16 research outputs found
Glutathione Responsive Hyaluronic Acid Nanocapsules Obtained by Bioorthogonal Interfacial “Click” Reaction
Azide-functionalized hyaluronic acid
and disulfide dialkyne have
been used for “click” reaction polymerization at the
miniemulsion droplets interface leading to glutathione responsive
nanocapsules (NCs). Inverse miniemulsion polymerization was chosen,
due to its excellent performance properties, for example, tuning of
size and size distribution, shell thickness/density, and high pay
loading efficiency. The obtained size, size distribution, and encapsulation
efficiency were checked via fluorescent spectroscopy, and the tripeptide
glutathione was used to release an encapsulated fluorescent dye after
cleavage of the nanocapsules shell. To show the glutathione-mediated
intracellular cleavage of disulfide-containing NC shells, CellTracker
was encapsulated into the nanocapsules. The cellular uptake in dendritic
cells and the cleavage of the nanocapsules in the cells were studied
using confocal laser scanning microscopy. Because of the mild reaction
conditions used during the interfacial polymerization and the excellent
cleavage properties, we believe that the synthesis of glutathione
responsive hyaluronic acid NCs reported herein are of high interest
for the encapsulation and release of sensitive compounds at high yields
Molecular Exchange Kinetics of Diblock Copolymer Micelles Monitored by Fluorescence Correlation Spectroscopy
We investigated the equilibrium chain-exchange
kinetics of amphiphilic
diblock copolymer micelles, using a new method based on fluorescence
correlation spectroscopy. The micelles were formed from polystyrene-<i>block</i>-poly[oligo(ethylene glycol) methyl ether methacrylate]
(PS–POEGMA) in different solvents and studied at various temperatures.
This linear-brush copolymer was chosen as a model system, forming
micelles with short and bulky corona. Depending on the applied solvent,
fast exchange could be observed even at temperatures well below the
nominal glass transition of the core-forming PS block. The effect
is caused by swelling of the core and allows extensive tuning of the
chain-exchange rate by adding to the system minor amounts of good
or bad solvent for the core block
Simplified metabolic network of the MiaPaCa-2 cell line as determined by proton NMR spectroscopy.
<p>The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.</p
Gene expression analysis of autophagy and apoptosis relevant genes by Western blot analysis.
<p>The Bad, Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP.</p
Identification of substances from a proton NMR spectrum of viable untreated MiaPaCa-2 cells.
<p>The cells were harvested from monolayer culture, kept and measured at 20°C. Measurement were performed by a 600 MHz Bruker spectrometer. For better visibility the part of the spectrum showing the protons of aliphatic groups is splitted into 2 parts - A and B. (A). aliphatic part I. The methyl and β- and γ- methylene groups of various fatty acids and amino acids are visible. In addition, isopropanol and tetrachlorethan (the external concentration standard) occured as pollutions. (B) Aliphatic part II. Phospholipid metabolites and the α-methylene groups of amino acids and lactate are visible. (C) Formula of OMP with numbering of the respective protons. The methyl groups (1–3, 9) and the methyl group (4) are covered by other metabolites in the aliphatic parts of the spectrum. In contrast, the aromatic protons are visible (H5, H8, H10). (D) Overlay of the aromatic parts of different spectra for intracellular identification of OMP. The singulet of the H5 proton and the dublets of the H8 and H10 protons can be identified when the medium and the cell spectra are compared to those without OMP treatment. Abbreviations: His - histidine, Tyr - tyrosine, Phe – phenylalanine, Leu, Ile, Val - Leucine, Isoleucine, Valine. Ala - Alanine. Glu - Glutamate, Gln – Glutamine, PC - phosphatidylcholine, Cho - Choline, GPC – glycerophosphocholine, Tau – taurine, Scyllo - scylloinositole.</p
Gene expression analysis of membranal transport and apoptosis relevant genes in MiaPaCa-2 cells.
<p>mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.</p
Electron microscopy of the ASPC-1 and MiaPaCa-2 cell lines treated or untreated with OMP.
<p>(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present. (E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).</p
Hormesis of low-dose 5-FU in pancreatic cancer cells and interaction with OMP.
<p>The cell lines ASPC-1, MiaPaCa-2, Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.</p
Box-Whisker plots of lysosomal acidity quantified by AO fluorescence microscopy and image analysis.
<p>Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU, OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.</p
Mean inhibitory concentrations (IC<sub>50</sub>) of OMP and 5-FU in various human pancreatic cancer cell lines.
<p>The IC<sub>50</sub> were determined by ATP-bioluminescence. All values are listed in µg/ml.</p><p>Abbreviations: conf. int. - 90% confidence intervals.</p