Szilvia Bősze

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

New fluorine-containing hydrazones active against MDR-tuberculosis

Several new fluorine-containing hydrazones were synthesized and screened for their in vitro antimycobacterial activity. Nine of these derivatives have shown a remarkable activity against MDR-TB strain with MIC 0.5 μg/mL and high value of selectivity index (SI). Compound 3h with the highest SI (1268.58) was used for stability evaluation with putative metabolites (ciprofloxacin and formylciprofloxacin) detection. Compound 3h was stable at pH 7.4 of aqueous buffer and rat plasma, in acidic buffers (at pH 3 and 5) slow decomposition was observed. Interestingly, no formylciprofloxacin was detected in the solution, and only slightly increased concentration of ciprofloxacin was observed instead. Trifluoromethyl hydrazones 3f and 3g exhibited the best activity also against two strains of Mycobacterium kansasii (MIC 1-4 μmol/L). All evaluated compounds were found to be non-cytotoxic.

New series of isoniazid hydrazones linked with electron-withdrawing substituents.

A series of new isoniazid hydrazones was synthesized by two procedures. In the first isoniazid was activated with diethoxymethyl acetate and condensed with the appropriate anilines. Alternatively, substituted anilines were activated by diethoxymethyl acetate and subsequently condensed with isoniazid. NMR study confirmed that both synthetic approaches gave the same tautomer. All compounds were screened for in vitro antimycobacterial activity. Most of them exhibited the same activity against Mycobacterium tuberculosis (MIC 1 μmol L(-1)) as isoniazid (INH), better activity against Mycobacterium kansasii 325/80 (MIC 0.125-0.250 μmol L(-1)), high value of selectivity index (SI) and IC(50) between 0.0218 and 0.326 mmol L(-1). Compound 2o with the best SI was used as a model compound for the stability test and was found to be stable at neutral pH, but under acidic conditions it slowly hydrolysed.

Complex forming competition and in-vitro toxicity studies on the applicability of di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) as a metal chelator

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is a potential candidate in chelation therapy as an iron chelator. This study showed that a combined treatment with 2μM easily available Fe(II), Cu(II) and Zn(II) each and 5μM Dp44mT on eight different cancer cell lines resulted in a 10-40-fold increase in the intracellular Cu content compared to control samples. The uptake of Cu and Cu-dependent cytotoxicity strictly depend on the Cu concentration of the culture medium. Even as low concentration of Dp44mT as 0.1μM can transport high amounts of copper inside the cells. The Cu accumulation and toxicity through Dp44mT can hardly be influenced by Fe. Copper uptake and toxicity triggered by 2μM extracellular Cu(II) and 5μM Dp44mT could not be influenced by Fe(II) extracellular concentrations even 50-times higher than that of Cu(II). A 50-times higher Co(II) extracellular concentration hindered the Cu(II) uptake almost completely and a 10-times higher Co(II) concentration already decreased the Dp44mT-mediated Cu toxicity. Conditional complex stability constant determinations for Dp44mT with Cu(II), Co(II), Fe(II), Ni(II) and Zn(II) revealed that the metal-to-ligand ratio is 1:1 in [Cu(II)Dp44mT] complex, while for Co(II), Fe(II) and Ni(II) is 1:2. The highest stability constant was obtained for Cu(II) (lg β=7.08±0.05) and Co(II) (lg β2=12.47±0.07). According to our results, Dp44mT in combination with Cu is highly toxic in vitro. Therefore, the use of Dp44mT as an iron chelator is limited if biologically available Cu is also present even at low concentrations.

Anthracycline-GnRH derivative bioconjugates with different linkages: Synthesis, in vitro drug release and cytostatic effect

To increase the selectivity and consequently to minimize the side effects of chemotherapeutic agents, receptor mediated tumor targeting approaches have been developed. In the present work, various anthracycline-GnRH derivative bioconjugates were synthesized with the aim of investigating the influence of (i) different anthracycline anticancer drugs, (ii) different linkages between the targeting moiety and the anticancer drug, and (iii) different targeting moieties (e.g., GnRH-III and [D-Lys⁶]-GnRH-I) on their in vitro drug release and cytostatic effect. The anthracyclines, daunorubicin or doxorubicin, were attached to the ε-amino group of Lys of GnRH-III or [D-Lys⁶]-GnRH-I through oxime, hydrazone or ester bonds. In another bioconjugate, a self-immolative p-aminobenzyloxycarbonyl spacer was used to link daunorubicin to GnRH-III. The in vitro degradation of the bioconjugates was investigated in the presence of rat liver lysosomal homogenate and cathepsin B. The cellular uptake of the compounds was evaluated by flow cytometry and their in vitro cytostatic effect was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. The results indicate that on the tested cancer cell lines there is no significant difference in the cellular uptake and in vitro cytostatic effect of bioconjugates containing GnRH-III or [D-Lys⁶]-GnRH-I as a targeting moiety. The bioconjugates containing ester bond, hydrazone bond and the self-immolative spacer exert the highest cytostatic effect, followed by oxime bond-linked compounds.

Nanoparticle Encapsulated Lipopeptide Conjugate of Antitubercular Drug Isoniazid: In Vitro Intracellular Activity and in Vivo Efficacy in a Guinea Pig Model of Tuberculosis

Considering that Mycobacterium tuberculosis (Mtb) can survive in host phagocytes for decades and currently applied drugs are largely ineffective in killing intracellular Mtb, novel targeted delivery approaches to improve tuberculosis chemotherapy are urgently needed. In order to enhance the efficacy of a clinically used antitubercular agent (isoniazid, INH) a novel lipopeptide carrier was designed based on the sequence of tuftsin, which has been reported as a macrophage-targeting molecule. The conjugate showed relevant in vitro activity on Mtb H37Rv culture with low cytotoxicity and hemolytic activity on human cells. The conjugate directly killed intracellular Mtb and shows much greater efficacy than free INH. To improve bioavailability, the conjugate was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles and tested in vivo in a guinea pig infection model. External clinical signs, detectable mycobacterial colonies in the organs, and the histopathological findings substantiate the potent chemotherapeutic effect of orally administered conjugate-loaded nanoparticles.

Enhanced cellular uptake of a new, in silico identified antitubercular candidate by peptide conjugation

Mycobacterium tuberculosis is a successful pathogen, and it can survive in infected macrophages in dormant phase for years and decades. The therapy of tuberculosis takes at least six months, and the slow-growing bacterium is resistant to many antibiotics. The development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies is urgently needed. In silico docking methods and structure-based drug design are useful bioinformatics tools for identifying new agents. A docking experiment to M. tuberculosis dUTPase enzyme, which plays a key role in the bacterial metabolism, has resulted in 10 new antitubercular drug candidates. The uptake of antituberculars by infected macrophages is limited by extracellular diffusion. The optimization of the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5 coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H37Rv culture in vitro; the chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5-OT20 tuftsin conjugate exhibited significant antimycobacterial activity on M. tuberculosis H37Rv infected MonoMac6 cells and inhibited intracellular bacteria.

Antimycobacterial activity of peptide conjugate of pyridopyrimidine derivative against Mycobacterium tuberculosis in a series of in vitro and in vivo models

New pyridopyrimidine derivatives were defined using a novel HTS in silico docking method (FRIGATE). The target protein was a dUTPase enzyme (EC 3.6.1.23; Rv2697) which plays a key role in nucleotide biosynthesis of Mycobacterium tuberculosis (Mtb). Top hit molecules were assayed in vitro for their antimycobacterial effect on Mtb H37Rv culture. In order to enhance the cellular uptake rate, the TB820 compound was conjugated to a peptid-based carrier and a nanoparticle type delivery system (polylactide-co-glycolide, PLGA) was applied. The conjugate had relevance to in vitro antitubercular activity with low in vitro and in vivo toxicity. In a Mtb H37Rv infected guinea pig model the in vivo efficacy of orally administrated PLGA encapsulated compound was proven: animals maintained a constant weight gain and no external clinical signs of tuberculosis were observed. All tissue homogenates from lung, liver and kidney were found negative for Mtb, and diagnostic autopsy showed that no significant malformations on the tissues occurred.

Synthesis and in vitro biological evaluation of 2-(phenylcarbamoyl)phenyl 4-substituted benzoates.

Based on the previously described antimicrobial activity of salicylanilide derivatives, we designed and synthesized novel 2-(phenylcarbamoyl)phenyl 4-substituted benzoates. The most active salicylanilides were selected for esterification by various 4-substituted benzoic acids. These compounds were evaluated in vitro against Mycobacterium tuberculosis, including multidrug-resistant strains, nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii), and eight bacterial and fungal strains. We also investigated the cytostatic and cytotoxic actions of the esters. The minimum inhibitory concentrations (MICs) against mycobacteria ranged from 0.125 to 8μM. Interestingly, the drug-resistant strains exhibited the highest susceptibility without any cross-resistance with established drugs. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 4-nitrobenzoate showed the most potent inhibition with MIC values ranging from 0.25 to 2μM. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited by two derivatives with MIC values of at least 0.49μM, whereas Gram-negative bacteria and most of the tested fungi did not display any marked susceptibility. Benzoates exhibited no cytotoxicity at concentrations up to 50μM but most caused significant cytostasis with IC50 values lower than 10μM. Some cytotoxicity-based selectivity indexes for drug-susceptible and drug-resistant M. tuberculosis as well as Staphylococci were higher than 100. These values indicate that some of these derivatives are promising candidates for future research.

A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces

Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Seri-DL-Alam)] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss® bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.