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Surface functionalization of the biological gold nanoparticles for micro-rna targeting
Pourali, Parastoo ; Benada, Oldřich ; Benson, Veronika
Among non-viral gene carriers with low toxicity and high transfection efficiency, the use of gold nanoparticles (AuNPs) is of particular interest due to their biocompatibility and special properties. This is the first time we attempted to functionalize the surface of the biological AuNPs in order to conjugate them with antimiR-135b through electrostatic interactions and knockdown the microRNA-135b gene expression inside the cells. A fungal strain, Fusarium oxysporum, was cultured in Sabouraud Dextrose Broth (SDB), centrifuged, and the mycelium-free supernatant was challenged with 1 mmol final concentration of HAuCl4.3H2O and incubated for 24 h at 37°C in a shake flask. AuNPs were characterized by visible spectrophotometry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDS), and a zetasizer. The washed and sterilized AuNPs were used for cytotoxicity and conjugation assays. First transferrin (Tf) and then polyethylenimine (PEI) were used to functionalize and change the surface charge of the AuNPs and then antimiR-135b was conjugated to the AuNPs trough electrostatic interactions. Their association was confirmed by visible spectrophotometry and electrophoresis. Confocal microscopy was used to investigate the internalization of the AuNPs-antimiR-135b complex. The results proved the formation of AuNPs with a maximum absorption peak at 528 nm, round and oval shapes (15-20 nm), and average zeta potential of -21.02 mV. The AuNPs-antimiR-135b showed delayed electrophoresis unlike antimiR-135b or AuNPs alone. Functionalized AuNPs did not cause any toxicity in cell culture and confocal microscopy showed successful transfection of AuNPs-antimiR-135b into the vast majority of 4T1 cells. We concluded that the biological AuNPs were non-toxic and they could carry antimiR-135b to enable gene silencing
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The Cell-Nanomaterial Interactions and Their Application in Biomedicine
Vrabcová, Lucie ; Hubálek Kalbáčová, Marie (advisor) ; Pešta, Martin (referee) ; Benson, Veronika (referee)
Nanomaterials entered the biomedicine already at the beginning of the millennium and they still bring new and unique advances and possibilities for treatment, diagnosis and regeneration, thus they continue to facilitate the development of personalized medicine. Interactions at nanoscale allow until then unconceivable opportunities to influence processes on molecular level. The completely new perspectives of nanomaterial applications jointly raise attention concerning health and environmental safety issues. Although a lot of novel biomedical applications of nanomaterials have emerged, the vast majority is still found to be at the stage of a concept. The consistent basic in vitro research of elemental interactions of nanomaterial with biological environment should represent an essential part of its development. The concern of this thesis was to describe the cytocompatibility and interactions of two types of nanomaterials with different human cells. First, ultra-fine grain titanium was tested for prospective use in implant development. We confirmed its positive effect mainly on the growth of osteoblasts and recommended the further pre-clinical trials of this material in a form of a bone or dental implant. Second, several types of ultra-small (˂ 5 nm) nanoparticles of different origin (silicon, gold...
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Targeted biocompatible nanoparticles for therapy and cancer diagnostics.
Neburková, Jitka ; Cígler, Petr (advisor) ; Benson, Veronika (referee) ; Schirhagl, Romana (referee)
Nanoparticles (NPs) have considerable potential in targeted medicine. NPs can merge various functions and serve as labels for imaging or as nanocarriers in therapy. Modification of NPs with targeting ligands can lead to highly specific interactions with targeted cancer cells. However, the efficacy of targeting depends on the ratio between specific and non-specific interactions of a NP with the cell. Non-specific interactions of NPs are unrelated to targeted receptors and need to be eliminated in order to decrease background noise during imaging and adverse effect of drugs on healthy tissues. In this thesis, surface modifications of NPs were explored mainly on biocompatible carbon NPs called nanodiamonds (NDs), which have exceptional fluorescent properties such as long fluorescence lifetime, no photobleaching and photoblinking and sensitivity of their fluorescence to electric and magnetic field. Main issues addressed in this thesis are low colloidal stability of NDs in buffers and media, their non-specific interactions with proteins and cells and limited approaches for ND surface modifications. These issues were solved by coating NDs with a layer of biocompatible, hydrophilic, and electroneutral poly(ethylene glycol) or poly[N-(2- hydroxypropyl) methacrylamide] polymers. Optimized polymer coating...
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Biocompatibility of porous NCD layers with neurons
Freislebenová, Hana ; Benson, Veronika (advisor) ; Rudajev, Vladimír (referee)
Nanodiamond is currently one of the most researched materials in the field of regenerative medicine in the study of treatment of neurodegenerative diseases. Due to direct interaction of this material with nerve tissue, it needs to be biocompatible with primary neurons. Furthemore, it is desirable for the used material to induce cell adhesion and stimulate the adherent cells to regeneration. This work evaluates the biocompatibility of porous boron-doped diamond layers with the culture of primary neurons. We compared the effect of diamond surface treatment by poly- D-lysine (PDL) coating on the adhesion and prosperity of the primary neuronal culture. We found that the tested nanodiamond layers are biocompatible with the primary neuronal culture. We further conclude that the PDL coating slightly increases the rate of cell adhesion to the surface but on the other hand induces greater adhesion of glial cells than the surface without PDL coating. Key words: nanodiamond, biocompatibility, adhesion, neurons
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Interactions of cells with nanoparticles for bio-medical applications
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Benson, Veronika (referee) ; Topinka, Jan (referee)
In the past decades, nanoparticles have been viewed as a potentially powerful platform for various applications in biomedical sciences. The possible application of nanoparticles varies from drug delivery agents to novel imaging platforms and surely, some application potential still remains hidden. Thus, it is necessary to broadly study their in vitro behavior in order to assess the precise theranostic potential as well as to distinguish possible threats to human health. Even though nanoparticles are getting more and more attention in current research, still only a limited amount of information is available, especially regarding interactions of ultra-small (< 5 nm) nanoparticles with biological environment and cells. The aim of the work presented herein is to provide the reader with information concerning interactions of various ultra-small nanoparticles (silicon-based, gold, nanodiamonds) with biological environment and human cells. Dose- and time-dependent influence of the various nanoparticles on behavior of different human cells (osteoblasts, monocytes, keratinocytes, mesenchymal stem cells) was established under different conditions, stressing out the importance of protein corona (a layer of proteins originating from cultivation medium attached to nanoparticles). Biocompatibility of two...
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Histone deacetylase inhibitors in plasma cell leukemia treatment: effect of the bone marrow microenvironment
Burianová, Ilona ; Stöckbauer, Petr (advisor) ; Pour, Luděk (referee) ; Benson, Veronika (referee)
Multiple myeloma and its aggressive variant, plasma cell leukemia, are still considered to be incurable diseases despite the progressive treatment approaches comprising novel drugs. This can be attributed to the presence of the bone marrow microenvironment which plays an important role in drug resistance of myeloma cells. Hematopoietic cell lines derived from hematologic malignancies are suitable models for the study of etiopathogenesis of these malignant diseases and for testing new potential drugs. Establishment of these cell lines is still considered to be coincidental and rare event. The first part of the thesis is focused on establishment and characterization of the cell line UHKT-944 derived from a patient with primary plasma cell leukemia, and on completion of characterization of the cell line UHKT-893 derived from a patient with multiple myeloma. Additional analysis of UHKT-893 cell line were performed including sequence analysis of IgVH gene rearrangements and cytogenetic analysis which contributed to more detailed characterization of this cell line. During cultivation of UHKT-944 cells, we monitored the cell growth and confirmed dependence on interleukin-6 (IL-6). Immunophenotype analysis revealed the presence of surface markers characteristic of malignant plasma cells. UHKT-944 cells...
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The study of signaling pathways that modulate multidrug resistance
Dvořák, Pavel ; Souček, Pavel (advisor) ; Daum, Ondřej (referee) ; Benson, Veronika (referee)
The study of signaling pathways that modulate multidrug resistance The theme of cancer cell resistance to anti-cancer drugs including the common mechanisms of resistance development and the theory of cancer stem cells was introduced in the Introduction to the doctoral thesis. The theoretical part was focused more deeply on the two topics - the role of ATP-binding cassette (ABC) transport proteins and chromosomal abnormalities in the development of cancer chemoresistance. The possible therapeutic potential for the treatment of cancer was stressed for both topics. The Results were composed of the commentaries on the five published works, which the author of the thesis conducted as the main author. The first work brought the evidence supporting the hypothesis of the existence of ABC gene expression profiles (signatures), which are common to multiple types of tumors and are associated with significant clinical consequences. These general ABC gene expression profiles could possibly form a new hallmark of cancer. The second work studied more closely a group of acute myeloid leukemia patients, who did not achieve complete cytogenetic remission after two attempts to maintain remission of the malignant disease. The new entity, consisting of patients with the translocation t(2;11)(p21;q23) without the...
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Use of nanodiamond carriers for gene therapy
Křivohlavá, Romana ; Benson, Veronika (advisor) ; Černý, Jan (referee)
Nanodiamonds (ND) serve as RNA carriers with potential for in vivo application. ND coatings and their administration strategy significantly change their fate, toxicity, and effectivity within a multicellular system. Our goal was to develop multiple ND coating for effective RNA delivery in vivo. Our final complex (NDA135b) consisted of ND, polymer, antisense RNA, and transferrin. We aimed (i) to assess if a tumor-specific coating promotes NDA135b tumor accumulation and effective inhibition of oncogenic microRNA-135b and (ii) to outline off-targets and immune cell interactions. First, we tested NDA135b toxicity and effectiveness in tumorospheres co- cultured with immune cells ex vivo. We found NDA135b to target tumor cells, but also to interact with granulocytes. Then, we followed with NDA135b intravenous and intratumoral applications in tumor-bearing animals in vivo. Application of NDA135b in vivo led to the effective knockdown of microRNA-135b in tumor tissue regardless of administration. Only intravenous application resulted in NDA135b circulation in peripheral blood and urine and it decreased granularity of splenocytes. Our data showed that localized intratumoral application of NDA135b represented a suitable and safe approach for in vivo application of nanodiamond- based constructs. Systemic...
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Biocompatibility of porous NCD layers with neurons
Freislebenová, Hana ; Benson, Veronika (advisor) ; Rudajev, Vladimír (referee)
Nanodiamond is currently one of the most researched materials in the field of regenerative medicine in the study of treatment of neurodegenerative diseases. Due to direct interaction of this material with nerve tissue, it needs to be biocompatible with primary neurons. Furthemore, it is desirable for the used material to induce cell adhesion and stimulate the adherent cells to regeneration. This work evaluates the biocompatibility of porous boron-doped diamond layers with the culture of primary neurons. We compared the effect of diamond surface treatment by poly- D-lysine (PDL) coating on the adhesion and prosperity of the primary neuronal culture. We found that the tested nanodiamond layers are biocompatible with the primary neuronal culture. We further conclude that the PDL coating slightly increases the rate of cell adhesion to the surface but on the other hand induces greater adhesion of glial cells than the surface without PDL coating. Key words: nanodiamond, biocompatibility, adhesion, neurons
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Interactions of cells with nanoparticles for bio-medical applications
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Benson, Veronika (referee) ; Topinka, Jan (referee)
In the past decades, nanoparticles have been viewed as a potentially powerful platform for various applications in biomedical sciences. The possible application of nanoparticles varies from drug delivery agents to novel imaging platforms and surely, some application potential still remains hidden. Thus, it is necessary to broadly study their in vitro behavior in order to assess the precise theranostic potential as well as to distinguish possible threats to human health. Even though nanoparticles are getting more and more attention in current research, still only a limited amount of information is available, especially regarding interactions of ultra-small (< 5 nm) nanoparticles with biological environment and cells. The aim of the work presented herein is to provide the reader with information concerning interactions of various ultra-small nanoparticles (silicon-based, gold, nanodiamonds) with biological environment and human cells. Dose- and time-dependent influence of the various nanoparticles on behavior of different human cells (osteoblasts, monocytes, keratinocytes, mesenchymal stem cells) was established under different conditions, stressing out the importance of protein corona (a layer of proteins originating from cultivation medium attached to nanoparticles). Biocompatibility of two...
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