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.

Joint forces of direct, single particle, CE- and HPLC-inductively coupled plasma mass spectrometry techniques for the examination of gold nanoparticle accumulation, distribution and changes inside human cells.

The intracellular localization and transformation of gold nanoparticles (AuNPs) are among the crucial aspects in future applications in cancer therapy. In the context of the study, inductively coupled plasma mass spectrometry (ICP-MS)-based techniques were effectively applied to reveal the fate of AuNPs internalized in cancerous MCF-7 cells. Direct ICP-MS was used to obtain quantitative information about the distribution rate of gold from the AuNPs in the cells, namely their membranes, cytosol as well as nuclei. Moreover, the combination of capillary electrophoresis and reversed-phase liquid chromatography with ICP-MS was used as a tool to probe and compare for the effective monitoring of the speciation changes of the gold-containing forms in the cytosol. The chemical nature (ionic vs. nano) of the metal detected in the cytosol was verified via ICP-MS in a single-particle mode, confirming the stability of the nanomaterials and the absence of ionic gold forms inside the 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.

An improved protocol for ICP-MS-based assessment of the cellular uptake of metal-based nanoparticles.

Along with a growing interest in biomedical applications of metal-based nanoparticles, there is a compelling need in systematic information on their behavior in human body systems, preferably at the cellular level. However, in most of the in-vitro uptake experiments, the nanomaterial was applied in its native form that in reality can hardly reach the cell. In this work, we developed an improved procedure in which prior to addition to the cells the particles are converted into the protein conjugates by incubation in human serum. The procedure was tested for gold nanoparticles of different size, chosen as a representative nanomaterial on multifunctional medicinal use, and MCF-7 cell line. Using ICP-MS to measure intracellular metal concentration, it was shown that an original state has significant effect on particle internalization. The protein corona significantly inhibits the uptake amount by MCF-7 cells, with the greatest influence (a 15-fold decrease compared to uncoated particles) being exerted over the smallest, 5-nm particles (3 pg Au/cell). Conjugates of larger particles (20 and 50 nm) are taken up more effectively (45 and 34 pg Au/cell, respectively). The advanced protocol makes the uptake results more reliable and its implementation may accelerate the preclinical development of metal-based nanoparticles as a viable theranostic implement.

2D Ti2C (MXene) as a novel highly efficient and selective agent for photothermal therapy.

Photothermal therapy (PTT) has shown significant potential for anti-cancer modality. In this report, according to our best knowledge, we explore for the first time Ti2C-based MXene as a novel, highly efficient and selective agent for photothermal therapy (PTT). Ti2C superficially modified with PEG was obtained from the layered, commercially available Ti2AlC MAX phase in the process of etching aluminum layers using concentrated HF, and characterized by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HREM) as well as X-ray photoelectron spectroscopy for chemical analysis (ESCA-XPS). The PEG-coated Ti2C flakes showed a satisfactory photothermal conversion efficacy (PTCE) and good biocompatibility in wide range of the tested concentrations. Through in vitro studies, the PEG-modified Ti2C demonstrated notable NIR-induced ability to cancerous cells' ablation with minimal impact on non-malignant cells up to the concentration of 37.5 µg mL-1. The applied doses of Ti2C_PEG in our work were even 24 times lower comparing other MXene-based photothermal agents. This work is expected to expand the utility of 2D MXenes to biomedical applications through the development of entirely novel agents for photothermal therapy. This work is expected to expand the utility of 2D MXenes to biomedical applications through the development of entirely novel agents for photothermal therapy.

The influence of selected ω-mercaptocarboxylate ligands on physicochemical properties and biological activity of Cd-free, zinc­copper­indium sulfide colloidal nanocrystals.

In this work core/shell cadmium-free zinc­copper­indium sulfide ZnCuInS/ZnS quantum dots (QDs) originally stabilized with hydrophilic alkanethiol were modified with 3-mercaptopropionic acid (MPA) or 6-mercaptohexanoic acid (MHA) via two-step ligand exchange method. The obtained QDs were further characterized by TEM, UV Vis, and fluorescence spectroscopy. Both types of QDs were non-toxic in a wide range of concentrations. To our knowledge, our studies are the first attempt to determine the type of cell death and reactive oxygen species production level as a result of incubation of cell cultures with ZnCuInS/ZnS QDs. Furthermore, the accumulation of QDs in vitro was examined on three human cell lines by fluorescence intensity measurements and visualized by confocal microscopy. The modification of QDs with a ligand characterized by slightly longer aliphatic chain (MHA), instead of typically used MPA turns out to be beneficial both from the point of colloidal stability, preservation of optical properties during ligand exchange as well as reflects in a higher cellular uptake. This contribution can be beneficial from the point of view of the selection of the optimal ligands and concentrations in the case of ZnCuInS/ZnS core-shell QDs for biological applications.

Studies on effectiveness of PTT on 3D tumor model under microfluidic conditions using aptamer-modified nanoshells.

Herein, we present the research focused on the synthesis and application of aptamer-modified gold nanoshells for photothermal therapy (PTT). NIR-absorbing hollow gold nanoshells were synthetized and conjugated with anti-MUC1 aptamer (HGNs@anti-MUC1). MUC1 (Mucin 1) is a transmembrane glycoprotein, which is overexpressed in a variety of epithelial cancers (eg. breast, lung, pancreatic). In order to evaluate the efficiency of PTT with HGNs@anti-MUC1 we used 3D cell culture model - multicellular spheroids. The selected cell culture model is considered as the best in vitro model for cancer research (similar morphology, metabolite and oxygen gradients, cellular interactions and cell growth kinetics in the spheroids are similar to the early stage of a nonvascular tumor). We conducted our research on human normal (MRC-5, MCF-10A) and tumor (A549, MCF-7) cell lines using a microfluidic system. Aptamer-modified nanoparticles were accumulated selectively in tumor cells (A549, MCF-7) and this fact contributed to the reduction of tumor spheroids viability and size. It should be underlined, that it is the first example of photothermal therapy carried out in a microsystem on multicellular spheroids.

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.

Studies on influence of polymer modifiers for fluorescent nanocrystals' cytotoxicity.

The presented studies aimed at investigation of the effect of CdSeS/ZnS quantum dots (QDs) stabilized with hyperbranched polyglycidol and its carboxylated derivative on adenocarcinomic human alveolar basal epithelial cells (A549). The first stage of studies concerned the modification of quantum dots with both types of the tested polymers with the use of pyridine as an intermediate agent. Subsequently, cytotoxic effect of the prepared nanoparticles was examined after various incubation time using MTT test (cell metabolic activity assay). Our studies revealed that CdSeS/ZnS with a diameter of 6nm, which were stabilized with hyperbranched polymers do not penetrate into cells, even after prolonged incubation time. Moreover, the cytotoxic effect of the tested QDs was observed over a range of tested concentrations (5-90µM of Cd(2+)). It was confirmed that tested nanoparticles had significant influence on cell culture viability. The examined cytotoxic effect of the tested quantum dots was dependent on the type of polymer applied and the experiments indicated, that the one bearing carboxylic moieties is more toxic to A549 cells.

Evaluation of biological activity of quantum dots in a microsystem.

The presented work aimed at systematic investigation of biological activity of CdSex S1- x /ZnS and CdSe/ZnS quantum dots (QDs), whose surface was modified with different ligands. For these studies, we used a microfluidic system combined with fluorescence microscopy techniques, which enabled analysis of cells' morphology, viability, and QDs uptake. PDMS and glass-based microfluidic system enabled the precise control of the cell environment, allowed to examine five replications of each tested QDs concentrations (statistically significant number), monitor multiple cellular events, and avoid manual preparation of QDs dilutions. We investigated the influence of the core composition and the type of surface modifiers on QDs toxicity. We also determined whether the examined nanoparticles penetrate into the cells. For all tested nanoparticles, the decrease of cells' viability was observed when increasing nanoparticles concentration. The decrease of live cells' number in microchambers and the accumulation of the nanoparticles around cultured cells were observed. The effect of hydrocarbon chain length of surface modifiers and QDs core composition on the cell viability was confirmed in our tests.

Multi-function microsystem for cells migration analysis and evaluation of photodynamic therapy procedure in coculture.

Cell migration is an important physiological process, which is involved in cancer metastasis. Therefore, the investigation of cell migration may lead to the development of novel therapeutic approaches. In this study, we have successfully developed a microsystem for culture of two cell types (non-malignant and carcinoma) and for analysis of cell migration dependence on distance between them. Finally, we studied quantitatively the influence of photodynamic therapy (PDT) procedures on the viability of pairs of non-malignant (MRC5 or Balb/3T3) and carcinoma (A549) cells coculture. The proposed geometry of the microsystem allowed for separate introduction of two cell lines and analysis of cells migration dependence on distance between the cells. We found that a length of connecting microchannel has an influence on cell migration and viability of non-malignant cells after PDT procedure. Summarizing, the developed microsystem can constitute a new tool for carrying out experiments, which offers a few functions: cell migration analysis, carcinoma and non-malignant cells coculture, and evaluation of PDT procedure in the various steps of cell migration.