Fluorescence Correlation Spectroscopy as a Quantitative Tool Applied to Drug Delivery Model Systems

The delivery of drugs to cells is a very active area of research. Drugs are commonly believed to ameliorate illnesses. The targeted, controlled and/or enhanced uptake of the drug into cells is facilitated by loading the drug into carrier vehicles (nanoparticles, e.g.. micelles).The distribution of a drug over the nanoparticles used to deliver the drug to the targeted cells is of vital importance. It makes a difference, whether all nanoparticles carry the same amount of drug or whether a little amount of the nanoparticles carries a high proportion of the drug material and most nanoparticles carry no material. This depends on the method used to load the drugs into the carriers. Furthermore, the absolute amount of drug present in the nanoparticles and the absolute number of nanoparticles loaded with drug molecules is very important, because "dosis venum facit" [Paracelsus]. We present an approach to resolve these issues by optical means.If the drug is fluorescent or labeled with a fluorescent label, fluorescence correlation spectroscopy (FCS) can be used to determine quantitative size distributions with a (confocal) microscope. In order to determine absolute concentrations the spatial dimensions of the confocal observation volume have to be known. These can be obtained from numerical simulations using vectorial diffraction theory. The theoretical results have been compared with experimental results from FCS using the calibration dye Rhodamine 6G. In a straightforward procedure the results were corrected for adsorption effects and excellent agreement to the theory was foun

Optimization of soil microbial fuel cell for sustainable bio-electricity production: combined effects of electrode material, electrode spacing, and substrate feeding frequency on power generation and microbial community diversity

BACKGROUND: Microbial fuel cells (MFCs) are among the leading research topics in the field of alternative energy sources due to their multifunctional potential. However, their low bio-energy production rate and unstable performance limit their application in the real world. Therefore, optimization is needed to deploy MFCs beyond laboratory-scale experiments. In this study, we investigated the combined influence of electrode material (EM), electrode spacing (ES), and substrate feeding interval (SFI) on microbial community diversity and the electrochemical behavior of a soil MFC (S-MFC) for sustainable bio-electricity generation. RESULTS: Two EMs (carbon felt (CF) and stainless steel/epoxy/carbon black composite (SEC)) were tested in an S-MFC under three levels of ES (2, 4, and 8 cm) and SFI (4, 6, and 8 days). After 30 days of operation, all MFCs achieved open-circuit voltage in the range of 782 + 12.2 mV regardless of the treatment. However, the maximum power of the SEC–MFC was 3.6 times higher than that of the CF–MFC under the same experimental conditions. The best solution, based on the interactive influence of the two discrete variables, was obtained with SEC at an ES of 4.31 cm and an SFI of 7.4 days during an operating period of 66 days. Analysis of the experimental treatment effects of the variables revealed the order SFI < ES < EM, indicating that EM is the most influential factor affecting the performance of S-MFC. The performance of S-MFC at a given ES value was found to be dependent on the levels of SFI with the SEC electrode, but this interactive influence was found to be insignificant with the CF electrode. The microbial bioinformatic analysis of the samples from the S-MFCs revealed that both electrodes (SEC and CF) supported the robust metabolism of electroactive microbes with similar morphological and compositional characteristics, independent of ES and SFI. The complex microbial community showed significant compositional changes at the anode and cathode over time. CONCLUSION: This study has demonstrated that the performance of S-MFC depends mainly on the electrode materials and not on the diversity of the constituent microbial communities. The performance of S-MFCs can be improved using electrode materials with pseudocapacitive properties and a larger surface area, instead of using unmodified CF electrodes commonly used in S-MFC systems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-022-02224-9

Noxic effects of polystyrene microparticles on murine macrophages and epithelial cells

Abstract Microplastic (MP) contamination has been identified as an ecological problem with an increasing impact on everyday life. Yet, possible effects of MP at the cellular level are still poorly understood. Here, the interaction of murine macrophages (J774A.1, ImKC) and epithelial cells (STC-1, BNL CL.2) with well-characterized poly(styrene) MP particles (MPP) of varying sizes (0.2–6.0 µm) was studied. Macrophages are expected to actively engulf particles which could be confirmed in this study, while epithelial cells are found in tissues with direct contact with ingested or inhaled MPP. Here, the epithelial cells from both investigated cell lines did not ingest MPP in significant numbers. Concomitantly, no cytotoxic effects nor any influence on cellular proliferation were observed. Cells from the two macrophage cell lines showed high ingestion of MPP of all sizes, but cytotoxic effects were observed only for one of them (ImKC) and only at MPP concentrations above 250 µg/mL. Indications of cellular stress as well as effects on cell proliferation were observed for cell populations with high particle cell interactions

Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current “gold standard”, namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol

In vitro cultivation of primary intestinal cells from Eisenia fetida as basis for ecotoxicological studies

The earthworm Eisenia fetida is a commonly used model organism for unspecific soil feeders in ecotoxicological studies. Its intestinal cells are the first to encounter possible pollutants co-ingested by the earthworm, which makes them prime candidates for studies of toxic effects of environmental pollutants on the cellular as compared to the organismic level. In this context, the aim of this study was to demonstrate the suitability of preparations of primary intestinal E. fetida cells for in vitro ecotoxicological studies. For this purpose, a suitable isolation and cultivation protocol was established. Cells were isolated directly from the intestine, maintaining >85% viability during subsequent cultivations (up to 144 h). Exposure to established pollutants and soil elutriates comprising silver nanoparticles and metal ions (Cu(2+), Cd(2+)) induced a significant decrease in the metabolic activity of the cells. In case of microplastic particles (MP particles), namely 0.2, 0.5, 2.0, and 3.0 µm diameter polystyrene (PS) beads as well as 0.5 and 2.0 µm diameter polylactic acid (PLA) beads, no active uptake was observed. Slight positive as well as negative dose and size dependent effects on the metabolism were seen, which to some extent might correlate with effects on the organismic level