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ABSTRACT: The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.
Langmuir 12/2009; 26(5):3453-61. · 4.19 Impact Factor
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ABSTRACT: This study evaluated novel structural motifs known as "plum pudding" gels as potential drug-eluting stent coatings. Controlled delivery of a HMG-CoA reductase inhibitor (statin) from the intravascular stent surface represents a potential therapeutic modality for prevention of in-stent restenosis (ISR). In this study, gels were comprised of fluvastatin-loaded thermoresponsive microgel particles containing the relatively hydrophilic N-isopropylacrylamide (NiPAAm), mixed with the more hydrophobic N-tert-butylacrylamide (NtBAAm) in different wt/wt ratios: 85/15, 65/35, and 50/50, randomly dispersed in a 65/35 or 85/15 NiPAAm/NtBAAm copolymer matrix. Fluvastatin release from 5 microm copolymer films was greatest from the most hydrophilic systems and least from the more hydrophobic systems. Release from hydrophobic matrices appeared to be via Fickian diffusion, enabling use of the Stokes-Einstein equation to determine diffusion coefficients. Release from hydrophilic matrices was non-Fickian. Eluted drug retained its bioactivity, assessed as selective inhibition of human coronary artery smooth muscle cell proliferation. When stainless steel stent wires were coated (25 microm thickness) with fluvastatin-loaded 65/35 microgels in an 85/15 copolymer matrix, drug elution into static and perfused flow environments followed similar elution profiles. In contrast to elution from copolymer films cast on flat surfaces, diffusion from stent wires coated with hydrophilic and hydrophobic systems both followed Fickian patterns, with slightly larger diffusion coefficients for elution from the flow system. We conclude that manipulation of the relative hydrophobicities of both microgel and matrix components of "plum pudding" gels results in tightly regulated release of fluvastatin over an extended time period relevant to initiation and propagation of ISR.
Journal of Biomedical Materials Research Part A 01/2007; 79(4):923-33. · 2.63 Impact Factor
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ABSTRACT: This study evaluated novel structural motifs known as “plum pudding” gels as potential drug-eluting stent coatings. Controlled delivery of a HMG-CoA reductase inhibitor (statin) from the intravascular stent surface represents a potential therapeutic modality for prevention of in-stent restenosis (ISR). In this study, gels were comprised of fluvastatin-loaded thermoresponsive microgel particles containing the relatively hydrophilic N-isopropylacrylamide (NiPAAm), mixed with the more hydrophobic N-tert-butylacrylamide (NtBAAm) in different wt/wt ratios: 85/15, 65/35, and 50/50, randomly dispersed in a 65/35 or 85/15 NiPAAm/NtBAAm copolymer matrix. Fluvastatin release from 5 μm copolymer films was greatest from the most hydrophilic systems and least from the more hydrophobic systems. Release from hydrophobic matrices appeared to be via Fickian diffusion, enabling use of the Stokes–Einstein equation to determine diffusion coefficients. Release from hydrophilic matrices was non-Fickian. Eluted drug retained its bioactivity, assessed as selective inhibition of human coronary artery smooth muscle cell proliferation. When stainless steel stent wires were coated (25 μm thickness) with fluvastatin-loaded 65/35 microgels in an 85/15 copolymer matrix, drug elution into static and perfused flow environments followed similar elution profiles. In contrast to elution from copolymer films cast on flat surfaces, diffusion from stent wires coated with hydrophilic and hydrophobic systems both followed Fickian patterns, with slightly larger diffusion coefficients for elution from the flow system. We conclude that manipulation of the relative hydrophobicities of both microgel and matrix components of “plum pudding” gels results in tightly regulated release of fluvastatin over an extended time period relevant to initiation and propagation of ISR. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Journal of Biomedical Materials Research Part A 12/2006; 79A(4):923 - 933. · 2.63 Impact Factor
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ABSTRACT: The release of two compositionally different solutes from a composite gel composed of two different populations of microgel particles embedded in a single bulk gel matrix is described, showing the potential of the "plum-pudding gel" as a multifunctional platform for controlled surface release. One hydrophobic solute (pyrene) and one hydrophobic and charged solute (rhodamine 123) were chosen as the solutes to be released. Hydrophobic microgels composed of 50% N-isopropylacrylamide (NIPAM) and 50% N-tert-butylacrylamide (BAM) were loaded with pyrene, and anionic microgels composed of 30% acrylic acid (AAc), 20% NIPAM, and 50% BAM were loaded with rhodamine 123. The two solute-loaded microgel populations were incorporated into a single bulk gel network, from which the two solutes were released simultaneously and independently. Using this structural motif, solutes that are mutually incompatible can be incorporated into a single matrix with which they may also be incompatible. The electrostatically incorporated solute was released much more slowly than the hydrophobically attracted solute, indicating that the microgel composition can be tailored to the specific solute, and thus control its release rate. The choice of bulk matrix was also found to influence the release rate much more than expected, offering a further control element to the system.
The Journal of Physical Chemistry B 05/2005; 109(13):6257-61. · 3.70 Impact Factor
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ABSTRACT: The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.
Journal of Biomedical Materials Research Part A 02/2005; 72(1):25-35. · 2.63 Impact Factor
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ABSTRACT: The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of an antimitotic agent to human vascular smooth muscle cells (HASMC) over an extended period of time. Copolymer films were prepared using a 50:50 (w/w) ratio of N-isopropylacrylamide (NiPAAm) monomer to the more hydrophobic N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (0.1 micromol per film) at room temperature. Colchicine release from films was sustained over a 14-day period. At 24 h postloading, the concentration of colchicine in the medium overlying films was 2.12 +/- 0.16 microM; this fell to 0.20 +/- 0.01 microM at 7 days and decreased further to 0.12 +/- 0.01 microM after 14 days. Colchicine released from copolymer films inhibited proliferation when subsequently placed on HASMC: at 0.1 microM, released colchicine reduced proliferation to 18.5 +/- 0.8% of control cells (p < 0.001, n = 9). The antiproliferative effect of released colchicine was comparable to that of native colchicine, as observed in separate experiments. Furthermore, colchicine released from 50:50 polymer films inhibited the proliferation of cells grown in the same environment as the copolymer. Inhibition of cell proliferation was not due to the release of cytotoxic particles from the copolymer because medium incubated with copolymer for 5 days and then applied to HASMC did not alter cell viability. In conclusion, this study demonstrates that 50:50 NiPAAm:NtBAAm copolymers can deliver bioactive concentrations of the antimitotic agent colchicine to human vascular cells over an extended period of time.
Journal of Biomedical Materials Research Part A 11/2003; 67(2):667-73. · 2.63 Impact Factor
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ABSTRACT: κ-Carrageenans are known to form strong but turbid gels in the presence of calcium ions. Nevertheless, the resulting gels show
no syneresis and a relatively low melting temperature, which makes them of great interest in food applications. To better
understand the mechanism of gelation of these polysaccharides, the effect of chemical modification of the remaining free secondary
hydroxyl groups was studied. The formation of hydroxyethyl derivatives increases the hydrophilicity, resulting in less turbid
but thermally and physically weaker gels. Alkylation of the κ-carrageenan polymer resulted in increased hydrophobicity, turbidity and brittleness of the gels. Differences in thermal stability
and physical strength of both types of modified κ-carrageenan gels were studied by rheology. Turbidity was assessed by UV—v is spectroscopy.
09/2001: pages 127-131;
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ABSTRACT: In previous work the binding of the cationic surfactant dodecyltrimethylammonium bromide to DNA was studied. The original
work has been extended to include data for the decyltrimethy-lammonium ion. Additionally, while previously considerations
of the complex structure were done within the framework of the dynamics of rigid rods, in the present case we have extended
this analysis to include a wide range of three-dimensional configurations. Furthermore, the secondary structure of the DNA
within the complex is taken into account. Examination of secondary structural changes on surfactant binding indicates that
there are no significant changes in the DNA secondary structure. Consideration of the hydrodynamic properties of the surfactant—DNA
complex and extension of the experimental data to include decyltrimethylammonium along with application of hydrody-namic modelling
allowed us to exclude highly bent or folded complex conformations. The magnitude of the DNA diffusion coefficient decrease
on surfactant binding was measured for surfactant molecules of two different tail lengths. The data showed that a rod covered
in a single surfactant layer can provide a simple explanation for the difference in the magnitude of the decrease between
the two surfactants. In order to account for the observed ratio of 0.8 surfactants per DNA phosphate observed on completion
of the first-stage binding, the surfactant headgroups should be located close to the DNA surface, within the condensation
volume. This would leave the tail groups projecting outwards, with lateral hydrophobic association between the tails.
01/2001: pages 226-231;
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ABSTRACT: We have investigated the solubility, thermal stability and structural transitions of DNA complexed with the cationic surfactant
dodecyltrimethylammonium bromide (DTAB). The study was done over a broad range of ethanol/water mixtures. The dependence of
solubility on surfactant and salt concentration was studied. For low salt concentrations, it was found that DNA-DTA complexes
are soluble at an ethanol content higher then 57% v/v; however, addition of excess DTAB increases the ethanol concentration
at which the complexes begin to solubilise. At higher ethanol concentrations (about 80% v/v) DNA-DTA complexes were soluble
over the entire range of DTAB concentrations investigated. Also at higher ethanol concentrations it was found that the DNA
underwent a helix-coil transition. Circular dichroism data indicated that excess surfactant inhibited the B-to-A transition.
In the presence of 5 mM NaBr, the DNA was found to be insoluble in the high-ethanol region, while the surfactant-dependent
solubility line was shifted to lower ethanol concentrations. We present the phase diagram describing the solubility and conformational
state of the DNA-DTA complex and discuss the influence salt has on its stability and solubility.
05/2000: pages 186-191;
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ABSTRACT: The shrinking kinetics of submillimetre spherical gels of composition 10:20:70 mol% N-tert-butylacrylamide: N,′N-dimethylacrylamide: N-isopropylacrylamide have been studied in detail. A description of the shrinking kinetics throughout the transition region
(34–8 °C) is given as, in the size range studied, the gels showed no shape distortion during temperature jumps to these temperatures.
All gels relaxed to their equilibrium size exponentially after temperature jumps to temperatures in the transition region.
Using this unique molar composition, we systematically studied the effect of changing the network structure on the shrinking
kinetics. The aim was to achieve rapid controlled collapse with potential application in medicine and medical devices. The
approaches used included grafting of freely mobile polymer chains onto the polymer backbone, introduction of poly(acrylamide)
and incorporation of a second component to form a composite gel matrix.
05/2000: pages 121-127;
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ABSTRACT: In this work we describe a simple two step separation procedure for the separation and purification of short DNA fragments. The first step involves precipitating the DNA using the cationic surfactant dodecyltrimethylammonium bromide. Dodecyltrimethylammonium bromide, unlike cetyltrimethylammonium bromide will not precipitate DNA before complexation is complete thus providing a high purity DNA. The second step involves dissolution of the DNA-dodecyltrimethylammonium complex in 75% ethanol, followed by precipitation of the Sodium-DNA salt, by titrating in a salt solution. This method is particularly suited to purification of short fragments as it does not require high salt concentrations in the ethanol precipitation step, which can be damaging for short DNA. The ability of dodecyltrimethylammonium bromide to remove ethidium bromide from intercalation sites on the DNA is also discussed
Bioseparation 02/2000; 9(5):307-13.
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ABSTRACT: DNA-DPPC complexes can be prepared by means of a single step procedure of mixing DNA solution and aqueous lipid dispersion in the presence of calcium ions. Interaction between DPPC and DNA brings about a biphasic shape of melting curves corresponding to the free lipid and the strongly bound one. The amount of the strongly bound lipid is 5 molecules per nucleotide which is close to the size of the first lipid monolayer around DNA molecule.
FEBS Letters 04/1999; 446(1):27-9. · 3.54 Impact Factor
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ABSTRACT: Due to the growing interest in the use of cationic surfactants for the construction of liposomal genetic delivery systems, the study of complex formation between DNA and quaternary ammonium detergents is of fundamental importance. In this context, we undertook the study of this complex formation using capillary zone electrophoresis (CZE) with suppressed electroosmotic flow, a technique that allowed us to both monitor the change in mobility of DNA as a function of added surfactant in a precise and reproducible manner and evaluate the potential of CZE to reflect the change in hydrodynamic friction upon binding. Nevertheless, CZE must be applied with caution for binding studies where strong cooperativity occurs, because of the presence of peak splitting at concentrations close to the half-point of binding. Also, a comparison between this experiment and Manning's polyelectrolyte transport properties theory on one hand and Tirado and Garcia de la Torre expression for hydrodynamic friction of rod-like molecules on the other hand is given.
Journal of Chromatography 09/1998; 817(1-2):263-71. · 4.53 Impact Factor
