78 research outputs found
pH-Sensitive ChitosanâHeparin Nanoparticles for Effective Delivery of Genetic Drugs into Epithelial Cells
Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosanâheparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosanâheparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosanâheparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosanâheparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors
Amphiphilic Polypeptides for VEGF siRNA Delivery into Retinal Epithelial Cells
Polyethyleneimine, poly-L-lysine, chitosan and some others cationic polymers have been thoroughly studied as nucleic acid delivery systems in gene therapy. However, the drug release from these systems proceeds at a very low rate due to extremely high binding between a carrier and gene material. To reduce these interactions and to enhance drug release, we developed a set of amphiphilic polypeptides containing positively and negatively charged amino acids as well as a hydrophobic one. The copolymers obtained were characterized by size-exclusion chromatography, static light scattering, HPLC amino acid analysis and (HNMR)-H-1 spectroscopy. All copolymers formed particles due to a self-assembly in aqueous media. Depending on polypeptide composition, the formation of particles with hydrodynamic diameters from 180 to 900 nm was observed. Stability of polymer particles, loading and release efficiency were carefully studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. The application of polymer carriers, containing siRNA, to vascular endothelial growth factor (VEGF-A165) silencing of ARPE-19 cells was successful. The gene silencing was confirmed by suppression of both messenger RNA and protein expression.Peer reviewe
Photosensitive poly-l-lysine/heparin interpolyelectrolyte complexes for delivery of genetic drugs
Photo-triggered release of biopharmaceutical drugs inside the cells is a challenging direction of modern science, which requires obtaining new polymeric systems. The interpolyelectrolyte complexes (IPECs) of poly-l-lysine with heparin capable of encapsulation of genetic constructions-such as model oligonucleotide, siRNA, and pDNA-were obtained. Poly-l-lysine to heparin ratios were optimized to provide the appropriate release kinetics of genetic material from the polyplex. In order to impart the obtained IPEC with photosensitive properties, the linker was synthesized as based on 4-brommethyl-3-nitrobenzoic acid. The conditions and kinetics of photosensitive linker destruction were carefully studied. The colloid particles of IPEC were modified with Cy3 probe and their cellular internalization was investigated by flow cytometry method. The efficacy of photosensitive IPECs as siRNA and pDNA delivery system was evaluated. © 2020 by the authors
From random to rational: A discovery approach to selective subnanomolar inhibitors of human carbonic anhydrase IV based on the Castagnoli-Cushman multicomponent reaction
Development of Fluorescent IsocoumarinâFused Oxacyclononyne â 1,2,3âTriazole Pairs
Fluorescent isocoumarin-fused cycloalkynes, which are reactive in SPAAC and give fluorescent triazoles regardless of the azide nature, have been developed. The key structural feature that converts the non-fluorescent cycloalkyne/triazole pair to its fluorescent counterpart is the pi-acceptor group (COOMe, CN) at the C6 position of the isocoumarin ring. The design of the fluorescent cycloalkyne/triazole pairs is based on the theoretical study of the S1 state deactivation mechanism of the non-fluorescent isocoumarin-fused cycloalkyne IC9O using multi-configurational ab initio and DFT methodologies. The calculations revealed that deactivation proceeds through the electrocyclic ring opening of the α-pyrone cycle and is accompanied by a redistribution of electron density in the fused benzene ring. We proposed that the S1 excited state deactivation barrier could be increased by introducing a pi-acceptor group into a position that is in direct conjugation with the formed C=O group and has a reduced electron density in the transition state. As a proof of concept, we designed and synthesized two fluorescent isocoumarin-fused cycloalkynes IC9O-COOMe and IC9O-CN bearing pi-acceptors at the C6 position. The importance of the nature of a pi-acceptor group was shown by the example of much less fluorescent CF-substituted cycloalkyne IC9O-CF
Mechanism of pure glucose-dependent insulinotropic activity of a novel imidazoline compound BL11282
We developed a novel, pure glucose-dependent, insulinotropic imidazoline
compound, BL11282, which directly affects the insulin exocytotic
machinery and does not block the KATP channel activity. BL11282 does not
induce insulin secretion at basal glucose concentrations, whereas it
stimulates insulin secretion at an elevated glucose level. Therefore this
imidazoline should not provoke hypoglycemic episodes as it has been
observed for the sulfonylureas. However, so far, the detailed biochemical
and pharmacological mechanisms underlying the insulinotropic effects of
BL11282 are not fully established. The overall objective of this study
was to investigate signaltransduction pathways involved in the pure
glucose-dependent activity of BL11282 on insulin release. Using SUR1(-/-)
mice, we unambiguously confirmed the previous notion that the
insulinotropic activity of BL11282 is unrelated to its interaction with
ATPdependent K+ channels. We have also shown that previously described
targets for imidazoline compounds like alpha2-adrenoreceptors,
imidazoline I1-receptors and monomeric G-protein Rhes are not involved in
the mechanisms of the insulinotropic action of BL11282. To clarify the
molecular mechanisms underlying the effects of BL11282 on insulin
secretion, we have used an approach involving desensitization of
beta-cells to the insulinotropic activity of BL11282 by prolonged
incubation with the compound. The data obtained show that overnight
pretreatment of pancreatic islets with BL11282 desensitizes the
subsequent islet response to this imidazoline. Islets pretreated for the
same time with efaroxan, another insulinotropic imidazoline, are
unresponsive to subsequent addition of efaroxan but preserve their
response to BL11282. The effect of pretreatment with BL11282 is
accompanied by an increased insulinotropic response to subsequent high
glucose concentration. Desensitization of islet response to BL11282 does
not eliminate subsequent islet response to GLP-1, but significantly
decreases the fold potentiation of insulin release by this peptide. The
latter effect points to the importance of GLP-1 stimulated
signal-transduction pathways for the insulinotropic activity of BL11282.
Our results support the involvement of the cAMP-GEFII·Rim2 pathway in
BL11282 stimulated insulin secretion. Indeed, expression of dominant
negative cAMP-GEFII and Rim2 mutant proteins in MIN6 cells lead to a
significant reduction in insulin secretion stimulated by the imidazoline.
To further investigate this direct mechanism of BL11282 on insulin
release, we turned our attention to calcium-independent PLA2 isoform
iPLA2beta, which is predominantly expressed in pancreatic islets and
plays an important role in insulin secretion in pancreatic islets. Our
observations indicate a deficiency in iPLA2beta isoform expression in
diabetic Goto-Kakizaki (GK) rat islets compared to Wistar rat islets,
this effect being in agreement with an impairment in the
glucose-stimulated insulin response in GK rat islets. Pharmacological
inhibition of iPLA2 and cytochrome P-450 enzymes completely abolished the
insulinotropic effect of BL11282. BL11282 stimulated arachidonic acid
release from the islets in the presence of high glucose concentration and
this effect was fully blocked by iPLA2 inhibitor, bromoenol lactone. The
data suggest that potentiation of glucose-induced insulin release by
BL11282, independent of concomitant changes in cytoplasmic free Ca2+
concentration, involves the release of arachidonic acid by iPLA2 and its
metabolism to epoxyeicosatrienoic acids through the cytochrome P-450
pathway
The transcriptional co-repressor myeloid translocation gene 16 inhibits glycolysis and stimulates mitochondrial respiration.
The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect
Chronic high glucose and pyruvate levels differentially affect mitochondrial bioenergetics and fuel-stimulated insulin secretion from clonal INS-1 832/13 cells.
Glucotoxicity in pancreatic ÎČ-cells is a well-established pathogenetic process in Type 2 Diabetes. It has been suggested that metabolism-derived reactive oxygen species perturb the ÎČ-cell transcriptional machi-nery. Less is known about altered mitochondrial function in this condition. We used INS-1 832/13 cells cultured for 48 h in 2.8 mM glucose (low-G), 16.7 mM glucose (high-G) or 2.8 mM glucose plus 13.7 mM pyruvate (high-P) to identify metabolic perturbations. High-G cells showed decreased responsiveness, relative to low-G cells, with respect to mitochondrial membrane hyperpolarization, plasma membrane depolarization and insulin secretion, when stimulated acutely with 16.7 mM glucose or 10 mM pyruvate. In contrast, high-P cells were functionally unimpaired, eliminating chronic provision of saturating mitochondrial substrate as a cause of glucotoxicity. Although cellular insulin content was depleted in high-G cells, relative to low-G and high-P cells, cellular functions were largely recovered following a further 24 h culture in low-G medium. After 2 h at 2.8 mM glucose, high-G cells did not retain increased levels of glycolytic or TCA-cycle intermediates, but nevertheless displayed increased glycolysis, increased respiration and an increased mitochondrial proton leak relative to low-G and high-P cells. This notwithstanding, titration of low-G cells with low protonophore concen-trations, monitoring respiration and insulin secretion in parallel, showed that the perturbed insulin secretion of high-G cells could not be accounted for by increased proton leak. The present study supports the idea that glucose-induced disturbances of stimulus-secretion coupling by extra-mitochondrial metabolism upstream of pyruvate, rather than exhaustion from metabolic overload, underlie glucotoxicity in insulin-producing cells
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