52 research outputs found
Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency
Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a âclassicalâ condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model systemâs silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the âstratified siRNA silencing vectorâ requires
Ibuprofen-loaded chitosanâlipid nanoconjugate hydrogel with gum arabic: Green synthesis, characterisation, in vitro kinetics mechanistic release study and PGE2 production test
Ibuprofen is a well-known non-steroidal anti-inflammatory (NSAID) medicine that is often used to treat inflammation in general. When given orally, it produces gastrointestinal issues which lead to lower patient compliance. Ibuprofen transdermal administration improves both patient compliance and the efficacy of the drug. Nanoconjugation hydrogels were proposed as a controlled transdermal delivery tool for ibuprofen. Six formulations were prepared using different compositions including chitosan, lipids, gum arabic, and polyvinyl alcohol, through ionic interaction, maturation, and freezeâthaw methods. The formulations were characterised by size, drug conjugation efficiency, differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Further analysis of optimised hydrogels was performed, including X-ray diffraction (XRD), rheology, gel fraction and swelling ability, in vitro drug release, and in vitro macrophage prostaglandin E2 (PGE2 ) production testing. The effects of ibuprofenâs electrostatic interaction with a lipid or polymer on the physicochemical and dissolution characterisation of ibuprofen hydrogels were evaluated. The results showed that the S3 (with lipid conjugation) hydrogel provided higher conjugation efficiency and prolonged drug release compared with the S6 hydrogel
Time and cell-dependent effects of endocytosis inhibitors on the internalization of biomolecule markers and nanomaterials
Endocytosis is an essential function of cells, with key roles in the internalisation of nutrients, signal molecules and also drugs. Endocytic processes are therefore widely investigated in the context of drug delivery, and inhibitors of endocytic pathways have been used to provide information regarding uptake mechanisms of drug carrier materials. Here we describe studies in which two established inhibitors of clathrin dependent and independent endocytosis, chlorpromazine and methylâβâcyclodextrin respectively, were employed to probe endocytic pathways of three cell lines chosen to represent tumourârelevant or associated phenotypes: 3 T3 (fibroblasts), HCT 116 (colon cancer) and MGLVAâ1 (gastric cancer). For clathrin mediated endocytosis the data highlight that chlorpromazine inhibition of transferrin internalization, via clathrin dependent endocytosis, is cell and time dependent. We also show that inhibition of uptake is transient with a resumption of transferrin internalization after a maximal inhibition period. The same endocytosis inhibitors were used to probe the internalization of 50 and 100 nm carboxylated polystyrene nanoparticles (CâPSâNPs) as model drug delivery carriers. Flow cytometry data indicated that internalisation of CâPSâNPs varied considerably with the incubation time of cells with chlorpromazine or methylâβâcyclodextrin, and that the effects were also markedly cellâline dependent. These data highlight that the effects of endocytosis inhibitors on the internalisation pathways even of relatively simple nanoparticles are complex and interdependent. We suggest that mechanistic investigations of the endocytic processes which govern practical applications of nanoparticles for diagnostic and therapeutic applications should be considered on a cell, time and concentration basis
Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery
The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyper-branched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. Here, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known âultra-facileâ thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 cells indicated that all the new hyper-branched polymers were well-tolerated but, surprisingly, the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyper-branched poly(lysine)s synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer stiffness, pKa and ability to form stable polyplexes with DNA. The results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyper-branched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy
Mechanisms of Nanoparticle Internalization and Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge
This
study investigated the effect of nanoparticle size (50 and
100 nm) and surface charge on their interaction with Caco-2 monolayers
as a model of the intestinal epithelium, including cell internalization
pathways and the level of transepithelial transport. Initially, toxicity
assays showed that cell viability and cell membrane integrity were
dependent on the surface charge and applied mass, number, and total
surface area of nanoparticles, as tested in two epithelial cell lines,
colon carcinoma Caco-2 and airway Calu-3. This also identified suitable
nanoparticle concentrations for subsequent cell uptake experiments.
Nanoparticle application at doses below half maximal effective concentration
(EC<sub>50</sub>) revealed that the transport efficiency (ratio of
transport to cell uptake) across Caco-2 cell monolayers is significantly
higher for negatively charged nanoparticles compared to their positively
charged counterparts (of similar size), despite the higher level of
internalization of positively charged systems. Cell internalization
pathways were hence probed using a panel of pharmacological inhibitors
aiming to establish whether the discrepancy in transport efficiency
is due to different uptake and transport pathways. Vesicular trans-monolayer
transport for both positively and negatively charged nanoparticles
was confirmed via inhibition of dynamin (by dynasore) and microtubule
network (via nocodazole), which significantly reduced the transport
of both nanoparticle systems. For positively charged nanoparticles
a significant decrease in internalization and transport (46% and 37%,
respectively) occurred in the presence of a clathrin pathway inhibitor
(chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(<i>N</i>-ethyl-<i>N</i>-isopropyi)-amiloride), and under
cholesterol depletion (38%; via methyl-β-cyclodextrin), but
remained unaffected by the inhibition of lipid raft associated uptake
(caveolae) by genistein. On the contrary, the most prominent reduction
in internalization and transport of negatively charged nanoparticles
(51% and 48%, respectively) followed the inhibition of lipid raft-associated
pathway (caveolae inhibition by genistein) but was not significantly
affected by the inhibition of clathrin pathway
Insulinotropic Potential of Moxifloxacin and Gemifloxacin: An In Vivo Rabbits Model Study Followed by Randomized Phase I Clinical Trial
Fluoroquinolones (FQs) have been reported to cause dysglycemia in both diabetic and non-diabetic patients. However, diabetic patients are usually on polypharmacy, so we cannot attribute the dysglycemia specifically to FQs. To answer the question as to whether Moxifloxacin and Gemifloxacin influence blood glucose levels and serum insulin levels or otherwise, rabbits were used as experimental animals in an in vivo model followed by a phase I randomized clinical trial in euglycemic healthy volunteers. The effects on the serum insulin and blood glucose levels in the Moxifloxacin and Gemifloxacin treated groups were, respectively, determined on the fifth day in both the in-vivo rabbits model and in the test subjects of the phase I clinical trial. The effects of these drugs were also checked on the histomorphology of the pancreas in the rabbits. The findings of our study suggest that Moxifloxacin and Gemifloxacin significantly (p < 0.05) reduced the blood glucose levels via a subsequent significant shift in the serum insulin levels both in the in vivo animal model and in the test subjects of the phase I clinical trial. No prominent effects on the beta cells histomorphology were noted in this study. Moxifloxacin showed a more significant effect than Gemifloxacin. The insulinotropic effect was comparable to the effect of Glibenclamide. It is concluded that Moxifloxacin and Gemifloxacin have a significant blood glucose lowering effect mediated through insulinotropic action. (Clinical Trials.gov identifier: NCT04692623)
Protective Effect of Salvianolic Acid B in Acetic Acid-Induced Experimental Colitis in a Mouse Model
In its prominent experimental studies salvianolic acid B (Sal B) is novel because of its well-defined, common physiological effects, which include anti-inflammatory, anti-depressant, cardioprotective, DNA protective, neuroprotective and hepatoprotective activity in experimental animals. Initially, Sal B was studied for its anti-inflammatory properties, used as a remedy for a wide range of disease conditions, but its specific efficacy on inflammatory bowel disease is still unclear. The aim of this current study was to understand the therapeutic potential of Sal B in an acetic acid (AA)âtriggered experimental mouse colitis model. Colitis was triggered by intrarectal injection of 5% AA, and then laboratory animals were given Sal B (10, 20 and 40 Îźg/kg) for seven days. The ulcerated colonic mucosa was assessed by clinical experiment, macroscopical, biological and histopathological analysis. The results showed depleted SOD, CAT, GSH levels and consequential elevated MPO and MDA levels and aberrant crypt foci and mast cells were seen in the AA-induced colonic mucosa of experimental animals. The data obtained from this study demonstrate that a dose of 40 Âľg/kg showed an efficacious anti-ulcer effect against AA-induced experimental colitis. Based on its antioxidant efficacy, Sal B may therefore be useful as a therapeutic approach for ulcerative colitis
Time-lapse live cell imaging to monitor doxorubicin release from DNA origami nanostructures
Synthesis of New Naphthyl Aceto Hydrazone-Based Metal Complexes: Micellar Interactions, DNA Binding, Antimicrobial, and Cancer Inhibition Studies
In the present study, naphthyl acetohydrazide (HL) ligand was prepared and used for the synthesis of new six amorphous transition metal (Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II)) complexes. All the compounds were characterized by elemental analysis, UV-vis, FT-IR, 1H- and 13C-NMR, and Matrix-Assisted Laser Desorption Ionization (MALDI). The solubilization study was carried out by estimating the interaction between the metal complexes with surfactants viz. sodium stearate (SS) and Cetyltrimethylammonium bromide (CTAB). UV-Visible spectroscopy was employed to determine partitioning and binding parameters, whereas electrical conductivity measurements were employed to estimate critical micellar concentration (CMC), the extent of dissociation, and free energy of micellization. The CT-DNA interaction of synthesized compounds with DNA represents the major groove binding. The synthesized ligand and metal complexes were also tested against bacterial and fungal strains and it has been observed that Cu(II) complex is active against all the strains except Candida albicans, while Cd(II) complex is active against all bacterial and fungal strains except Pseudomonas. Among all compounds, only the Pd(II) complex shows reasonable activity against cervical cancer HeLa cell lines, representing 97% inhibition
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