Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain the DNA of living cells. To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.

Degeneration of articular cartilage (AC) is a common healthcare issue that can result in significantly impaired function and mobility for affected patients. The avascular nature of the tissue strongly burdens its regenerative capacity contributing to the development of more serious conditions such as osteoarthritis. Recent advances in bioprinting have prompted the development of alternative tissue engineering therapies for the generation of AC. Particular interest has been dedicated to scaffold-based strategies where 3D substrates are used to guide cellular function and tissue ingrowth. Despite its extensive use in bioprinting, the application of polycaprolactone (PCL) in AC is, however, restricted by properties that inhibit pro-chondrogenic cell phenotypes. This study proposes the use of a new bioprintable poly(ester urea) (PEU) material as an alternative to PCL for the generation of an in vitro model of early chondrogenesis. The polymer was successfully printed into 3D constructs displaying adequate substrate stiffness and increased hydrophilicity compared to PCL. Human chondrocytes cultured on the scaffolds exhibited higher cell viability and improved chondrogenic phenotype with upregulation of genes associated with type II collagen and aggrecan synthesis. Bioprinted PEU scaffolds could, therefore, provide a potential platform for the fabrication of bespoke, pro-chondrogenic tissue engineering constructs.

A new library of biocompatible AAA-PEAs with tunable physicochemical properties is proposed and validated for use as polymeric materials in the additive manufacturing (AM) of tissue engineering scaffolds.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain DNA of living cells. To do so, it needs to interact with and permeate through cell membranes. The pH of biological media may vary quite significantly and this influences H33342 ionization state, and may change its rate of permeation through biomembranes. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics simulations. While probe insertion occurs for all ionization states, with similar overall equilibrium location, orientation was found to depend on the probe charge, with the neutral form lying mostly parallel and the doubly charged displaying a significant tilt relative to the membrane plane. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy of interaction is negative and is larger at lower pH values. This suggests efficient hydrogen bonding between the probe and the lipid, namely for the protonated +2 form. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from water and octanol partition. This highlights the distinction between lipophilicity and hydrophobicity, and the importance of considering association with lipid bilayers when predicting the affinity for biomembranes.

Introduction Despite the existence of new therapeutic options, breast (BC) and colorectal (CC) cancers remain the leading causes of cancer death and the most commonly diagnosed in worldwide. Studies have reported that imidazole derivates show anticancer, antimicrobial, antibacterial, antifungal and antioxidant activities. Furthermore, recently it has been found that the association between imidazole ligands and copper increases their DNA binding affinity giving potential anticancer activity. Therefore, we synthesised three novel Cu(II) complexes using heterocyclic nitroimidazole derivatives as ligands. The aim of this study is to evaluate the cytotoxic activity of these complexes in two BC and two CC cell lines. Material and methods Nitroimidazole derived ligands containing cyclohexylamine, morpholine and piperidine and the respective Cu(II) complexes were synthesised. MCF-7, HCC1806, LS1034 and WiDr cell lines were cultured and grown in proper conditions. To evaluate the cytotoxic activity of these Cu(II) complexes on four cell lines, MTT colorimetric assay was used. Cells were seeded in 48 well-plates and then were treated with increasing concentrations of the complexes, from 0.5 to 200 µM. After 48 hour of incubation, medium was removed and MTT was added. Two hours later, isopropanol was added in order to dissolve formazan crystals. The absorbance was read at 570 and 620 nm and IC₅₀ (half maximal inhibitory concentration) values determined. Results and discussions Some Cu(II) complexes exhibit anticancer activity in cell lines. Studies show that for the MCF-7 and LS1034 cell lines, the nitroimidazole derived complex containing cyclohexylamine presents IC₅₀ values of 22.2 and 23.9 µM, respectively. For WiDr cell line, the same complex has an IC₅₀ of 50.5 µM. The best IC₅₀ value (2.9 µM) for this complex occurs in the HCC1806 cell line, a chemoresistant BC cell line. Curiously, the complex containing piperidine presents a IC₅₀ of 3.8 µM in HCC1806 cell line, while in the other BC cell line (MCF-7) there is no anticancer activity. The nitroimidazole derived complex containing cyclohexylamine is, consequently, the most promising compound in four cell lines. Conclusion Cu(II) complexes derived from nitroimidazole presented anticancer activity against all cell lines. The complex containing cyclohexylamine revealed to be the most promising, especially in HCC1806, basaloid triple-negative breast cancer, known as therapy-resistant.