Anna Woźniak

Assessment of polydopamine coated magnetic nanoparticles in doxorubicin delivery

Magnetic nanoparticles (MNP) coated with bioinspired polydopamine (PDA) were obtained via a co-precipitation method and oxidative polymerization of dopamine. Nanoparticles were investigated by FTIR, TEM and SQUID. Loading capacity of anticancer drug doxorubicin was determined by UV-Vis spectroscopy. The nanocomposites exhibit a high drug loading capacity of 0.46 mg mg−1. Anticancer activity of the nanocomposites was proved in profound in vitro tests on HeLa cells. Cytotoxicity and internalization of nanoparticles were checked using various method, i.e. proliferation assay (WST-1), a two-colour fluorescence cell viability assay, and fluorescent and confocal microscopy.

Size and shape-dependent cytotoxicity profile of gold nanoparticles for biomedical applications

Metallic nanoparticles, in particular gold nanoparticles (AuNPs), offer a wide spectrum of applications in biomedicine. A crucial issue is their cytotoxicity, which depends greatly on various factors, including morphology of nanoparticles. Because metallic nanoparticles have an effect on cell membrane integrity, their shape and size may affect the viability of cells, due to their different geometries as well as physical and chemical interactions with cell membranes. Variations in the size and shape of gold nanoparticles may indicate particular nanoparticle morphologies that provide strong cytotoxicity effects. Synthesis of different sized and shaped bare AuNPs was performed with spherical (~ 10 nm), nanoflowers (~ 370 nm), nanorods (~ 41 nm), nanoprisms (~ 160 nm) and nanostars (~ 240 nm) morphologies. These nanostructures were characterized and interacting with cancer (HeLa) and normal (HEK293T) cell lines and cell viability tests were performed by WST-1 tests and fluorescent live/dead cell imaging experiments. It was shown that various shapes and sizes of gold nanostructures may affect the viability of the cells. Gold nanospheres and nanorods proved to be more toxic than star, flower and prism gold nanostructures. This may be attributed to their small size and aggregation process. This is the first report concerning a comparison of cytotoxic profile in vitro with a wide spectrum of bare AuNPs morphology. The findings show their possible use in biomedical applications.Graphical Abstract

Impact of Globulins Derived from Genetically Modified and Conventional Soybean on Swine Lymphocyte Proliferation in in vitro Cultures

Impact of Globulins Derived from Genetically Modified and Conventional Soybean on Swine Lymphocyte Proliferation in in vitro Cultures The majority of the global feed market is dominated by the Roundup Ready 40-3-2 transgenic soybean varieties developed and marketed by Monsanto Company, which are characterized by tolerance to glyphosate, the active ingredient of the Roundup herbicide. It should be remembered, however, that soybean is one of the major allergens which may affect animal health. The aim of the study was to compare allergenic properties of globulins derived from genetically modified (GM) soybean imported from the USA and conventional soybean developed in Poland. Analyses were performed by measuring porcine lymphocyte proliferation in in vitro cultures. It turned out that both genetically modified and conventional soybean proteins caused immune response at the level of negative control. A slight increase in relation to the negative control was observed in the case of 7S and 11S fractions derived from the GM meal and 7S fraction isolated from Nawiko meal. Wpływ globulin soi genetycznie modyfikowanej i konwencjonalnej na proliferację limfocytów świńskich w kulturach in vitro Większość globalnego rynku zdominowała soja transgeniczna Roundup Ready 40-3-2, wprowadzona przez firmę Monsanto, cechująca się tolerancją na glifosat, składnik aktywny herbicydu Roundup. Uprawia się również w niewielkich ilościach soję konwencjonalną. Należy jednak pamiętać, że soja należy do jednego z głównych alergenów, który może mieć wpływ na zdrowotność zwierząt. Celem pracy było porównanie właściwości alergennych globulin soi modyfikowanej genetycznie i konwencjonalnej wyhodowanej w Polsce. Analizy dokonano mierząc proliferację limfocytów świńskich w kulturach in vitro. Okazało się, iż soja modyfikowana genetycznie oraz konwencjonalna wywołują odpowiedź immunologiczną na poziomie kontroli negatywnej. Nieznaczny wzrost w stosunku do kontroli negatywnej obserwowano w przypadku frakcji 7S i 11S pochodzących ze śruty GM oraz frakcji 7S izolowanej ze śruty Nawiko.

In vitro genotoxicity and cytotoxicity of polydopamine-coated magnetic nanostructures

Synthesis of magnetic nanoparticles and magnetic nanoclusters was performed by the co-precipitation method or solvothermal synthesis, respectively, followed by oxidative polymerization of dopamine, resulting in a polydopamine (PDA) shell. The nanomaterials obtained were described using TEM, FTIR and magnetic measurements. For the first time, cyto- and genotoxicity studies of polydopamine-coated nanostructures were performed on cancer and normal cell lines, providing in-depth insight into the toxicity of such materials. The tests conducted, e.g. ROS, apoptosis and DNA double-break of the nanomaterials obtained revealed the low toxicity of these structures. Thus, these results prove the biocompatibility and low genotoxicity of these materials and provide new data on the toxicity of PDA-coated materials, which is of great importance for their biomedical application.

ZnO@Gd2O3 core/shell nanoparticles for biomedical applications: Physicochemical, in vitro and in vivo characterization

The chemical composition of nanoparticles (NPs) may be so designed as to provide measurability for numerous imaging techniques in order to achieve synergistic advantages. Innovative and unique structure of the core/shell ZnO@Gd2O3 NPs possesses luminescent and magnetic properties, and is expected that they will become a new generation of contrast agents for Magnetic Resonance Imaging (MRI) and nanocarriers for theranostics. Thus, by surface biofunctionalization, it is possible to indicate particular nanoparticle compositions which provide efficient imaging, targeted drug delivery, and biocompatibility. Novel ZnO@Gd2O3 NPs were synthesized and biofunctionalized by folic acid (FA) and doxorubicin (Doxo) to provide target and anticancer functions. Physicochemical analyses of the nanoparticles were performed. The biological study included a cytotoxicity in vitro, cellular distribution evaluation, as well as toxicity analyses, performed for the first time, on the in vivo zebrafish (Danio rerio) model. Nanoparticles were found to be effective double-function biomarkers (MRI T2 contrast agents, fluorescent imaging). The biological study showed that ZnO@Gd2O3 and ZnO@Gd2O3@OA-polySi@FA NPs are biocompatible in a particular concentration ranges. Conjugation with folic acid and/or doxorubicin resulted in effective drug delivery targeting. The in vivo results described the toxicology profile toward the zebrafish embryo/larvae, including new data concerning the survival, hatching ratio, and developmental malformations.

Release and cytotoxicity studies of magnetite/Ag/antibiotic nanoparticles: An interdependent relationship

Though the cytotoxic properties of magnetite nanoparticles (NPs) are rather well investigated and known to be dose dependent and rather low, surface functionalization can drastically change their properties. To determine whether the cytotoxicity of magnetite/Ag/antibiotic NPs may be associated, among other things, with iron, silver and antibiotic release, this study investigates the release profiles and cytotoxicity of magnetite/Ag/rifampicin and magnetite/Ag/doxycycline NPs compares it similar profiles from magnetite, magnetite/Ag NPs and antibiotics. It was established that the studied NPs released not only water-soluble substances, such as antibiotics, but also poorly-soluble ones, such as iron and silver. The deposition of silver on the magnetite surface promotes the release of iron by the formation of a galvanic couple. Antibiotic adsorbed on the magnetite/Ag surface plays a dual role in the galvanic corrosion processes: as a corrosion inhibitor for iron oxides and as a corrosion promoter for silver. Magnetite/Ag/rifampicin and magnetite/Ag/doxycycline. NPs were found to have greater cytotoxicity towards the HEK293T cell line than magnetite NPs. These results were attributed to the combined toxic action of the released iron, silver ions and antibiotics. Intensive and simultaneous release of the NP components caused cell stress and suppressed their growth.

Cytotoxicity and imaging studies of β-NaGdF4:Yb3+Er3+@PEG-Mo nanorods

Multimodal imaging based on nanostructures has become a subject of interest for numerous biomedical laboratories. The main focus was placed on applying nanocrystals for the purpose of two types of clinical imaging (contrast and fluorescent agents) due to their excellent luminescence and/or paramagnetic properties. Such systems should also be characterized by low toxicity and high cellular uptake efficiency. Since bare rare earth fluoride nanocrystals influence the cell membrane integrity, it is expected that their coatings will improve biocompatibility profile, as well as increase hydrophilicity, dispersion and chemical stability. Hence, by synthesis of β-NaGdF4:Yb3+Er3+ nanorods (NRs) coated with noncovalently bounded polyethylene glycol monooleate (PEG-Mo), it should be possible to obtain multimodal imaging biomarkers meeting established criteria. Synthesis of β-NaGdF4:Yb3+Er3+@PEG-Mo NRs was performed by the co-precipitation method. These nanostructures were characterized in terms of their size, morphology, zeta potential, magnetic and optical properties as well as their cytotoxicity profile and cellular internalization was evaluated. It was shown that the shape and size of nanocrystals, namely 20 nm nanorods, present generally accepted parameters for biomedical purpose. Ligand attraction of PEG-Mo 860 resulted in the encapsulation of oleic acid coated NRs and formation of hydrophilic bilayer. Superparamagnetic and luminescence properties were highly efficient. Cytotoxic profiles of normal and cancer cell lines were low and determined by dose and time. Cellular uptake was confirmed by the presence of upconversion luminescence in cell interior. These findings are showing multimodal imaging properties of rod shaped β-NaGdF4:Yb3+Er3+@PEG-Mo NRs which may be useful in some biomedical applications.

Cytotoxicity of thermo-responsive polymeric nanoparticles based on N-isopropylacrylamide for potential application as a bioscaffold.

Polymeric nanoparticles based on poly-N-isopropylacrylamide (pNiPAM NPs) and their bio-medical applications have been widely investigated in recent years. These tunable nanoparticles are considered to be great candidates for drug delivery systems, biosensors and bioanalytical devices. Thus, the biocompatibility and toxicity of these nanoparticles is clearly a crucial issue. In this work, the cytotoxicity of thermo-responsive pNiPAM nanoparticles was studied, followed by a detailed analysis of the NPs morphology in growing cell cultures and their 3D structure. Cytotoxic examination was conducted for two cell cultures - HeLa (cervical cancer cell line) and HeK293 (human embryonic kidney cell line), employing MTT (3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) assay and viability tests. We used Cryo-SEM (scanning electron microscopy) and fluorescence microscopy (IN Cell Analyzer) in order to investigate the morphological structure of the polymer network. We show that pNiPAM nanoparticles do not exhibit any cytotoxicity effects on the investigated cell lines. Additionally, we report that the pNiPAM nanoparticle based scaffold promotes cell growth.

EPR Oximetry Sensor—Developing a TAM Derivative for In Vivo Studies

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.

Characterization of three generations of transgenic pigs expressing the HLA-E gene

Abstract The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.