Monitoring cell endocytosis of liposomes by real-time electrical impedance spectroscopy.

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)-sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. Graphical abstract.

Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs.

In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.

3D Carbon Microelectrodes with Bio-Functionalized Graphene for Electrochemical Biosensing.

An enzyme-based electrochemical biosensor has been developed with 3D pyrolytic carbon microelectrodes that have been coated with bio-functionalized reduced graphene oxide (RGO). The 3D carbon working electrode was microfabricated using the pyrolysis of photoresist precursor structures, which were subsequently functionalized with graphene oxide and enzymes. Glucose detection was used to compare the sensor performance achieved with the 3D carbon microelectrodes (3DCMEs) to the 2D electrode configuration. The 3DCMEs provided an approximately two-fold higher sensitivity of 23.56 µA·mM-1·cm-2 compared to 10.19 µA mM-1·cm-2 for 2D carbon in glucose detection using cyclic voltammetry (CV). In amperometric measurements, the sensitivity was more than 4 times higher with 0.39 µA·mM-1·cm-2 for 3D electrodes and 0.09 µA·mM-1·cm-2 for the 2D configuration. The stability analysis of the enzymes on the 3D carbon showed reproducible results over 7 days. The selectivity of the electrode was evaluated with solutions of glucose, uric acid, cholesterol and ascorbic acid, which showed a significantly higher response for glucose.

Three-dimensional fabrication of thick and densely populated soft constructs with complex and actively perfused channel network.

One of the fundamental steps needed to design functional tissues and, ultimately organs is the ability to fabricate thick and densely populated tissue constructs with controlled vasculature and microenvironment. To date, bioprinting methods have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry, where the majority lacks the ability to be directly perfused. Moreover, it appears to be difficult to fabricate vascular tissue constructs targeting the stiffness of soft tissues such as the liver. Here we present a method for the fabrication of thick (e.g. 1 cm) and densely populated (e.g. 10 million cells·mL-1) tissue constructs with a three-dimensional (3D) four arm branch network and stiffness in the range of soft tissues (1-10 kPa), which can be directly perfused on a fluidic platform for long time periods (>14 days). Specifically, we co-print a 3D four-arm branch using water-soluble Poly(vinyl alcohol) (PVA) as main material and Poly(lactic acid) (PLA) as the support structure. The PLA support structure was selectively removed, and the water soluble PVA structure was used for creating a 3D vascular network within a customized extracellular matrix (ECM) targeting the stiffness of the liver and with encapsulated hepatocellular carcinoma (HepG2) cells. These constructs were directly perfused with medium inducing the proliferation of HepG2 cells and the formation of spheroids. The highest spheroid density was obtained with perfusion, but overall the tissue construct displayed two distinct zones, one of rapid proliferation and one with almost no cell division and high cell death. The created model, therefore, simulate gradients in tissues of necrotic regions in tumors. This versatile method could represent a fundamental step in the fabrication of large functional and complex tissues and finally organs. STATEMENT OF SIGNIFICANCE: Vascularization within hydrogels with mechanical properties in the range of soft tissues remains a challenge. To date, bioprinting have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry that are most of the time not perfused. This study shows the creation of densely populated tissue constructs with a 3D four arm branch network and stiffness in the range of soft tissues, which can be directly perfused. The cells encapsulated within the construct showed proliferation as a function of the vasculature distance, and the control of the micro-environment induced the encapsulated cells to aggregate in spheroids in specific positions. This method could be used for modeling tumors and for fabricating more complex and densely populated tissue constructs with translational potential.

Multichannel bipotentiostat integrated with a microfluidic platform for electrochemical real-time monitoring of cell cultures.

An electrochemical detection system specifically designed for multi-parameter real-time monitoring of stem cell culturing/differentiation in a microfluidic system is presented. It is composed of a very compact 24-channel electronic board, compatible with arrays of microelectrodes and coupled to a microfluidic cell culture system. A versatile data acquisition software enables performing amperometry, cyclic voltammetry and impedance spectroscopy in each of the 12 independent chambers over a 100 kHz bandwidth with current resolution down to 5 pA for 100 ms measuring time. The design of the platform, its realization and experimental characterization are reported, with emphasis on the analysis of impact of input capacitance (i.e., microelectrode size) and microfluidic pump operation on current noise. Programmable sequences of successive injections of analytes (ferricyanide and dopamine) and rinsing buffer solution as well as the impedimetric continuous tracking for seven days of the proliferation of a colony of PC12 cells are successfully demonstrated.