.0; and EFV 10.6 ± 2.7 μg and continued until day 28 (all AR ≥ 0.9 μg). Free drugs (25 μg of each drug in 25 μL ethanol) added to PBMCs served as control were eliminated by 2 days. Fluorescence microscopy and flow cytometry demonstrated phagocytosis of NP into monocytes-derived macrophages (MDMs). Cellular MTT assay performed on MDMs demonstrated that NPs are not significantly cytotoxic.</p> <p>Conclusion</p> <p>These results demonstrated AR NPs could be fabricated containing three antiretroviral drugs (RTV, LPV, EFV). Sustained release of AR from PLGA NP show high drug levels in PBMCs until day 28 without cytotoxicity.</p

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier

Microfluidic In Vitro Platform for (Nano)Safety and (Nano)Drug Efficiency Screening

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal‐free risk assessment of new chemicals and drugs. Microfluidic cell‐based devices allow high‐throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal‐free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo‐like in vitro cell cultivation. It is equipped with a wafer‐based silicon chip including integrated electrodes and a microcavity. A proof‐of‐concept using different relevant cell models shows its suitability for label‐free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label‐free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole‐body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments

High Viral Fitness during Acute HIV-1 Infection

Several clinical studies have shown that, relative to disease progression, HIV-1 isolates that are less fit are also less pathogenic. The aim of the present study was to investigate the relationship between viral fitness and control of viral load (VL) in acute and early HIV-1 infection. Samples were obtained from subjects participating in two clinical studies. In the PULSE study, antiretroviral therapy (ART) was initiated before, or no later than six months following seroconversion. Subjects then underwent multiple structured treatment interruptions (STIs). The PHAEDRA study enrolled and monitored a cohort of individuals with documented evidence of primary infection. The subset chosen were individuals identified no later than 12 months following seroconversion to HIV-1, who were not receiving ART. The relative fitness of primary isolates obtained from study participants was investigated ex vivo. Viral DNA production was quantified using a novel real time PCR assay. Following intermittent ART, the fitness of isolates obtained from 5 of 6 PULSE subjects decreased over time. In contrast, in the absence of ART the fitness of paired isolates obtained from 7 of 9 PHAEDRA subjects increased over time. However, viral fitness did not correlate with plasma VL. Most unexpected was the high relative fitness of isolates obtained at Baseline from PULSE subjects, before initiating ART. It is widely thought that the fitness of strains present during the acute phase is low relative to strains present during chronic HIV-1 infection, due to the bottleneck imposed upon transmission. The results of this study provide evidence that the relative fitness of strains present during acute HIV-1 infection may be higher than previously thought. Furthermore, that viral fitness may represent an important clinical parameter to be considered when deciding whether to initiate ART during early HIV-1 infection

Service-sector Guilds and the Challenge of Liberalization: The organization of maritime-cargo handling in Barcelona, c.1760-1840

[eng] This thesis examines the guilds comprising the sub-sector of maritime cargo handling in Barcelona from 1760-1840, a period defined in economic terms by the progressive advance of liberal reforms and attempts at abolishing the guilds. Guilds in the service sector were responsible for activities that were vital to economic development, yet the organization of that labor remains an under-represented subject of investigation. By studying the totality of maritime-cargo handling trades of Barcelona, the thesis aims to contribute to the relatively limited scholarship of service-sector guilds and of port labor during the artisan phase. With this aim, the thesis discusses the historiography of guild and port labor studies, passing to a description of the geographic, infrastructural and socio-spatial delineations of the port based on objective and subjective considerations. By comparing the case of Barcelona to a number of other European ports, the study highlights relationships between port types and objective needs of cargo handling with the different organizational models that developed in the sub-sector. Thereafter, the investigation examines in detail the different guilds and the socio-judicial determination of responsibilities, privileges, organizational models and internal composition. Having covered these issues, the study then focuses on the various attempts at reforming or abolishing the guilds and the respective responses of these organizations to structural economic policy changes. During the period studied, the port infrastructure remained relatively unchanged and there was no mechanization of cargo-handling activities, facilitating the focus on changes in economic policy as factors in the application of liberal measures and the organizational responses of the guilds to these important changes. As such, the period studied covers the flexibilization of the labor market through reforms of monopolistic guild privileges and the eventual abolition of Spanish guilds in 1836. The proper handling of cargo and the mode of work influenced – but did not determine – occupational and organizational cultures: some guilds operated cooperatively, others, individually. These corporations were organized horizontally – there were no apprentices or journeymen in these trades, only masters – and developed alternative methods for organizing work processes, determining recruitment qualification, and for developing and transmitting skills. The investigation looks at the internal and external considerations that influenced the organizational responses to liberalism. The traditional (and arguably still generally dominant) historiography of guilds centers on discontinuity between the guilds and later organizations of labor and capital (trade unions and owners’ associations). This investigation highlights a number of direct continuities, contributing to the study of the development of capitalism. The principal conclusions of the research are focused on a number of related considerations, including: port types and infrastructures; the objective, cargo-based necessities of handling; work cultures, organizational cultures, and organizational models, (especially collaborative or individualistic activities); leveling mechanisms and the socio-economic composition of the guilds (vis à vis internal and external employment schemes), and the responses to liberalization (which is shown to have developed progressively over decades).[spa] Esta tesis tiene como objetivo elucidar el sub-sector del manejo de la carga marítima en Barcelona (1760-1840) para mejor entender la función de los gremios de servicios. En especial analiza como reaccionaron estos gremios al desarrollo progresivo del liberalismo, que representó un reto considerable al sistema gremial. De esta manera, la tesis contribuye a la historiografía de los estudios de gremios europeos y de la historia de trabajo en el ámbito marítimo. Es notable que el objeto de estudio se encuentre como un outlier (o, caso atípico) en varios campos, ya que en los estudios de gremios hay poca atención puesta en el sector de servicios, y en los estudios de trabajo marítimo hay muy pocas investigaciones sobre la época artesanal. A nivel de historia local, existen unas pocas publicaciones sobre los gremios tratados aquí, pero en su mayoría se enfocan en un solo gremio, desconectado del resto de componentes del universo del trabajo portuario. Entre los temas tratados se encuentran: el análisis del sistema gremial europeo en general y la diferenciación de éstos gremios de servicios; un acercamiento al entendimiento socio-espacial del puerto (con casos comparativos); la definición socio-jurídica y económica de los gremios tratados; el avance progresivo del liberalismo en España y particularmente en Barcelona; y las estrategias de los diferentes gremios del sub-sector del manejo de la carga marítima frente las reformas y aboliciones liberales del sistema gremial. Basado en literatura secundaria principalmente en castellano, catalán e inglés, y en fuentes primarias mayoritariamente en castellano, la tesis se presenta en inglés, con un resumen exhaustivo en castellano. Las conclusiones se analizan a partir del objetivo y su relevancia a varios campos académicos

Julia J Von Briesen's Quick Files

The Quick Files feature was discontinued and it’s files were migrated into this Project on March 11, 2022. The file URL’s will still resolve properly, and the Quick Files logs are available in the Project’s Recent Activity

Specific targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified human serum albumin nanoparticles

Specific targeting of tumor cells to achieve higher drug levels in tumor tissue and to overcome cardiotoxic and other secondary effects is the major goal in cancer therapy. With trastuzumab as a humanized monoclonal antibody binding, the HER2 receptor specific targeting is possible. In the present study, target-oriented nanoparticles based on biodegradable human serum albumin (HSA) loaded with cytostatic drug doxorubicin were developed. The surface of the nanoparticles was modified by covalent attachment of trastuzumab. HER2 overexpressing breast cancer cells showed a good cellular binding and uptake of these nanoparticles. The specific transport of the cytostatic drug doxorubicin with this nanoparticulate formulation into the HER2 overexpressing breast cancer cells, their release, and biological activity was demonstrated. The results indicate that these cell-type specific drug-loaded nanoparticles could achieve an improvement in cancer therapy. To our knowledge, this is the first study demonstrating a specific trastuzumab-based targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded nanoparticles