Marcin Drozd

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.

Cytotoxicity studies of CdSeS/ZnS quantum dots on cell culture in microfluidic system

Quantum dots (QDs) semi-conducting nanocrystals have found numerous applications in many fields of science. Nowadays one can observe a growing perspective to use them in biomedicine. Thanks to QDs unique fluorescence properties (narrow emission spectra, high extinction coefficients, high quantum yields, photostability) and possibility to form conjugates with bioactive molecules, they can become a chance for better cancer cells imaging in cancer therapy. Therefore there is a need for better understanding of biological interactions between QDs and cancer cells in vitro. For this purpose we performed cytotoxicity tests of CdSeS/ZnS quantum dots stabilized with mercaptopropionic acid (MPA) ligand, on human lung cancer cell line (A549) in vitro in macro- (96-well plate) and micro-scale (a specially designed and fabricated microfluidic device). The results obtained demonstrated a little extent of cytotoxic effect of selected solutions of QDs to A549 cells.

Cytotoxicity studies of selected cadmium-based quantum dots on 2D vs. 3D cell cultures

In our work, the cytotoxicity of selected, cadmium-based quantum dots with various surface architectures was studied on 3D spheroids. A specially designed microsystem as a tool for three-dimensional cell culture and nanoparticle toxicity evaluation was used for this aim. Two types of hydrophilic quantum dots with different surface charges at physiological pH were examined: CdTe-capped with ω-mercaptocarboxylic acid (COO−-terminated) and CdSeS/ZnS-glucosamine (–OH-terminated). We studied the influence of five different concentrations of nanoparticles within the range of 5 to 100 μM in order to assess dose-dependent toxic effects of the selected quantum dots on cellular spheroids as a more realistic (in vivo-like) model of human tissue. The obtained results of cytotoxicity were compared with the results for a standard, two-dimensional model – a cell monolayer. In the case of three-dimensional structures of various cell lines (normal MRC-5 and tumor A549) no significant differences in cytotoxicity caused by the tested nanoparticles have been noticed. The comparative studies revealed the enhanced biocompatibility of CdSeS/ZnS-OH quantum dots resulting from the presence of an uncharged ligand of biomimetic character on their surface. It was also found that the cytotoxicity of the same quantum dots depends on the cell culture model on which the tests were conducted. Significantly higher cytotoxicity of the tested nanomaterial was observed when experiments were carried out with the use of cell monolayers. Based on the obtained results, we claim that cytotoxicity studies of nanomaterials conducted on standard 2D cell monolayers are overestimated. In our opinion, reliable in vitro studies on the biological activity of nanoparticles require application of 3D cell cultures.

SPRi-Based Biosensing Platforms for Detection of Specific DNA Sequences Using Thiolate and Dithiocarbamate Assemblies

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0.66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 · 1012 molecules · cm−2 (with the calculated area occupied by single nanoparticle label of ~132.7 nm2), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

Comparative studies of biological activity of cadmium-based quantum dots with different surface modifications

This paper presents a modification of the surface of CdS/ZnS and CdSexS1−x/ZnS quantum dots (QDs) with 3-mercaptopropionic and 6-mercaptohexanoic acid. The obtained QDs were characterized using TEM, DLS, UV–Vis, and fluorescence spectroscopy. Flow cytometry was applied to evaluate the cytotoxicity of QDs and examine the type of death caused by the tested nanoparticles. In addition, the generation of reactive oxygen species after incubation of the tested cells with CdSexS1−x/ZnS–MPA and CdSexS1−x/ZnS–MHA QDs was evaluated. The study was conducted on three cell lines: adherent (A549 and MRC-5) and suspension ones (K562). The conducted research demonstrated that the tested nanoparticles exhibit concentration-dependent toxicity. It was observed that the surface modification influences the toxicity level of the examined QDs, and modification of their surface with the use of the ligand of longer carbon chain (MHA) reduces the toxicity in comparison with QDs–MPA. It was also found that all tested QDs caused the death of cells in the course of necrosis. Based on obtained results, it was concluded that the cytotoxicity of QDs is to a large extent related to reactive oxygen species (ROS) generation.

Revisiting catechol derivatives as robust chromogenic hydrogen donors working in alkaline media for peroxidase mimetics

Colloidal noble metal-based nanoparticles are able to catalyze oxidation of chromogenic substrates by H2O2, similarly to peroxidases, even in basic media. However, lack of robust chromogens, which work in high pH impedes their real applications. Herein we demonstrate the applicability of selected catechol derivatives: bromopyrogallol red (BPR) and pyrogallol (PG) as chromogenic substrates for peroxidase-like activity assays, which are capable of working over wide range of pH, covering also basic values. Hyperbranched polyglycidol-stabilized gold nanoparticles (HBPG@AuNPs) were used as model enzyme mimetics. Efficiency of several methods of improving stability of substrates in alkaline media by means of selective suppression of their autoxidation by molecular oxygen was evaluated. In a framework of presented studies the impact of borate anion, applied as complexing agent for PG and BPR, on their stability and reactivity towards oxidation mediated by catalytic AuNPs was investigated. The key role of high concentration of hydrogen peroxide in elimination of non-catalytic oxidation of PG and improvement of optical properties of BPR in alkaline media containing borate was underlined. Described methods of peroxidase-like activity characterization with the use of BPR and PG can become universal tools for characterization of nanozymes, which gain various applications, among others, they are used as catalytic labels in bioassays and biosensors.

Studies on voltammetric determination of cadmium in samples containing native and digested proteins.

This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples.