Kantajan ja endosytoosireitin merkitys viruksettomassa geeninsiirrossa

Gene therapy offers promise for the treatment of both inherited and acquired diseases through the introduction of genetic material into target cells. The primary challenge for gene therapy is to develop a safe and efficient method for the delivery of therapeutic genetic material to the specific intracellular target. Non-viral carriers have received significant attention because of their potential to overcome the limitations of viral-based systems. However, their relatively low efficacy is a major obstacle to their clinical application. A thorough understanding of the key factors affecting the gene delivery process will provide clues on how to develop more effective carriers. This thesis focuses on the role of the carrier and the endocytic pathway in non-viral gene delivery, and also suggests improvements in the experimental methodology that would make it possible to obtain more reliable results in nanoparticle uptake studies. More effective carriers for gene delivery are very much needed. We tested the novel pentaspheric lysine-based dendrimer and its analog, modified with fatty acid residues, for their gene delivery capacity. We demonstrated that despite their relatively low in vitro transfection efficacy, lysine dendrimers have good plasmid DNA (pDNA) binding and protective properties, and can therefore be used as the basis for the development of more effective carriers. A detailed understanding of the cellular kinetics of gene delivery systems is critical to the further development of more effective carriers. We studied the impact of the carrier and of the endocytic pathway on cell uptake and the intracellular processing of genetic material (pDNA). The highly sensitive method of quantitative real-time PCR was applied to the study of the intracellular kinetics of pDNA introduced by the cationic polymer PEI, cationic lipid DOTAP, and CaP precipitates at multiple time points after transfection. The results obtained indicate that the carrier affects the cell uptake and the intracellular kinetics, and therefore predetermines the main transfection-limiting step. Furthermore, we demonstrated the important role of the post-nuclear processes in efficient non-viral gene delivery. The impact of a specific endocytic pathway was studied by the inhibition of either the clathrin- or dynamin-mediated endocytosis. Selective blockage of endocytosis was achieved by two approaches, namely the genetically manipulated cell lines and the chemical inhibitors of endocytosis. Analysis of the intracellular kinetics of pDNA in the genetically blocked cells revealed that neither the amount of pDNA taken up by the cell, nor the intracellular pDNA elimination, but the amount of pDNA delivered to the nucleus was indicative of the significance of the particular pathway in the resulting efficacy of the carrier. A comparison of chemical and genetic means for blocking endocytosis revealed the limitations of both these methods. A careful optimization of the method and the use of several alternative approaches is recommended in order to obtain more reliable data. We suggest that the characterization of in vitro cell models for the expression and activity of specific endocytic pathways (endocytic profiling) would facilitate the interpretation of the data obtained in nanoparticle uptake studies. Endothelial and epithelial cells are widely utilized in such studies because they form substantial barriers en route from the administration site to the target tissue. We performed endocytic profiling of the epithelial CaCo-2 cells and the endothelial hCMEC/D3 cells at different stages of differentiation. The expression of genes involved in specific endocytic pathways was analysed at the mRNA level by quantitative real-time PCR, and at the protein level by Western blotting. The endocytic activities of the cells were analyzed by flow cytometry. We concluded that the mRNA expression of the endogenous proteins involved in particular endocytic pathways can be indicative of the expression and activity of these pathways. Furthermore, we showed that the differentiation status of the cells affects their endocytic activity, and must therefore be taken into account when designing nanoparticle uptake and transcellular permeability experiments. A comparison of the endocytic profiles of cell lines with primary cells revealed clear discrepancies, pointing to the importance of careful selection of in vitro models for endocytosis-related studies. Overall, our study has improved the mechanistic understanding of the non-viral gene delivery process. We have described how the carrier and endocytic pathway both affect intracellular kinetics and the efficacy of gene transfer. Furthermore, we have demonstrated the importance of method optimization and the endocytic profiling of in vitro cell models in improving the quality of the obtained data.Geeniterapiassa sairautta hoidetaan korjaamalla proteiinituotannon häiriö DNA- tai RNA-tasolla. Geeniterapia onkin lupaava hoitomuoto erilaisten sekä perinnöllisten että ei-perinnöllisten sairauksien hoidossa. Geeniterapiassa geneettinen materiaali kuten DNA täytyy kuljettaa kohdekudokseen ja edelleen kohdesolujen sisällä kohdeorganelliin. Tämä ei onnistu ilman kantajaa, jonka tehtävänä on geneettisen materiaalin suojaus ja kuljetus kohteeseen. Tällä hetkellä geeniterapian suurimpana haasteena onkin löytää tehokas ja turvallinen kantaja. Viruspohjaiset kantajat ovat tehokkaita, mutta niiden turvallisuuteen ja laajamittaiseen tuotantoon liittyy paljon ongelmia. Laajasti tutkituilla viruksettomilla geeninkuljettimilla on turvallisuuteen ja tuotantoon liittyviä etuja viruspohjaisiin kuljettimiin nähden, mutta niiden kliinistä käyttöä on rajoittanut tehottomuus. Tästä syystä geeninsiirron mekanismien syvällinen ymmärtäminen on tärkeää ja mahdollistaa tehokkaampien kuljettimien kehittämisen. Tässä väitöskirjatyössä selvitettiin kantajan ja soluunotossa keskeisten endosytoosireittien merkitystä viruksettomassa geeninsiirrossa sekä vertailtiin erilaisia kokeellisia menetelmiä nanopartikkelien solunotonmekanismien selvittämisessä. Väitöskirjatyössä testattiin uusia rasvahappo tähteillä muokattuja lysiinipohjaisia denrimeerejä ja niiden johdannaisia geeninkuljettimina. Kokeet osoittivat tutkittujen kantajien sitovan ja suojaavan hyvin DNA:ta itse geeninsiirtotehokkuuden jäädessä kuitenkin suhteellisen heikoksi. Saatua tietoa voidaan kuitenkin hyödyntää tehokkaampien kantajien kehittämisessä. Kantajien geeninsiirron solunsisäisen kinetiikan ymmärtäminen on tärkeää. Väitöskirjatyössä selvitettiin erilaisten yleisesti käytettyjen viruksettomien kantajien (positiivinen polymeeri PEI, positiivinen liposomi DOTAP ja kalsiumfosfaattikiteet) solunoton ja solunsisäisen kulkeutumisen kinetiikkaa mittaamalla siirtogeenin määrää eri solunosissa erittäin herkällä reaaliaikaisella PCR menetelmällä. Tulokset osoittivat kantajien määrittävän solunsisäisen käyttäytymisen ja tätä kautta geeninsiirtotehokkuuden. Lisäksi itse siirtogeenin ilmentymisen todettiin olevan geeninsiirron tehokkuutta rajoittava tekijä. Samassa yhteydessä tutkittiin endosytoosireitin merkitystä geeninsiirron solunsisäiseen kinetiikkaan inhiboimalla reittejä spesifisesti joko hyödyntämällä geneettisesti manipuloituja solulinjoja tai altistamalla solut kemiallisille inhibiittoreille. Molempien menetelmien käytössä todettiin puutteita ja tästä syystä menetelmien huolellinen optimointi ja useiden menetelmien käyttö mahdollistaa luotettavien tulosten saamisen. Solut ilmentävät erilaisia endosytoosireittejä eri tasolla ja tästä syystä nanopartikkelien soluunottotulosten tulkinnan helpottamiseksi on tärkeää karakterisoida kokeissa käytetyt solumallit endosytoosireittien ilmentymisen ja toiminnallisuuden suhteen. Epiteeli- ja endoteelisolumallit ovat tärkeitä työkaluja tutkittaessa nanopartikkelien kulkeutumista. Tässä väitöskirjatyössä karakterisoitiin epiteelisolumallin (CaCo-2) ja endoteelisolumallin (hCMEC/D3) endosytoosireittien ilmentyminen että toiminnallusuus solujen erilaistumisen eri vaiheissa. Ilmentymistutkimukset tehtiin sekä mRNA- että proteiinitasolla. RNA-tason mittaukset antoivat melko hyvän kuvan eri reittien toiminnallisuudesta ja tästä syystä ne soveltuvat hyvin endosytoosireittien nopeaan seulontaan. Solujen erilaistumisasteella on suuri vaikutus endosytoosireittien aktiivisuuteen. Tämä on tärkeää ottaa huomioon soluunottokokeita suunniteltaessa

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.Peer reviewe

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition

Synthesis, Structure and Biological Activity of Novel 4,5-dihydro-1H-imidazol-2-yl-phthalazine Derivatives and Their Copper(II) Complexes

As a continuation of our previous investigations aimed at the synthesis of novel nitrogen-containing heterocycles and their metal complexes, we have now prepared two series of compounds incorporating a phthalazine ring at the position C2 of 4,5-dihydro-1H-imidazole. The starting phthalazine (I) in the reaction with 2-chloroimidazoline (II) gives rise to the formation of pseudobase III. Then, compound III upon treatment with HOSA yields betaine which under basic conditions gives 2-(4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-imine (IV). In turn, the reactions of compound IV with a variety of acyl and sulfonyl chlorides lead to the formation of benzamides (V) and benzenesulfonamides (VI). Moreover, compounds V and VI can be transformed into corresponding 2-(4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-one derivatives VII and VIII. Such ligands are susceptible to the reaction with CuCl2 giving rise to the formation of corresponding copper(II) complexes: dichloro[2-(4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-imine]copper(II) (1), dichloro[2-(1-benzoyl-4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-one]copper(II) (2) and dichloro{bis-[2-(1-(phenylsulfonyl)-4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-one]}copper(II) (3). The most promising results of biological studies were obtained for complex 1 towards the HeLa cell line (IC50 = 2.13 μM) without a toxic effect against fibroblasts BALB/3T3 (IC50 = 135.30 μM), which pointed towards its selectivity as a potential antitumor agent. It should be pointed out, that corresponding free ligand 2-(4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-imine (IV) was less active than its metal complex (IC50 = 87.74 μM)

Separation of fiber bundles from willow bark using sodium bicarbonate and their novel use in yarns for superior UV protection and antibacterial performance

The development of a mild and green method for separating natural fiber bundles from willow bark is an essential step in exploring and preserving their natural functions. The isolation of well-oriented fiber bundles from the bark of a fast-growing willow hybrid solely using sodium bicarbonate under mild conditions was successfully demonstrated. Additionally, Lyocell fibers were mixed with an equal amount of the willow bark fiber bundles and proved their ability to convert into spun yarns, which provided excellent protection for ultraviolet radiation (UPF > 140). Moreover, these yarns demonstrated strong antibacterial activity (A > 8) against the Gram-positive pathogen Staphylococcus aureus, resulting in complete eradication of viable bacteria after 24 -h incubation with the material. A laundering treatment had no effect on the UV protection or the antibacterial performance. Utilizing these inherent properties from natural fibers for technical textile applications is very promising.Peer reviewe

Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs

Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC50 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure–activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed

Effect of Hybrid Type and Harvesting Season on Phytochemistry and Antibacterial Activity of Extracted Metabolites from Salix Bark

Hundreds of different fast-growing Salix hybrids have been developed mainly for energy crops. In this paper, we studied water extracts from the bark of 15 willow hybrids and species as potential antimicrobial additives. Treatment of ground bark in water under mild conditions extracted 12-25% of the dry material. Preparative high-performance liquid chromatography is proven here as a fast and highly efficient tool in the small-scale recovery of raffinose from Salix bark crude extracts for structural elucidation. Less than half of the dissolved material was assigned by chromatographic (gas chromatography and liquid chromatography) and spectroscopic (mass spectrometry and nuclear magnetic resonance spectroscopy) techniques for low-molecular-weight compounds, including mono- and oligosaccharides (sucrose, raffinose, and stachyose) and aromatic phytochemicals (triandrin, catechin, salicin, and picein). The composition of the extracts varied greatly depending on the hybrid or species and the harvesting season. This information generated new scientific knowledge on the variation in the content and composition of the extracts between Salix hybrids and harvesting season depending on the desired molecule. The extracts showed high antibacterial activity on Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.6-0.8 mg/mL; however, no inhibition was observed against Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. MIC of triandrin (i.e., 1.25 mg/mL) is reported for the first time. Although antibacterial triandrin and (+)-catechin were present in extracts, clear correlation between the antibacterial effect and the chemical composition was not established, which indicates that antibacterial activity of the extracts mainly originates from some not yet elucidated substances. Aquatic toxicity and mutagenicity assessments showed the safe usage of Salix water extracts as possible antibacterial additives.Peer reviewe

Phototoxicity of BODIPY in long-term imaging can be reduced by intramolecular motion

For long-term live-cell fluorescence imaging and biosensing, it is crucial to work with a dye that has high fluorescence quantum yield and photostability without being detrimental to the cells. In this paper, we demonstrate that neutral boron-dipyrromethene (BODIPY)-based molecular rotors have great properties for high-light-dosage demanding live-cell fluorescence imaging applications that require repetitive illuminations. In molecular rotors, an intramolecular rotation (IMR) allows an alternative route for the decay of the singlet excited state (S1) via the formation of an intramolecular charge transfer state (CT). The occurrence of IMR reduces the probability of the formation of a triplet state (T1) which could further react with molecular oxygen (3O2) to form cytotoxic reactive oxygen species, e.g., singlet oxygen (1O2). We demonstrate that the oxygen-related nature of the phototoxicity for BODIPY derivatives can be significantly reduced if a neutral molecular rotor is used as a probe. The studied neutral molecular rotor probe shows remarkably lower phototoxicity when compared with both the non-rotating BODIPY derivative and the cationic BODIPY-based molecular rotor in different light dosages and dye concentrations. It is also evident that the charge and localization of the fluorescent probe are as significant as the IMR in terms of the phototoxicity in a long-term live-cell imaging. Graphical abstract: [Figure not available: see fulltext.].publishedVersionPeer reviewe

Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives

Peer reviewe