Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery

DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced by coaxial EHDS had delivery efficiencies up to 2.6 times higher than those produced by bulk mixing. The N/P ratio and the structure of the EHDS used to make the polyplexes were crucial factors in determining the delivery efficiency. Biotechnol. Bioeng. 2010. 105: 834–841. © 2009 Wiley Periodicals, Inc.     

Relationships between the physicochemical properties of an amphiphilic triblock copolymers/DNA complexes and their intramuscular transfection efficiency

Poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer (PEO-PPO-PEO) based plasmid delivery systems are increasingly drawing attention in the field of nonviral gene transfer because of their proven in vivo transfection capability. They result from the simple association of DNA molecules with uncharged polymers. We examined the physicochemical properties of PEO-PPO-PEO/DNA mixtures, in which the PEO-PPO-PEO is Lutrol® (PEO₇₅-PPO₃₀-PEO₇₅), formulated under various conditions. We found that interactions between PEO-PPO-PEO and DNA are mediated by the central hydrophobic block within the block copolymer. Dynamic light scattering and cryo-electron microscopy showed that the mean diameter of transfecting particles as well as their stability depended on the PEO-PPO-PEO/DNA ratio and on the ionic composition of the formulating medium. The most active formulation promoting a good tissue-distribution and an optimal transfection was characterized by a reduced electrophoretic mobility, a mean hydrodynamic diameter of ~250–300 nm and by a conserved B-DNA form as shown by circular dichroism studies. Our study also revealed that the stability of these formulations strongly depended on a concentration balance between the DNA and the PEO-PPO-PEO, over which the DNA conformation was modified, micron-sized particles were generated, and the transgene expression was declined. We showed that the physicochemical properties of PEO-PPO-PEO/DNA formulations directly impact the level of gene expression in transfected muscles.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Synthesis and characterization of biocompatible thermo-responsive gelators based on ABA triblock copolymers

The synthesis of biocompatible, thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(N-isopropylacrylamide) and the central B block is poly(2-methacryloyloxyethyl phosphorylcholine) is achieved using atom transfer radical polymerization with a commercially available bifunctional initiator. These novel triblock copolymers are water-soluble in dilute aqueous solution at 20 degrees C and pH 7.4 but form free-standing physical gels at 37 degrees C due to hydrophobic interactions between the poly(N-isopropylacrylamide) blocks. This gelation is reversible, and the gels are believed to contain nanosized micellar domains; this suggests possible applications in drug delivery and tissue engineering.     

Synthesis of biocompatible,stimuli-responsive,physical gels based on ABA triblock copolymers

ABA triblock copolymers [A = 2-(diisopropylamino)ethyl methacrylate), DPA or 2-(diethylamino)ethyl methacrylate), DEA; B = 2-methacryloyloxyethyl phosphorylcholine, MPC] prepared using atom transfer radical polymerization dissolve in acidic solution but form biocompatible free-standing gels at around neutral pH in moderately concentrated aqueous solution (above approximately 10 w/v % copolymer). Proton NMR studies indicate that physical gelation occurs because the deprotonated outer DPA (or DEA) blocks become hydrophobic, which leads to attractive interactions between the chains: addition of acid leads to immediate dissolution of the micellar gel. Release studies using dipyridamole as a model hydrophobic drug indicate that sustained release profiles can be obtained from these gels under physiologically relevant conditions. More concentrated DPA-MPC-DPA gels give slower release profiles, as expected. At lower pH, fast, triggered release can also be achieved, because gel dissolution occurs under these conditions. Furthermore, the nature of the outer block also plays a role; the more hydrophobic DPA-MPC-DPA triblock gels are formed at lower copolymer concentrations and retain the drug longer than the DEA-MPC-DEA triblock gels.     

Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer

Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.     

Effect of polymer ionization on the interaction with DNA in nonviral gene delivery systems

The optimization of DNA-cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA-cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, K(obs) increased from 7.8 x 10(5) to 20.4 x 10(5) M(-1). More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.     

Poly(ethylene oxide) grafted with short polyethylenimine gives DNA polyplexes with superior colloidal stability, low cytotoxicity, and potent in vitro gene transfection under serum conditions

Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ～ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.     

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.     

Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA–chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA–chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from ∼10 to ∼1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.     

Chitosan grafted with macrocyclic polyamines on C-2 and C-6 positions as nonviral gene vectors: preparation, characterization, and in vitro transfection studies

Chitosan grafted with macrocyclic polyamines (Cs-g-MCPA) on the C-2 or the C-6 position was synthesized by a simple method. Four copolymers prepared were characterized by (1)H NMR, (13)C NMR, Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), and gel permeation chromatography (GPC). Circular dichroism spectra (CD), fluorescence spectra and agarose gel electrophoresis assay showed that Cs-g-MCPA copolymers had good binding ability to DNA. By modification of the MCPAs, the copolymers showed low cytotoxicity and high transfection efficiency as new gene vectors. It was found that Cs-g-MCPA copolymers with different grafted positions showed different properties: copolymers grafted on the C-2 position showed higher cytotoxicity and higher transfection efficiency than those grafted on position C-6.     

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

DNA compaction into new DNA vectors based on cyclodextrin polymer: surface enhanced Raman spectroscopy characterization

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.     

Au-NPs enhanced SPR biosensor based on hairpin DNA without the effect of nonspecific adsorption

Gold nanoparticles (Au-NPs) are usually used to amplify surface plasmon resonance (SPR) signals, however, the serious nonspecific adsorption has largely limited their practical applications. Here, we developed a novel Au-NPs enhanced biosensor without the effect of nonspecific adsorption: cutting Au-NPs off from the biosensor surface by RsaI endonuclease. In order to further improve the sensitivity, the probe DNA was designed specially. After the cleavage reaction, the residual probe DNA formed hairpin structure, which also resulted in a great change in SPR dip shift. Then, with the coaction of Au-NPs and conformation change of probe DNA, the SPR signal was amplified greatly. Using this method, we monitored the process of DNA cleavage in real-time and achieved a detection level of 5×10(-8) M. Moreover, the result of X-ray photoelectron spectroscopy (XPS) experiment further confirmed that large nonspecific adsorption existed. However, because SPR recorded a process in which the Au-NPs were cut off, the serious nonspecific adsorption did not affect the experimental result.     

Effect of DNA damage on the expression of the chloramphenicol acetyltransferase gene after transfection into diploid human fibroblasts.

The activity of the chloramphenicol acetyltransferase (cat) gene after transfection into human fibroblasts has been measured following treatment of the plasmid pRSVcat with either restriction enzymes or ultraviolet light. Restriction enzymes producing single cuts in the plasmid inactivated the expression of the cat gene whether the enzymes cut the plasmid inside the coding region of the gene or several kilobases away from the gene. Ultraviolet light produced a dose-dependent inactivation of the gene. The inactivation curve was steeper if the recipient cell strain was derived from a patient with xeroderma pigmentosum. The findings with this transient expression system contrast with previously reported results of experiments using plasmids which transform cells stably by integrating into the cellular genomic DNA.     

Synthesis, characterization, and DNA adsorption studies of ampholytic model conetworks based on cross-linked star copolymers

Five model conetworks based on cross-linked star ampholytic copolymers were synthesized by group transfer polymerization. The ampholytic copolymers were based on two hydrophilic monomers: the positively ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the negatively ionizable methacrylic acid (MAA). Ethylene glycol dimethacrylate was used as the cross-linker. These five ampholytic model conetworks were isomers based on equimolar DMAEMA-MAA copolymer stars of different architectures: heteroarm (two), star block (two), and statistical. The two networks based on the homopolymer stars were also synthesized. The MAA units were introduced via the polymerization of tetrahydropyranyl methacrylate and the acid hydrolysis of the latter after network formation. All the precursors to the (co)networks were characterized in terms of their molecular weights using gel permeation chromatography (GPC). The mass of the extractables from the (co)networks was measured and characterized in terms of molecular weight and composition using GPC and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The degrees of swelling (DS) of all the ampholytic conetworks were measured as a function of pH and were found to present a minimum at a pH value which was taken as the isoelectric point, pI. The DS and the pI values did not present a dependence on conetwork architecture. Finally, DNA adsorption studies onto the ampholyte conetworks indicated that DNA binding was governed by electrostatics.     

An ensemble method for gene discovery based on DNA microarray data

The advent of DNA microarray technology has offered the promise of casting new insights onto deciphering secrets of life by monitoring activities of thousands of genes simultaneously. Current analyses of microarray data focus on precise classification of biological types, for example, tumor versus normal tissues. A further scientific challenging task is to extract disease-relevant genes from the bewildering amounts of raw data, which is one of the most critical themes in the post-genomic era, but it is generally ignored due to lack of an efficient approach. In this paper, we present a novel ensemble method for gene extraction that can be tailored to fulfill multiple biological tasks including (i) precise classification of biological types; (ii) disease gene mining; and (iii) target-driven gene networking. We also give a numerical application for(i) and (ii) using a public microarrary data set and set aside a separate paper to address (iii).