Stimulus-sensitive polymer drug conjugates based on high molecular weight N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers carrying doxorubicin via a pH-dependent cleavable bond (pHPMA-Dox) were previously shown to be able to overcome multi-drug resistance. Nevertheless, a tumor type dependent differential response was observed. Although an improved and more selective tumor accumulation of pHPMA-Dox is generally achieved due to the enhanced permeability and retention (EPR) effect, little is known about the fate of these conjugates upon entering the tumor tissue, which could explain the different responses. In this study, we compared in vitro and in vivo accumulation and Dox-activation of pHPMA-Dox in three cancer cell line models (1411HP, A2780cis, HT29) and derived xenograft tumors using a near-infrared fluorescence-labeled pHPMA-Dox conjugate. Firstly, cytotoxicity assays using different pH conditions proved a stepwise, pH-dependent increase in cytotoxic activity and revealed comparable sensitivity among the cell lines. Using multispectral fluorescence microscopy, we were able to track the distribution of drug and polymeric carrier simultaneously on cellular and histological levels. Microscopic analyses of cell monolayers confirmed the assumed mechanism of cell internalization of the whole conjugate followed by intracellular cleavage and nuclear accumulation of Dox in all three cell lines. In contrast, intratumoral distribution and drug release in xenograft tumors were completely different and were associated with different tissue substructures and microenvironments analyzed by Azan- and Hypoxisense®-staining. In 1411HP tumors, large vessels and less hypoxic/acidic microenvironments were associated with a pattern resulting from consistent tissue distribution and cellular uptake as whole conjugate followed by intracellular drug release. In A2780cis tumors, an inconsistent pattern of distribution partly resulting from premature drug release was associated with a more hypoxic/acidic microenvironment, compacted tumor tissue with compressed vessels and specific pre-damaged tissue structures. A completely different distribution pattern was observed in HT29 tumors, resulting from high accumulation of polymer in abundant fibrotic structures, with small embedded vessels featuring this tumor type together with pronounced premature drug release due to the strongly hypoxic/acidic microenvironment. In conclusion, the pattern of intratumoral distribution and drug release strongly depends on the tumor substructure and microenvironment and may result in different degrees of therapeutic efficacy. This reflects the pronounced heterogeneity observed in the clinical application of nanomedicines and can be exploited for the future design of such conjugates.
- Klíčová slova
- HPMA copolymer, chemotherapy resistance, pH-sensitive drug release, polymer drug conjugates, tumor microenvironment,
- MeSH
- buňky HT-29 MeSH
- doxorubicin aplikace a dávkování chemie farmakokinetika MeSH
- fluorescenční barviva chemie MeSH
- karbocyaniny chemie MeSH
- koncentrace vodíkových iontů MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- methakryláty chemie MeSH
- molekulová hmotnost MeSH
- myši nahé MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí MeSH
- nosiče léků aplikace a dávkování chemie farmakokinetika MeSH
- protinádorové látky aplikace a dávkování chemie farmakokinetika MeSH
- tkáňová distribuce MeSH
- uvolňování léčiv MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- doxorubicin MeSH
- fluorescenční barviva MeSH
- hydroxypropyl methacrylate MeSH Prohlížeč
- indotricarbocyanine MeSH Prohlížeč
- karbocyaniny MeSH
- methakryláty MeSH
- nosiče léků MeSH
- protinádorové látky MeSH
Visualization of membrane domains like lipid rafts in natural or artificial membranes is a crucial task for cell biology. For this purpose, fluorescence microscopy is often used. Since fluorescing probes in lipid membranes partition specifically in e.g. local liquid disordered or liquid ordered environments, the consequent changes in their orientation and location are both theoretically and experimentally of interest. Here we focused on a liquid disordered membrane phase and performed molecular dynamics (MD) simulations of the indocarbocyanine DiD probes by varying the length of the attached alkyl tails and also the length of the cyanine backbone. From the probed compounds in a DOPC lipid bilayer at ambient temperature, a varying orientation of the transition dipole moment was observed, which is crucial for fluorescence microscopy and which, through photoselection, was found to be surprisingly more effective for asymmetric probes than for the symmetric ones. Furthermore, we observed that the orientation of the probes was dependent on the tail length; with the methyls or propyls attached, DiD oriented with its tails facing the water, contrary to the ones with longer tails. With advanced hybrid QM/MM calculations we show that the different local environment for differently oriented probes affected the one-photon absorption spectra, that was blue-shifted for the short-tailed DiD with respect to the DiDs with longer tails. We show here that the presented probes can be successfully used for fluorescence microscopy and we believe that the described properties bring further insight for the experimental use of these probes.
- Klíčová slova
- Absorption, Cyanine probe, DiD, Fluorescence spectroscopy, Hybrid quantum mechanics – molecular mechanics, Lipid bilayer,
- MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie MeSH
- karbocyaniny chemie MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3,3'-dioctadecylindocarbocyanine MeSH Prohlížeč
- fluorescenční barviva MeSH
- karbocyaniny MeSH
- lipidové dvojvrstvy MeSH
The fluorescent probes based on Tröger's base motive with both coumarin and cyanine substitution 11-13 have been synthesized by multi-step synthesis in high overall yields. Intracellular localization of prepared probes have been tested using four different cell lines (HF-P4, BLM, U-2 OS and A-2058). Prepared probes have intensive green and red fluorescence. Co-localization with commercial lysosome specific marker LysoTracker Blue DND 22 has been confirmed that all prepared fluorescent probes labeled lysosomal compartment with high selectivity and probes show excellent brightness at low concentration.
- Klíčová slova
- Coumarin, Cyanine dye, Fluorescent probe, Lysosomal probe, Tröger’s base,
- MeSH
- fluorescenční barviva chemická syntéza chemie MeSH
- fluorescenční mikroskopie MeSH
- karbocyaniny chemie MeSH
- kultivované buňky MeSH
- kumariny chemická syntéza chemie MeSH
- lidé MeSH
- lyzozomy chemie MeSH
- molekulární struktura MeSH
- optické zobrazování * MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorescenční barviva MeSH
- karbocyaniny MeSH
- kumariny MeSH
Cancer cells preferentially utilize glycolysis for ATP production even in aerobic conditions (the Warburg effect) and adapt mitochondrial processes to their specific needs. Recent studies indicate that altered mitochondrial activities in cancer represent an actionable target for therapy. We previously showed that salt 1-3C, a quinoxaline unit (with cytotoxic activity) incorporated into a meso-substituted pentamethinium salt (with mitochondrial selectivity and fluorescence properties), displayed potent cytotoxic effects in vitro and in vivo, without significant toxic effects to normal tissues. Here, we investigated the cytotoxic mechanism of salt 1-3C compared to its analogue, salt 1-8C, with an extended side carbon chain. Live cell imaging demonstrated that salt 1-3C, but not 1-8C, is rapidly incorporated into mitochondria, correlating with increased cytotoxicity of salt 1-3C. The accumulation in mitochondria led to their fragmentation and loss of function, accompanied by increased autophagy/mitophagy. Salt 1-3C preferentially activated AMP-activated kinase and inhibited mammalian target of rapamycin (mTOR) signaling pathways, sensors of cellular metabolism, but did not induce apoptosis. These data indicate that salt 1-3C cytotoxicity involves mitochondrial perturbation and disintegration, and such compounds are promising candidates for targeting mitochondria as a weak spot of cancer.
- Klíčová slova
- autophagy, cancer therapy, glucose metabolism, mitochondria,
- MeSH
- chinazoliny chemie farmakologie MeSH
- karbocyaniny chemie MeSH
- kinasy AMP aktivovaných proteinkinas MeSH
- kvartérní amoniové sloučeniny chemie farmakologie MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- mitofagie * MeSH
- nádorové buněčné linie MeSH
- proteinkinasy metabolismus MeSH
- protinádorové látky chemie farmakologie MeSH
- TOR serin-threoninkinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chinazoliny MeSH
- karbocyaniny MeSH
- kinasy AMP aktivovaných proteinkinas MeSH
- kvartérní amoniové sloučeniny MeSH
- proteinkinasy MeSH
- protinádorové látky MeSH
- TOR serin-threoninkinasy MeSH
Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.
- Klíčová slova
- 6-[(3-methylbut-2-en-1-yl)amino]purine, ARR5:GUS, Competitive receptor bioassay, Cytokinin, Fluorescent label, Fluorescent probe, Isoprenoid, Linker, Live cell confocal microscopy, N(6)-isopentenyladenine,
- MeSH
- 4-chlor-7-nitrobenzofurazan farmakologie MeSH
- adenin analogy a deriváty chemie MeSH
- Arabidopsis metabolismus MeSH
- barvicí látky chemie MeSH
- cytokininy chemie farmakologie MeSH
- fluorescenční barviva chemie MeSH
- isopentenyladenosin chemická syntéza chemie farmakologie MeSH
- karbocyaniny chemie MeSH
- konfokální mikroskopie MeSH
- kukuřice setá metabolismus MeSH
- molekulární struktura MeSH
- proteiny huseníčku metabolismus MeSH
- puriny chemie MeSH
- receptory cytokinové antagonisté a inhibitory chemie MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus MeSH
- rhodaminy chemie MeSH
- semenáček metabolismus MeSH
- terpeny metabolismus MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 4-chlor-7-nitrobenzofurazan MeSH
- 6-((3-methylbut-2-en-1-yl)amino)purine MeSH Prohlížeč
- adenin MeSH
- barvicí látky MeSH
- cyanine dye 5 MeSH Prohlížeč
- cytokininy MeSH
- fluorescenční barviva MeSH
- isopentenyladenosin MeSH
- karbocyaniny MeSH
- N(6)-(delta(2)-isopentenyl)adenine MeSH Prohlížeč
- N(6),N(6)-dimethyladenine MeSH Prohlížeč
- proteiny huseníčku MeSH
- purine MeSH Prohlížeč
- puriny MeSH
- receptory cytokinové MeSH
- regulátory růstu rostlin MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy MeSH
- terpeny MeSH
Targeted biocompatible nanostructures with controlled plasmonic and morphological parameters are promising materials for cancer treatment based on selective thermal ablation of cells. Here, core-shell plasmonic nanodiamonds consisting of a silica-encapsulated diamond nanocrystal coated in a gold shell are designed and synthesized. The architecture of particles is analyzed and confirmed in detail using electron tomography. The particles are biocompatibilized using a PEG polymer terminated with bioorthogonally reactive alkyne groups. Azide-modified transferrin is attached to these particles, and their high colloidal stability and successful targeting to cancer cells overexpressing the transferrin receptor are demonstrated. The particles are nontoxic to the cells and they are readily internalized upon binding to the transferrin receptor. The high plasmonic cross section of the particles in the near-infrared region is utilized to quantitatively ablate the cancer cells with a short, one-minute irradiation by a pulse 750-nm laser.
- Klíčová slova
- ablation, cancer, gold, nanodiamonds, plasmonics,
- MeSH
- ablace metody MeSH
- biokompatibilní materiály farmakokinetika MeSH
- cílená molekulární terapie metody MeSH
- HeLa buňky účinky léků MeSH
- indukovaná hypertermie metody MeSH
- karbocyaniny chemie MeSH
- laserová terapie metody MeSH
- lidé MeSH
- nanočástice chemie MeSH
- nanodiamanty chemie MeSH
- nanoslupky chemie MeSH
- polyethylenglykoly chemie MeSH
- receptory transferinu metabolismus MeSH
- transferin chemie farmakologie MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- Alexa Fluor 647 MeSH Prohlížeč
- biokompatibilní materiály MeSH
- karbocyaniny MeSH
- nanodiamanty MeSH
- polyethylenglykoly MeSH
- receptory transferinu MeSH
- transferin MeSH
- zlato MeSH
In this work, we studied indolium and benzothiazolium pentamethine salts 1-3 as novel type of receptors for the recognition of sulphated signalling molecules (sulphated steroids: oestrone, pregnenolone and cholesterol sulphate). A recognition study was performed in an aqueous medium (1mM phosphate buffer (H2O:MeOH; 99:1 (v/v))) at pH 7.34. The tested salts displayed a high affinity for these sulphated analytes, mainly for cholesterol sulphate. However, no interaction between the salts and control, non-sulphated sterol analytes (cholesterol and bile acid) was observed. The highest affinity for the sulphated steroids was observed for benzothiazole salt 1. This salt also displayed different spectral behaviour from that observed for carbocyanine salts 2 and 3. In this presence of cholesterol sulphate, benzothiazole salt 1 displayed significant spectral changes depending on the medium used: a blue shift in the aqueous medium and a red shift in the methanolic one (H2O:MeOH; 2:1 (v/v)). Subsequently preliminary in vivo study showed that, salt 1 significantly inhibits a growth of breast carcinoma on Nu/nu mice model.
- Klíčová slova
- Molecular recognition, Polymethinium salts, Sulphated sterols, Synthetic receptors,
- MeSH
- benzothiazoly chemie MeSH
- estery cholesterolu chemie farmakologie MeSH
- estron analogy a deriváty chemie farmakologie MeSH
- heterocyklické sloučeniny chemie farmakologie MeSH
- karbocyaniny chemie MeSH
- myši nahé MeSH
- nádory prsu farmakoterapie MeSH
- pregnenolon chemie farmakologie MeSH
- protinádorové látky farmakologie MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- benzothiazole MeSH Prohlížeč
- benzothiazoly MeSH
- cholesteryl sulfate MeSH Prohlížeč
- estery cholesterolu MeSH
- estron MeSH
- estrone sulfate MeSH Prohlížeč
- heterocyklické sloučeniny MeSH
- karbocyaniny MeSH
- pregnenolon MeSH
- protinádorové látky MeSH
Here we present a fluorometric method for direct determination of supernatant-free fluorescence spectra generated from fluorescently stained cells in suspension. The key element in the new technique is the design of an adapter to a standard cuvette holder that makes it possible to measure front-face fluorescence spectra from thin layers of cells spun down to the bottom of a spectrofluorometric cuvette. We have demonstrated the applicability of this approach and its analytical potential using the suspensions of yeast cells stained with the potentiometric dye of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), and with the specific cell-wall marker calcofluor.
Cy3 and Cy5 cyanine dyes terminally attached to the 5'C end (C1) of the DNA oligonucleotide were studied by metadynamics (MTD), molecular dynamics (MD), and density-functional methods with dispersion corrections (DFT-D). MTD simulations explored the free energy surface (FES) of the dye-DNA interactions, which included stacking and major groove binding motifs and unstacked structures. Dynamics of the stacked structures was studied by the MD simulations. All possible combinations of stacking interactions between the two indole rings of the dyes and the neighbor guanine and cytosine rings were observed. The most probable interaction included the stacking between the dye's distal indole ring and the guanine base. In ∼10% of the structures the delocalized π-electrons of the dyes' polymethine linkers played a key role in the dye-DNA dispersion interactions. The stacked conformers of the Cy3 dye were confirmed as true minima by DFT-D full optimizations. The stacked dye decreased flexibility up to two neighbor base pairs.
- MeSH
- chemické modely MeSH
- DNA chemie MeSH
- elektrony MeSH
- entropie MeSH
- karbocyaniny chemie MeSH
- molekulární struktura MeSH
- ohebnost (fyzika) MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cyanine dye 3 MeSH Prohlížeč
- cyanine dye 5 MeSH Prohlížeč
- DNA MeSH
- karbocyaniny MeSH
Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.
- Klíčová slova
- Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
- MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie metody MeSH
- karbocyaniny chemie MeSH
- membránové potenciály * MeSH
- Saccharomyces cerevisiae chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
- fluorescenční barviva MeSH
- karbocyaniny MeSH
