Release of plasmid DNA from intravascular stents coated with ultrathin multilayered polyelectrolyte films

Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein (EGFP) sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 degrees C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions.     

Force-induced structural transitions in cross-linked DNA films

We report on the preparation and characterization of wet-spun films of sodium DNA in which intermolecular cross-links were introduced following formaldehyde treatment. Raman scattering shows that the DNA in moderately cross-linked films is mainly in the B conformation. Stretching experiments show a transition from plastic to elastomeric behavior with increasing exposure to the cross-linking agent. Elastomeric DNA films are strongly disordered. X-ray diffraction shows that stretching of moderately cross-linked films under controlled high humidity conditions results in increased molecular orientation as well as the appearance of meridional reflections at 7.4-7.8 and 8.2 A. These reflections are not observed for any of the classical conformations associated with mixed sequence DNA, and may arise from extended base-pair stacking in a stretched DNA structure.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

Iron/dextran sulfate multilayered microcapsules for controlled release of 10-hydroxycamptothecin

Stable 10-hydroxycamptothecin (HCPT) microcrystals with a length of about 5-10 μm and a ζ-potential of −38.5 mV were produced by pH-induced reprecipitation in presence of a stabilizer hydroxypropylmethylcellulose. Sequential layer growth was achieved by the layer-by-layer (LbL) assembly of Fe³⁺ and dextran sulfate (DS) on the surface of HCPT microcrystals via both electrostatic interaction and chemical complexation process. The satisfactory drug loading content (67.2 ± 0.82%) as well as high encapsulation efficiency (60.56 ± 0.82%) for four bilayers of Fe³⁺/DS coating was achieved. Both in vitro and in vivo release study revealed that the release time increased as the number of deposited Fe³⁺/DS bilayers increased. These results indicated that such iron-polysaccharide multilayered microcapsules can be a promising approach for the construction of an effective controlled release delivery system of HCPT as well as other drugs with potential cytotoxicity or short half-life time.     

Qualitative characteristics of histamine release from human basophils by covalently cross-linked IgE

We have studied the effects of permanent oligomers of human IgE produced using the cross-linking reagent, dimethyl suberimidate, on histamine release from human basophils. IgE dimers were found to be sufficient stimuli for both release and desensitization of these cells; monomeric IgE had no effect. Histamine release was augmented by deuterium oxide (D2O) in the medium, but D2O was not an absolute requirement to observe release. Desensitization by the dimeric IgE was specific in that the response to anti-IgE was not affected by preincubation of the leukocytes with the IgE dimer under suboptimal releasing conditions. IgE trimers and higher oligomers of IgE also caused both release and desensitization. IgE trimers were 3- to 4-fold more effective than IgE dimers with regard to the amount required for 50% histamine release. Dilution studies with monomeric IgE suggested that the difference was due to the presence of more "active" dimers in the trimeric IgE fractions. We conclude that dimeric IgE, by juxtaposing 2 receptors on the basophil membrane, is the "unit signal" for both release and desensitization of these cells.     

Mechanisms of controlled release from silk fibroin films

The controlled release of fluorescein-iso-thio-cyanate (FITC)-labeled dextrans from methanol-treated and untreated silk fibroin films was modeled to characterize the release kinetics and mechanisms. Silk films were prepared with FITC-dextrans of various molecular weights (4, 10, 20, 40 kDa). Methanol treatment was used to promote crystallinity. The release data were assessed with two different models, an empirical exponential equation commonly fit to release data and a mechanism-based semiempirical model derived from Fickian diffusion through a porous film. The FITC-dextran release kinetics were evaluated as a function of molecular weight and compared between the untreated- and methanol-treated films. For the empirical model, the estimated values of the model parameters decreased with the molecular weight of the analyte and showed no significant difference between untreated- and methanol-treated films. For the diffusion-based model, the estimated diffusion coefficient was smaller for the methanol-treated films than for the untreated films. Also, the diffusion coefficient was observed to decrease linearly with increasing molecular weight of the analyte. The percent of FITC-dextran loading entrapped and not released was less for the methanol-treated films than for untreated films and linearly increased with molecular weight. A linear regression was fit to the relationship between molecular weight and the percent of entrapped FITC-dextran particles. Using these defined linear relationships, we present an updated version of the diffusion model for simulating release of FITC-dextran of varied molecular weights from methanol-treated and untreated silk films.     

Thermally modulated insulin release from microgel thin films

We describe investigations of thermally triggered insulin release from poly(N-isopropylacrylamide-co-acrylic acid) microgel thin films prepared by layer-by-layer (LbL) polyelectrolyte assembly. The thermoresponsivity of these films was confirmed using light scattering techniques. Simultaneous monitoring of film collapse and insulin release kinetics shows that deswelling of the films is partially decoupled from macromolecule release and that release is mainly governed by partitioning effects. We hypothesize, however, that film thermoresponsivity plays an important role in that subjection to many thermal cycles enables the embedded peptide to solubilize and subsequently partition through film layers. Direct pulsatile and extended release studies confirm the capability of these films to release bursts of insulin over many cycles, and confirm that the magnitude of the release can be controlled based on film thickness. These insulin-impregnated films are extremely stable with the potential to release constant pulses of peptide for more than 1 month at a time.     

Doxorubicin uptake and release from microgel thin films

We report investigations on the thermally regulated uptake and release of the chemotherapeutic drug doxorubicin from microgel thin films. A spin coating, layer-by-layer (scLbL) assembly approach was used to prepare thin films composed of thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) microgels by alternatively exposing a 3-aminopropyltrimethoxysilane (APTMS) functionalized glass substrate to polyanionic pNIPAm-AAc microgels and polycationic poly(allylamine hydrochloride) (PAH). Using this method, 10, 20, and 30 microgel layer films were constructed with uniform layer buildup, as confirmed by quartz crystal microgravimetry (QCM). The films were subsequently loaded with doxorubicin by cycling the temperature of the film in an aqueous doxorubicin solution between 25 and 50 degrees C. Release characteristics were then examined using UV-vis spectroscopy, which revealed temperature-dependent release properties.     

Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules.

The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.     

Layer-by-layer self-assembly aluminum Keggin ions/Prussian blue nanoparticles ultrathin films towards multifunctional sensing applications

In this study we described the nanocomposites films of specially synthesized inorganic Prussian blue (PB) nanoparticles and polyoxocation Al₁₃ Keggin ions that possess the excellent sensing activities. Film fabrication using layer-by-layer (LBL) self-assembly technique was followed by electrochemical characterization. The assembled multilayer Al₁₃/PB films as sensor devices for both the catalytic reduction of H₂O₂ and detecting the change of relative humidity were also investigated. The sensitivity of the biosensor was 0.886 mA cm⁻² mM⁻¹, and about two orders of magnitude change in resistance was observed as the relative humidity increasing from 5 to 95%. Both sensors exhibited good reproducibility, wide linear range. The performance and multifunctional abilities of these nanocomposites promise potential applications in biosensors, environmental controlling system and biomedical devices.     

Influence of phosphated cross-linked high amylose on in vitro release of different drugs

The influence of structural characteristics of high amylose cross-linked at different degrees on the release of drugs with important molecular differences, namely sodium diclophenac (SD) and nicotinamide (NI), was assessed in vitro from non-compacted systems. The release profiles were related with classical kinetic mathematical models for better understanding of the release mechanism. An increase in polymer cross-linking degree resulted in longer release time for both drugs, although SD generally was released slower than NI. SD release from samples cross-linked at 2% of basis was driven mainly by Fickian diffusion, while from samples cross-linked at 4% of basis follows anomalous mechanism. Inversely, anomalous mechanism was responsible for NI release from 2% samples and Fickian diffusion from 4% samples. Results suggest that the performance of cross-linked high amylose as excipient for controlled drug release not only depends on cross-linking degree but also is highly influenced by structural characteristics of the drug.     

Loading/release behavior of (chitosan/DNA)n layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage

In the present work, positively charged chitosan (CS) and negatively charged DNA were alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes, forming (CS/DNA)(n) layer-by-layer films. Cyclic voltammetry (CV) results showed that negatively charged electroactive probe, 9,10-anthraquinone-2,6-disulfonate (AQDS), could be loaded into the (CS/DNA)(n) films from its solution (1 mM at pH 7.0, containing 0.1 M NaCl), designated as (CS/DNA)(n)-AQDS, and then released from the films in blank buffers. The loading/release behavior of (CS/DNA)(n) films toward AQDS was found to be obviously different between double-stranded (dsDNA) and single-stranded DNA (ssDNA). The release rate of AQDS from (CS/dsDNA)(n) films was much slower than that from the ssDNA counterparts mainly because AQDS could be intercalated into the double helix structure of dsDNA despite the repulsion between likely charged AQDS and DNA. The loading/release behavior of (CS/DNA)(n) films toward AQDS in recognition of dsDNA and ssDNA was then successfully applied to electrochemically detect the damage of natural DNA caused by Fenton reaction. To further understand the essence of the interactions involved in the AQDS loading/release process for (CS/DNA)(n) films, comparison experiments were performed, in which either positively charged intercalator brilliant cresyl blue (BCB) was used to replace AQDS as the redox probe, or poly(diallyldimethylammonium) (PDDA) with relatively high positive charge density was used to replace CS as the constituent of layer-by-layer films with DNA. The loading/release behavior of DNA films toward electroactive intercalator may open new possibilities for dsDNA/ssDNA recognition and of DNA damage detection by electrochemistry.     

Measurement of DNA methylase activity by tritium release from DNA cytosine

The advantages of assaying of DNA methylase by measuring the transfer to water of tritium from the 5 position of DNA cytosine, rather than the transfer to DNA of labeled methyl groups are discussed.     

Electrodissolution of inorganic ions/DNA multilayer film for tunable DNA release

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr(4+)) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G₂/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.Key words: chromosome condensation, DNA damage, G₂/M checkpoint, ionizing radiation, PCC syndrome, primary microcephaly, repair foci     

In vitro release of thymine from DNA by neocarzinostatin.

$\text{In vitro}$ degradation of DNA to acid soluble products was induced by the combined action of neocarzinostatin and sulfhydryl agent as 2-mercaptoethanol, dithiothreitol, or reduced glutathione, but not other reducting agent as ascorbic acid or NaBH₄. From the analysis by Sephadex G-10 gel filtration, acid soluble products were found to be thymine and oligonucleotide, but not thymidylic acid and thymidine. Release of adenine or guanine from DNA was not detected.From these results, it is suggested that DNA chain breakage by the combined action of neocarzinostatin and 2-mercaptoethanol may be due to an indirect phosphodiester bond breakage with release of thymine.     

Glucocorticoids inhibit trypsin-induced DNA release from phytohemagglutinin-stimulated blood lymphocytes.

Trypsin-induced DNA release from phytohemagglutinin (PHA)-stimulated human lymphocytes is inhibited by different glucocorticosteroid compounds at low pharmacologic concentrations, in a dose-dependent manner, and in order of the known anti-inflammatory potency of the different preparations. In contrast, PHA-stimulated cell growth is 100- to 1000-fold less sensitive to inhibition by the same glucocorticoids. Nonglucocorticoid steroids have little effect on either DNA release or cell growth except at high concentrations. Inhibition of DNA release appears to be mediated through glucocorticoid receptors since progesterone, which is ineffective alone, competitively inhibits the effect of dexamethasone. The glucocorticoid effect on DNA release is tightly coupled to the initial, PHA-induced stimulus. Glucocorticoids are maximally effective when added to cultures 1 hr before PHA. When added 6 hr after PHA, their effect is minimal or absent, even though they are then continuously present until DNA release is measured 5 days later. Lymphocytes from certain donors in these studies were resistant to glucocorticoids; these individuals all had allergies, including asthma, allergic rhinitis, and bee sting hypersensitivity.