Iron oxide nanoparticles with supramolecular ureido-pyrimidinone coating for antimicrobial peptide delivery

Antimicrobial peptides (AMPs) can kill bacteria by disrupting their cytoplasmic membrane, which reduces the tendency of antibacterial resistance compared to conventional antibiotics. Their possible toxicity to human cells, however, limits their applicability. The combination of magnetically controlled drug delivery and supramolecular engineering can help to reduce the dosage of AMPs, control the delivery, and improve their cytocompatibility. Lasioglossin III (LL) is a natural AMP form bee venom that is highly antimicrobial. Here, superparamagnetic iron oxide nanoparticles (IONs) with a supramolecular ureido-pyrimidinone (UPy) coating were investigated as a drug carrier for LL for a controlled delivery to a specific target. Binding to IONs can improve the antimicrobial activity of the peptide. Different transmission electron microscopy (TEM) techniques showed that the particles have a crystalline iron oxide core with a UPy shell and UPy fibers. Cytocompatibility and internalization experiments were carried out with two different cell types, phagocytic and nonphagocytic cells. The drug carrier system showed good cytocompatibility (>70%) with human kidney cells (HK-2) and concentration-dependent toxicity to macrophagic cells (THP-1). The particles were internalized by both cell types, giving them the potential for effective delivery of AMPs into mammalian cells. By self-assembly, the UPy-coated nanoparticles can bind UPy-functionalized LL (UPy-LL) highly efficiently (99%), leading to a drug loading of 0.68 g g−1 . The binding of UPy-LL on the supramolecular nanoparticle system increased its antimicrobial activity against E. coli (MIC 3.53 µM to 1.77 µM) and improved its cytocompatible dosage for HK-2 cells from 5.40 µM to 10.6 µM. The system showed higher cytotoxicity (5.4 µM) to the macrophages. The high drug loading, efficient binding, enhanced antimicrobial behavior, and reduced cytotoxicity makes ION@UPy-NH2 an interesting drug carrier for AMPs. The combination with superparamagnetic IONs allows potential magnetically controlled drug delivery and reduced drug amount of the system to address intracellular infections or improve cancer treatment.</p

Anticoagulant action of low, physiologic, and high albumin levels in whole blood

<div><p>Albumin is the most abundant plasma protein. Critical illness is often associated with altered, predominately decreased, serum albumin levels. This hypoalbuminaemia is usually corrected by administration of exogenous albumin. This study aimed to track the concentration-dependent influence of albumin on blood coagulation in vitro. Whole blood (WB) samples from 25 volunteers were prepared to contain low (19.3 ± 7.7 g/L), physiological (45.2 ± 7.8 g/L), and high (67.5 ± 18.1 g/L) levels of albumin. Haemostatic profiling was performed using a platelet function analyzer (PFA) 200, impedance aggregometry, a Cone and Platelet analyzer (CPA), calibrated automated thrombogram, and thrombelastometry (TEM). Platelet aggregation-associated ATP release was assessed via HPLC analysis. In the low albumin group, when compared to the physiological albumin group, we found: i) shortened PFA 200-derived closure times indicating increased primary haemostasis; ii) increased impedance aggregometry-derived amplitudes, slopes, ATP release, as well as CPA-derived average size indicating improved platelet aggregation; iii) increased TEM-derived maximum clot firmness and alpha angles indicating enhanced clot formation. TEM measurements indicated impaired clot formation in the high albumin group compared with the physiological albumin group. Thus, albumin exerted significant anticoagulant action. Therefore, low albumin levels, often present in cancer or critically ill patients, might contribute to the frequently occurring venous thromboembolism.</p></div

Ethyl pyruvate inhibits oxidation of LDL in vitro and attenuates oxLDL toxicity in EA.hy926 cells

<div><p>Background</p><p>Ethyl pyruvate (EP) exerts anti-inflammatory and anti-oxidative properties. The aim of our study was to investigate whether EP is capable of inhibiting the oxidation of LDL, a crucial step in atherogenesis. Additionally, we examined whether EP attenuates the cytotoxic effects of highly oxidized LDL in the human vascular endothelial cell line EA.hy926.</p><p>Methods</p><p>Native LDL (nLDL) was oxidized using Cu²⁺ ions in the presence of increasing amounts of EP. The degree of LDL oxidation was quantified by measuring lipid hydroperoxide (LPO) and malondialdehyde (MDA) concentrations, relative electrophoretic mobilities (REMs), and oxidation-specific immune epitopes. The cytotoxicity of these oxLDLs on EA.hy926 cells was assessed by measuring cell viability and superoxide levels. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells under increasing concentrations of EP in the media was assessed including measurements of high energy phosphates (ATP).</p><p>Results</p><p>Oxidation of nLDL using Cu²⁺ ions was remarkably inhibited by EP in a concentration-dependent manner, reflected by decreased levels of LPO, MDA, REM, oxidation-specific epitopes, and diminished cytotoxicity of the obtained oxLDLs in EA.hy926 cells. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells was remarkably attenuated by EP added to the media in a concentration-dependent manner reflected by a decrease in superoxide and an increase in viability and ATP levels.</p><p>Conclusions</p><p>EP has the potential for an anti-atherosclerotic drug by attenuating both, the oxidation of LDL and the cytotoxic effect of (already formed) oxLDL in EA.hy926 cells. Chronic administration of EP might be beneficial to impede the development of atherosclerotic lesions.</p></div

Effects of low, physiologic, and high albumin concentrations on primary haemostasis.

<p>Effects of low, physiologic, and high albumin concentrations on primary haemostasis.</p

Effects of low, physiologic, and high albumin concentrations on platelet aggregation.

<p>Effects of low, physiologic, and high albumin concentrations on platelet aggregation.</p

Effects of low, physiologic, and high albumin concentrations on platelet adhesion/aggregation assessed by means of CPA.

<p>Effects of low, physiologic, and high albumin concentrations on platelet adhesion/aggregation assessed by means of CPA.</p

Effects of low, physiologic, and high albumin concentrations on thrombin generation assessed by means of CAT and by F1+2 plasma levels.

<p>Effects of low, physiologic, and high albumin concentrations on thrombin generation assessed by means of CAT and by F1+2 plasma levels.</p

Cytotoxic effects of highly oxidized LDL on EA.hy926 cells in the presence of increasing medium concentrations of EP.

<p>nLDL (1.5 mg/mL) was oxidized by addition of 10 μmol/L CuCl₂ in the absence of EP in order to obtain the highly cytotoxic form of oxLDL. EP added to the culture medium concentration-dependently attenuated the cytotoxic effect of this oxLDL on EA.hy926 cells, reflected in increased viability <b>(A)</b> and decreased levels of superoxide radical anion formation <b>(B)</b>. Data represent mean ± SD (n = 6 for metabolic activity, n = 4 for superoxide levels), * p < 0.05, *** p < 0.001.</p

LDL oxidized in the presence of EP does not impact on cell monolayer integrity and barrier function of EA.hy926 cells.

<p><b>(A)</b> EA.hy926 cells grown on 12 well plates were treated with 0.3 mg/mL of either nLDL (a), oxLDL (b), or LDL which was oxidized in the presence of EP (500 μg/mL (c) and 1000 μg/mL (d)) for 4 h. After washing with serum-free culture medium cells were subjected to inverted light microscopy. <b>(B)</b> EA.hy926 cells grown on gold microelectrodes were treated as described above. Impedance was continuously monitored at 4 and 64 kHz starting after baseline stabilization (20 min post treatment start). Data represent mean ± SD (n = 4).</p

ATP levels and EC in EA.hy926 cells treated with highly oxidized LDL in the presence of increasing medium concentrations of EP.

<p>EP concentration-dependently attenuated the cytotoxic effects of highly Cu²⁺-oxidized LDL demonstrated by the increase of ATP levels <b>(A)</b> and EC <b>(B)</b> in EA.hy926 cells. Data represent mean ± SD (n = 6), ** p < 0.05, *** p < 0.001.</p