Excitation-Emission Matrix Fluorescence Spectroscopy Coupled with PARAFAC Modeling for Viability Prediction of Cells.

Cell-based sensors and assays have great potential in bioanalysis, drug discovery screening, and biochemical mechanisms research. The cell viability tests should be fast, safe, reliable, and time- and cost-effective. Although methods stated as "gold standards", such as MTT, XTT, and LDH assays, usually fulfill these assumptions, they also show some limitations. They can be time-consuming, labor-intensive, and prone to errors and interference. Moreover, they do not enable the observation of the cell viability changes in real-time, continuously, and nondestructively. Therefore, we propose an alternative method of viability testing: native excitation-emission matrix fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC), which is especially advantageous for cell monitoring due to its noninvasiveness and nondestructiveness and because there is no need for labeling and sample preparation. We demonstrate that our approach provides accurate results with even better sensitivity than the standard MTT test. With PARAFAC, it is possible to study the mechanism of the observed cell viability changes, which can be directly linked to increasing/decreasing fluorophores in the cell culture medium. The resulting parameters of the PARAFAC model are also helpful in establishing a reliable regression model for accurate and precise determination of the viability in A375 and HaCaT-adherent cell cultures treated with oxaliplatin.

New Route to Glycosylated Porphyrins via Aromatic Nucleophilic Substitution (SNAr)-Synthesis and Cellular Uptake Studies.

Glycoporphyrins are group of compounds of high value for the purpose of photodynamic therapy and other biomedical applications. Despite great progress in the field, new diversity-oriented syntheses of carbohydrate-porphyrin hybrids are increasingly desired. Herein, we present efficient, mild, and metal-free conditions for synthesis of glycoporphyrins. The versatile nature of the SNAr procedure is presented in 16 examples. Preliminary biological studies have been conducted on the cytotoxicity and cellular uptake of the final molecules.

Excitation-emission matrix fluorescence spectroscopy for cell viability testing in UV-treated cell culture.

Monitoring of cells viability is essential in a number of biomedical applications, including cell-based sensors, cell-based microsystems, and cell-based assays. The use of spectroscopic techniques for such purposes is especially advantageous since they are non-invasive, label-free, and non-destructive. However, such an approach must include chemometric analysis of the data to assess the information on cells viability. In the presented article we demonstrate, that excitation-emission matrix (EEM) fluorescence spectroscopy can be applied for reliable determination of cells viability due to the high correlation of EEM fluorescence data with the MTT test data. A375 cells (malignant melanoma) were exposed to UV radiation as a physical stress factor, resulting in a decrease of viability up to ca. 20%, confirmed by the standard MTT test. They were also characterized by means of EEM fluorescence spectroscopy coupled with unfolded partial least squares (UPLS) regression. Statistical evaluation revealed high accordance of the two methods of viability testing in terms of accuracy, precision, and correlation. The presented results are very promising for the development of spectroscopic soft sensors that can be applied for drug screening, biocompatibility testing, tissue engineering, and pharmacodynamic studies.

Nanoparticles of chosen noble metals as reactive oxygen species scavengers.

Reactive oxygen species (ROS) play an important role in various physiological processes of living organisms. However, their increased concentration is usually considered as a threat for our health. Plants, invertebrates, and vertebrates including humans have various enzymatic and non-enzymatic defence systems against ROS. Unfortunately, both bad condition of surrounding environment and unhealthy lifestyle can interfere with an activity of enzymes responsible for a regulation of ROS levels. Therefore, it is important to look for alternative ROS scavengers, which could be administrated to chosen tissues to prevent pathological processes such as distortion of DNA or RNA structures and oxidation of proteins and lipids. One of the most recently proposed solutions is the application of nanozymes, which could mimic the activity of essential enzymes and prevent excessive activity of ROS. In this work, nanoparticles of Au, Pt, Pd, Ru and Rh were synthesized and studied in this regard. Peroxidase-, catalase (CAT)- and superoxide dismutase (SOD)-like activity of obtained nanoparticles were tested and compared using different methods. The influence of bovine and human albumins on CAT- and peroxidase-like activity was examined. Moreover, in the case of CAT-like activity, an influence of pH and temperature was examined and compared. Determination of SOD-like activity using the methods described for the examination of the activity of native enzyme was not fully successful. Moreover, cytotoxicity of chosen nanoparticles was studied on both regular and tumor cells.

Characterization of quantum dots in cancer cytosol using ICP-MS-based combined techniques.

Knowledge of the intracellular behavior of quantum dots (QDs), which encompasses the antiproliferative effect on living cells, is still limited. For this reason, the transformations of CdSeS/ZnS-based QDs in cancer cytosol were examined using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma MS (ICP-MS). CE-ICP-MS method revealed the dose- and time-dependent speciation changes of QDs in the cytosol, while HPLC-ICP-MS (in the size-exclusion chromatography mode) allowed further characterization of the resulting Cd species. In such an appraisal, the decent CE advantage of high resolution is well complemented by higher sensitivity of HPLC (LOD 4.0 × 10-10 and 5.4 × 10-12 mol/L Cd, respectively). Additionally, the influence of serum protein corona on the surface of QDs on their uptake by Hep G2 cancer cells was investigated by direct ICP-MS analysis that revealed that the conjugated proteins greatly reduce the particle internalization.

Cytotoxicity studies of quantum dots with the electroporation method.

In this study, the cytotoxicity of CdTe quantum dots (QDs) of various dimensions was examined using the electroporation method. The influence of the size of QDs on normal and tumour cell viability after 24 h of incubation with nanomaterials was examined. The three human cell lines were chosen for the tests: A549 (a tumour cell line derived from the lung), MRC-5 (normal fibroblasts from the lung) and HaCaT (normal keratinocytes from the skin). Accordingly, we modelled the effect of nanocrystals on various human tissues because nanoparticles can be introduced into an organism through different routes. We were also able to study which cells are more sensitive to nanoparticles: normal or tumour cells. The nanoparticles were introduced into cells through pores in the cell membranes that were generated by electrical pulses. The effectiveness of introducing nanocrystals into cells was determined as a function of the nanocrystal dimensions and accumulation locations. Moreover, the cytotoxicity of quantum dots was tested, and cell viability after electroporation was evaluated. We also investigated whether the introduced nanocrystals released cadmium ions.