17 research outputs found
Interaction of Different Divalent Metal Ions with Lipid Bilayer: Impact on the Encapsulation of Doxorubicin by Lipid Bilayer and Lipoplex Mediated Deintercalation
In this article,
we investigate the influence of different metal
ions (Ca<sup>2+</sup>, Mg<sup>2+</sup>, and Zn<sup>2+</sup>) on binding
of an anticancer drug doxorubicin (DOX) to DMPC bilayer and lipoplex
mediated deintercalation of DOX from DOX-DNA complex. Our study reveals
that lipid bilayer in the presence of different metal ions displays
much higher binding affinity toward DOX than bare lipid bilayer does.
Further, this affinity for a particular metal ion increases linearly
with metal ion concentration. The steady state and time-resolved fluorescence
studies reveal that binding of DOX with lipid bilayer in the presence
of different metal ions varies in the order of Ca<sup>2+</sup>>
Mg<sup>2+</sup>> Zn<sup>2+</sup>. The rotational relaxation of
DOX in the
presence of different metal ions takes place in the same order. We
explain these phenomena in the light of alteration of the physical
properties brought about by metal ions. Moreover, we find that binding
pattern of metal ions with lipid head groups influences the intake
of DOX in lipid bilayer. We exploit the binding of DOX with bilayer
to study the deintercalation of DOX from DOX-DNA complex. We observe
that with increase in metal ion concentration the deintercalation
increases. Among all metal ions, Ca<sup>2+</sup> appears to be most
effective in deintercalation compared to other metal ions. The time-resolved
fluorescence anisotropy and circular dichroism measurements indicate
that in the presence of Ca<sup>2+</sup>, lipid bilayer offer strongest
interaction with DNA while the same is weakest for Zn<sup>2+</sup>. This explains the highest percentage of deintercalation of DOX
from drugâDNA complex in the presence of Ca<sup>2+</sup>. Overall
the present study demonstrates a new strategy that binding of drug
molecules with lipid bilayer and deintercalation of the same from
drugâDNA complex can be tuned by modulation of lipid bilayer
with different metal ions and their concentration
Lipoplex-Mediated Deintercalation of Doxorubicin from Calf Thymus DNAâDoxorubicin Complex
In
this paper, we report the lipoplex-mediated deintercalation
of anticancer drug doxorubicin (DOX) from the DOXâDNA complex
under controlled experimental conditions. We used three zwitterionic
liposomes, namely, 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine
(DPPC), 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine
(DMPC), and 2-oleoyl-1-palmitoyl-<i>sn</i>-glycero-3-phosphocholine
(POPC), which are widely different in their phase transition temperatures
to form a lipoplex with calf thymus DNA in the presence of Ca<sup>2+</sup> ions. The study revealed that DPPC being in solâgel
phase was more effective in releasing the drug from the DOXâDNA
complex compared with liposomes that remain in liquid crystalline
phase (DMPC and POPC). The higher extent of drug release in the case
of DPPC liposomes was attributed to the stronger lipoplex formation
with DNA as compared with that of other liposomes. Owing to the relatively
smaller head group area, the DPPC liposomes in their solâgel
phase can absorb a larger number of Ca<sup>2+</sup> ions and hence
offer a strong electrostatic interaction with DNA. This interaction
was confirmed by time-resolved anisotropy and circular dichroism spectroscopy.
Apart from the electrostatic interaction, the possible hydrophobic
interaction between the liposomes and DNA was also taken into account
for the observed deintercalation. The successful uptake of drug molecules
by liposomes from the drugâDNA complex in the post-release
period was also confirmed using confocal laser scanning microscopy
(CLSM)
Design of Flexible MetalâOrganic Framework-Based Superprotonic Conductors and Their Fabrication with a Polymer into Proton Exchange Membranes
In
recent times, the deployment of metalâorganic frameworks
(MOFs) to develop efficient proton conductors has gained immense popularity
in the arena of sustainable energy research due to the ease of structural
and functional tunability in MOFs. In this work, we have focused on
developing âflexible MOFâ-based proton
conductors with Fe-MIL-53-NH2 and Fe-MIL-88B-NH2 MOFs using
postsynthetic modification (PSM) as the tool. Taking advantage of
the porous nature of these frameworks, we have carried out PSM on
the primary amine groups present on the MOFs and converted them to
âNH(CH2CH2CH2SO3H) groups. The PSM increased the number of labile protons in the
channels of the modified MOFs as well as the extent of H-bonded networks
inside the framework. The modified Fe-MIL-53-NH2 and Fe-MIL-88B-NH2 MOFs, named hereafter as 53-S and 88B-S, respectively, showed proton conductivity of 1.298 Ă 10â2 and 1.687 Ă 10â2 S cmâ1 at âŒ80 °C and 98% relative humidity (RH),
respectively. This reflects âŒ10-fold and âŒ5-fold increases
in their proton conductivity than their respective parent MOFs. Since
MOFs as such are difficult to make directly into flexible membranes,
and these are essential for practical applications as proton conductors,
we have incorporated 53-S and 88B-S as fillers
into a robust imidazole-based polymer matrix, namely, OPBI [poly(4,4âČ-diphenylether-5,5âČ-bibenzimidazole)].
The resulting polymerâMOF mixed matrix membranes (MMMs) after
doping with phosphoric acid (PA) performed as flexible proton exchange
membranes (PEMs) above 100 °C under anhydrous conditions and
were found to be much more efficient and stable than the pristine
OPBI membrane (devoid of any filler loading). By optimizing the amount
of filler loading in the membrane, we obtained the highest proton
conductivity
of 0.304 S cmâ1 at 160 °C under anhydrous conditions
Physical, Electrochemical, and Solvent Permeation Properties of Amphiphilic Conetwork Membranes Formed through Interlinking of Poly(vinylidene fluoride)-<i>Graft</i>-Poly[(2-dimethylamino)ethyl Methacrylate] with Telechelic Poly(ethylene glycol) and Small Molecular Weight Cross-Linkers
We report the preparation of dense and porous amphiphilic
conetwork
(APCN) membranes through the covalent interconnection of poly(vinylidene
fluoride)-graft-poly[(2-dimethylamino)ethyl methacrylate]
(PVDF-g-PDMAEMA) copolymers with telechelic poly(ethylene
glycol) (PEG) or α,α-dichloro-p-xylene
(XDC). The dense APCN membranes exhibit varying solvent swelling and
mechanical properties depending on the compositions and overall crystallinity.
The crystallinity of both PVDF (20â47%) and PEG (9â17%)
is significantly suppressed in the dense APCNs prepared through the
interconnection of PVDF-g-PDMAEMA with reactive PEG
as compared to the APCN membranes (48â53%) prepared with XDC
as well as mechanical blend of PVDF-g-PDMAEMA plus
nonreactive PEG. The dense APCN membranes exhibit a good transport
number of monovalent ions and ionic conductivity. The APCN membrane
interconnected with PEG and containing binary ionic liquids exhibits
a room-temperature lithium ion conductivity of 0.52 mS/cm. On the
other hand, APCN ultrafiltration (UF) membranes exhibit organic solvent-resistant
behavior. The UF membrane obtained by interconnecting PVDF-g-PDMAEMA with telechelic PEG shows low protein fouling
propensity, higher hydrophilicity, and water flux as compared to membranes
prepared using XDC as the interconnecting agent. The significant effect
of the covalent interconnection of the amphiphilic graft copolymers
with telechelic PEG or XDC on the overall properties provides a good
opportunity to modulate the properties and performance of APCN membranes
First Evidence of the Liposome-Mediated Deintercalation of Anticancer Drug Doxorubicin from the DrugâDNA Complex: A Spectroscopic Approach
Biocompatible
liposomes were used for the first time to study the
deintercalation process of a prominent anticancer drug, doxorubicin
(DOX), from doxorubicin-intercalated DNA (DOXâDNA complex)
under controlled experimental conditions. The study revealed that
anionic liposomes (DMPG liposomes) appeared to be the most effective
to bring in the highest percentage of drug release while cationic
liposomes (DOTAP liposomes) scored the lowest percentage of release.
The drug release was primarily attributed to the electrostatic interaction
between liposomes and drug molecules. Apart from this interaction,
changes in the hydrophobicity of the medium upon addition of liposomes
to the DNAâdrug solution accompanied by lipoplex formation
between DNA and liposomes were also attributed to the observed deintercalation.
The CD and the time-resolved rotational relaxation studies confirmed
that lipoplex formation took place between liposomes and DNA owing
to electrostatic interaction. The confocal study revealed that in
the postrelease period, DOX binds with liposomes. The reason behind
the binding is electrostatic interaction as well as the unique bilayer
structure of liposomes which helps it to act as a âhydrophobic
sinkâ for DOX. The study overall highlighted a novel strategy
for deintercalation of drug using biocompatible liposomes, as the
release of the drug can be controlled over a period of time by varying
the concentration and composition of the liposomes
Endocytic phenotypes in mutant primary hemocytes from <i>Drosophila</i>.
<p>(AâD) dsRNA treated S2R+ cells phenocopy corresponding allelic mutants in primary hemocyte cultures in a secondary assay. Scatter plots (A, B) show normalized fold change in fluorescence intensity of dextran that was pulsed (A) or chased (B) in S2R+ cells treated with different dsRNAs (y axis) or in hemocytes (x axis) from the corresponding mutant flies. In all cases, representative values were normalized to those from negative controls (CS hemocytes or zeo dsRNA treated S2R+ cells) and are plotted as mean± SEM. (n>30 for hemocyte assays, n>200 for S2R+ assays in all cases). For the chase assay in (B), we utilized <i>dor<sup>4</sup></i> and <i>car<sup>1</sup></i> mutant hemocytes as positive controls (shown in light blue; Sriram et al., 2003). (C) Representative micrographs of hemocyte cultures from flies carrying hypomorphic alleles of <i>vps35</i>, <i>epac</i>, <i>α-cop</i> and <i>CG1418</i> assayed as in (B). (D) Summary of the experiment in (AâB) displaying statistically significant (Student's T-test, p<0.05) changes in uptake/retention of mutant hemocytes or gene-depleted S2R+ cells as colour coded maps. Scale bar in (C)â=â5 ”m.</p
Quantitative profiling of two endocytic routes at single cell resolution.
<p>(A) Experimental workflow outline for cell seeding, transfection and multiplex endocytic assays to obtain multifeature data across 7131 gene depletions. The entire procedure was performed on a cell array (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s001" target="_blank">Figure S1A</a>; details in SOM) and the positions of negative and positive (dsRNA against <i>sec23</i>, <i>arf1</i>, <i>shi</i>) controls are highlighted in their respective colours. (B) Table grouping the 27 quantitative features into categories. The top half of the table contains direct measurements of intensity, while the lower half contains geometric parameters of the cell, endosomes and nucleus. Various measurements are made from each fluorescent channel, including those utilizing different pixel radii for local background subtraction while detecting endosomes. (C) Representative brightfield (bf) and fluorescent micrographs of a field of view of individual cells (zoomed in insets) labeled with four different fluorescent probes: Hoechst; FITC-Dextran (Fdex); Alexa568-Tf (Tf); Alexa647-αOkt9 (Okt9); (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s018" target="_blank">Methods S1</a> for details). The psuedocolour merge image is a composite of the Fdex (green), TfR (red) and Okt9 (blue) channels. Scale barâ=â15 ”m; insetâ=â3Ă. (D) Grayscale heatmap representing the fraction of four control genes (<i>arf1</i> (<i>arf79f</i>); <i>shi</i>; <i>sec23</i>; <i>chc</i>) picked up as hits (above a Z-score threshold of 3) across all 27 features in the entire dataset. Higher values on the grayscale bar denote higher pick-up rates. The features with higher pick-up rates correspond to the known endocytic roles of these genes.</p
Primary hits validated in a secondary classification assay.
<p>(AâB) Schema (A) and positional patterning (B) on cell arrays of secondary endocytic classification assays carried out for all CG features (upper schema) or a subset of CD features (lower schema).All the test genes were surrounded with local positive controls, and negative controls (see legend in (B)). With this patterning, each gene was tested in triplicate, with three local positive controls and six local negative controls. (C) Heatmap representing raw mean fluorescence intensities (in the pulse channel) across a test cell array used to validate the CG secondary endocytic assay described in (A). Only the means of control wells are shown in the top panel and the inter-control variation in means is representative of a typical experiment. For comparison, the lower panel depicts the mean fluorescence intensities of test genes. (D) The green bars show the fraction of genes predicted as hits for each feature in the primary screen that were also picked up as hits for that feature in the secondary. The gray bars show the fraction of genes not predicted as a hit for each feature in the primary screen that were nevertheless picked up as hits for that feature in the secondary. With a single exception (Tnum) we find that the green bars exceed the gray (p-value 5Ă10<sup>â6</sup> for 22 fair coin flips) demonstrating the selectivity and reproducibility of our primary assay. (E) Psuedocoloured fluorescence micrographs of representative control and <i>drab5</i>- and <i>dvps4</i>- dsRNA treated populations of cells that were subjected to the CG pulse-chase assay from (<b>A</b>). Both Drab5 and Dvps4 depleted cells were affected in the chase (with Fdex, green) portion of the assay, while the pulse portion (with Rdex, red) was unaffected (see quantitation in bar graphs on the right, normalized to control). Scale barâ=â10 ”m.</p
Role of lysosomal genes.
<p>(A) Network map depicting known and predicted interactions (green lines: genetic; blue lines: physical; brown lines: predicted based on conserved data) between the âGranule groupâ set of eye colour mutants (pink) and selected hits (gray). In this network, genes encoding Carnation (<i>car</i>; the fly homolog of VPS33), Deep orange (<i>dor</i>), Carmine (<i>cm</i>) and Rab7 were identified with roles in CG endocytosis in this study (denoted by black asterisks), while White (<i>w</i>) depletion affected at least one Tf pathway feature (white asterisk). (B) Localization of Carnation on early fluid endosomes. <i>Drosophila</i> S2R+ cells were pulsed with TMR-Dextran for two minutes and fixed and labeled with antibodies to Carnation (αCar). Micrographs show a representative cell imaged in two channels and a pseudo colour merge image (labeled TMRdex and αCar), in red, green and merge respectively). Carnation (green) is seen enriched on peripheral, small, early fluid endosomes (red). Three examples of such endosomes (white arrows in merge panel) are shown in the magnified inset. (C) Fluorescent micrographs depict the levels of fluid uptake in representative S2R+ cells treated with dsRNA against <i>car</i> (first lower panel) or <i>syx1A</i> (last lower panel) or in hemocytes from <i>car<sup>1</sup></i> mutant flies (middle lower panel), with their respective controls (upper panels). Bar graph represents mean and SD of normalized fluorescent integrated intensity per cell from 2â3 experiments, with 100â150 cells per treatment (S2R+ cells) or 40 cells per genotype (hemocytes). (D) Representative fluorescent micrographs depict fluid uptake measured in hemocytes as in (C), in flies that were: homozygous for a mutant allele of <i>car</i> (<i>car<sup>1</sup></i>); a hetero-allelic combination of <i>car<sup>1</sup></i>/+;<i>syx1</i>/+;or wild type (CS). Also tested were flies heterozygous for <i>syx1</i>/+ and <i>car<sup>1</sup></i>/+. Bar graph represents mean and SD of normalized fluorescent integrated intensity per hemocyte from 2â3 experiments with 40 cells per genotype. (E) Representative micrographs show human AGS cells treated with control siRNA or siRNA to hSYX1A and hVPS33A/B and pulsed with FITC-Dextran for 5 min. Right panel - Bar graphs show population averaged mean fluorescence intensity uptake per cell (representative experiment with n>50 cells per replicate, 2 replicates). Scale bar in (BâE) main panelâ=â5 ”m, insetâ=â1 ”m. Double asterisks denote significance <i>p</i> values lower than 0.01 with the Student's T-Test.</p
Experimental setup.
<p>(A) Experimental evolution of prototrophic, isogenic populations of different ploidy (haploid (VK111), diploid (VK145) and tetraploid (VK202)) for increased ethanol tolerance was performed in a turbidostat. Every 25 generations, the ethanol concentration in the media was increased in a stepwise manner (starting at 6% (v/v) and reaching 12% at 200 generations). Increasing the ethanol concentration from 10% to 11% dramatically reduced growth rate of evolving cells. Therefore, instead of increasing ethanol levels, we first reduced the ethanol level to 10.7% after 100 generations. (B) Red circles represent sampling points (indicated as number of generations) for which whole-genome sequencing was performed. For each circle, heterogeneous populations as well as three evolved, ethanol tolerant clones were sequenced. Sequencing of the population sample of reactor 4 at 200 generations failed, so this data is omitted from the manuscript. For generation 80 of reactor 1, only population data is available.</p