Interaction of DNA/polycation complexes with phospholipids: stabilizing strategy for gene delivery

The interaction between negatively charged lipid vesicles and positively charged DNA/polylysine complexes was studied. The interaction does not lead to release of DNA from the initial complexes. The particles formed are easy to prepare, they have slight negative charge, small dimensions and show good stability in physiological NaCl solution. Such properties might indicate that stabilization of the particles by lipid coating might be a potent strategy, alternative to PEGylation of DNA/polycation complexes. Interaction of DNA/polycation complex particles with lipid vesicles.     

Structural and conformational characteristics of DNA complexes with polycations of different structure

The formation of DNA complexes with polycations of different structure (homopolymers: N,N-dimethylaminoethyl and methylsulfate salt of N,N,N-trimethylaminoethylmethacrylate, along with a copolymer of N,N-dimethylaminoethyl methacrylate with N-methacryloylaminoglucose) in water-salt solutions, was studied by the method of static and dynamic light scattering. It was shown that, depending on the value of ϕ = [mol polycation]/[mole of DNA], complexes of different structure are formed: in field ϕ < 6, they are dense and compact formations; when ϕ > 6 in the presence of a large excess of polycations, loose structures are formed. The extreme dependence of the molecular weight of formed supramolecular structures on the values of ϕ was established.     

Cationic PLA nanoparticles for DNA delivery: comparison of three surface polycations for DNA binding, protection and transfection properties

Biodegradable cationic nanoparticles (cNP) made of poly(lactide) (PLA) have been shown to be promising carrier systems for in vivo DNA delivery and immunization. In previous work, we have described a versatile approach for the elaboration of cationic PLA cNP based on the use of pre-formed particles and subsequent adsorption of a model polycation, the poly(ethylenimine) (PEI). Here, we evaluated two more polycations, chitosan and poly(2-dimethyl-amino)ethyl methacrylate (pDMAEMA)) to determine the most suitable one for the development of PLA cNP as DNA carriers. Cationic PLA-PEI, PLA-chitosan and PLA-pDMAEMA nanoparticles were compared for interaction with plasmid DNA and, more importantly, with regards to the biological properties of bound DNA. pDMAEMA coating yielded the most positively charged nanoparticles with the highest DNA binding capacity (32 mg/g). Loaded with DNA, all three cNP were in the same size range ( approximately 500 nm) and had a negative zeta potential (-50 mV). PLA-chitosan was the only cNP that released DNA at pH 7; the two others required higher pH. Adsorption and release from cNP did not alter structural and functional integrity of plasmid DNA. Moreover, DNA coated onto cNP was partially protected from nuclease degradation, although this protection was less efficient for PLA-chitosan than others. The highest transfection efficiency in cell culture was obtained with PLA-pDMAEMA carriers. We have shown that at least three different cationic polymers (chitosan, PEI, pDMAEMA) can be used for the production of PLA-based particulate DNA carriers and most probably other cationic polymers can also be used in the same purpose. PLA-pDMAEMA cNP were the most promising system for DNA delivery in this in vitro study. Our future work will focus on the in vivo evaluation of these gene delivery systems.     

Formation of DNA complexes with diblock copolymers of poly(N-(2-hydroxypropyl) methacrylamide) and polycations

The self-assembly of calf thymus DNA with diblock copolymers of poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) and polycations (PC) with amino or quaternary ammonium groups in their side chains was tested by static and dynamic light scattering methods in aqueous 0.15 M NaCl. We have observed formation of well defined clusters of single DNA interpolyelectrolyte complexes with hydrodynamic radii of about 300 nm. Long-term size stability of clusters has been found to be a function of chemical structure of PC and of diblock copolymer composition. The most stable clusters were obtained by DNA association with diblock copolymers consisting of a poly(HPMA) block (A) and polycation block (B) with quaternary ammonium groups in their side chains, with molecular weight of A block higher than that of B. In the opposite case, lower molecular weight of A block, slow aggregation of SIPEC clusters was observed. The micelle-like structure was proposed for these clusters.     

Amorphous linear aliphatic polyesters for the facile preparation of tunable rapidly degrading elastomeric devices and delivery vectors

A versatile method for preparing amorphous degradable elastomers with tunable properties that can be easily fabricated into a wide variety of shape-specific devices was investigated. Completely amorphous, liquid poly(ester ether) prepolymers with number-average molecular weights between 4 and 6 x 10(3) g/mol were prepared via condensation polymerization. These liquid prepolymers were then thermally cross-linked to form degradable elastomeric structures. The ability to vary the composition of these liquid prepolymers allows for easy control of the mechanical and degradation properties of the resulting elastomeric structures. Materials can be designed to completely degrade in vitro over a range of 30 days to 6 months, while the Young's modulus can be varied over 3 orders of magnitude (G = 0.02-20 MPa). Also, the liquid nature of these prepolymers makes them amenable to a wide variety of fabrication techniques. Using traditional and modified imprint lithography techniques, we have fabricated devices that demonstrate a wide variety of biologically applicable topologies, which could easily be extended to fabricate devices with more complex geometries. Until now, no method has combined this ease and speed of fabrication with the ability to control the mechanical and degradation properties of the resulting elastomers over such a broad range.     

Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.     

Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40?U?mL(-1) and a detection limit of 0.2?U?mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01?U?mL(-1).     

Influence of biological media on the structure and behavior of ferrocene-containing cationic lipid/DNA complexes used for DNA delivery

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li(2)SO(4)) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately -35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about -20 mV in OptiMEM and -10 mV in Li(2)SO(4)), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.     

Polycationic amphiphilic cyclodextrins as gene vectors: effect of the macrocyclic ring size on the DNA complexing and delivery properties

A collection of homologous monodisperse facial amphiphiles consisting of an α-, β- or γ-cyclodextrin (α, β or γCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hexanoyl chains at the secondary hydroxyls have been prepared to assess the influence of the cyclooligosaccharide core size in their ability to complex, compact and protect pDNA and in the efficiency of the resulting nanocondensates (CDplexes) to deliver DNA into cells and promote transfection in the presence of serum. All the polycationic amphiphilic CDs (paCDs) were able to self-assemble in the presence of the plasmid and produce transfectious nanoparticles at nitrogen/phosphorous ratios ≥5. CDplexes obtained from βCD derivatives generally exhibited higher transfection capabilities, which can be ascribed to their ability to form inclusion complexes with cholesterol, thereby enhancing biological membrane permeability. The presence of thiourea moieties as well as increasing the number of primary amino groups then favour cooperative complexation of the polyphosphate chain, enhancing the stability of the complex and improving transfection. In the α and γCD series, however, only the presence of tertiary amino groups in the cationic clusters translates into a significant improvement of the transfection efficiency, probably by activating endosome escape by the proton sponge mechanism. This set of results illustrates the potential of this strategy for the rational design and optimisation of nonviral gene vectors.     

Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer

A highly efficient drug vector for photodynamic therapy (PDT) drug delivery was developed by synthesizing PEGylated gold nanoparticle conjugates, which act as a water-soluble and biocompatible "cage" that allows delivery of a hydrophobic drug to its site of PDT action. The dynamics of drug release in vitro in a two-phase solution system and in vivo in cancer-bearing mice indicates that the process of drug delivery is highly efficient, and passive targeting prefers the tumor site. With the Au NP-Pc 4 conjugates, the drug delivery time required for PDT has been greatly reduced to less than 2 h, compared to 2 days for the free drug.     

Quantum-chemical study of isocyanate reactions with linear methanol associates: IX. Methyl isocyanate reaction with methanol-phenol complexes

Quantum-chemical method B3LYP/6-311++G(df,p) was applied to the study of complex formation between phenol and methanol. The complexes of molecules bound by the hydrogen bond possess enhanced donoracceptor and acid-base properties as compared to monomer molecules. The reactions of phenol and methanol complexes with methyl isocyanate proceed through a concerted late asymmetric transition state by the type of nucleophilic addition. The most kinetically and thermodynamically favorable transformation is the process involving the complex “phenol-acceptor, methanol-donor.” Phenol compounds are able to catalyze the addition of alcohols to isocyanates.     

Microdetermination of double-stranded DNA by linear sweep voltammetry with phenosafranine.

A novel voltammetric method for the determination of microamounts of fish sperm double-stranded (ds) DNA based on its interaction with phenosafranine (PSF) is proposed in this paper. In a pH 3.5 Britton-Robinson (B-R) buffer solution, PSF had a well-defined second-order derivative linear-sweep voltammetric reductive peak at -0.32 V (vs. SCE) on a mercury electrode. After the addition of dsDNA into the PSF solution, the reductive peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experiment results showed that a new supramolecular complex was formed after the interaction of dsDNA with PSF, which resulted in a decrease of the diffusion coefficient, and then a decrease of the reductive peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. Under the optimal conditions, the decrease of the peak current was proportional to the dsDNA concentration in the range 1.0 - 40.0 microg/mL with the linear regression equation DeltaI(p)'(nA) = 32.59C(microg/mL) - 4.03 (n = 13, gamma = 0.998) and a detection limit of 0.25 microg/mL (3 sigma). The interaction mechanism was considered based on the aggregation of the dsDNA-PSF supramolecular complex; the stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of 3 and a binding constant of 2.76 x 10(12). This method was successfully applied to the determination of synthetic samples and the polymerase chain reaction (PCR) product of the nopaline synthase gene (NOS) DNA from genetically modified organisms (GMOs) with satisfactory results.     

Synthesis of novel amphiphilic conjugates with a biological recognition function for developing targeted triggered liposomal delivery systems

Several novel amphiphilic lipid derivatives were synthesized consisting of a lipid anchor connected to the hydrophilic moiety via a disulfide or glycoside bond and biotin linked to the hydrophilic part. Disulfide bonds were established by the help of 4-phenyltriazol-3,5-dione. Dansyl or fluorescein was covalently linked as fluorescent marker to some of the conjugates, allowing spectroscopic and microscopic detection. The conjugates represent first amphiphilic lipids carrying all four functions, i.e., lipophilic, hydrophilic, recognition, and disulfide cleavage group in one molecule, which are necessary for targeted, triggered drug delivery from phospholipid liposomes on demand.     

Immune complexes in blood: a new strategy for the analysis of their antigen portion.

A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two-dimensional gel electrophoresis.     

Elementary steps for charge transport in DNA: thermal activation vs. tunneling

Using stacks of Watson–Crick base pairs as an important example of multichromophoric molecular assemblies, we studied charge migration in DNA with special emphasis on the mechanism of hole hopping between neighboring guanines (G) connected by the adenine–thymine (AT) bridge. The tight-binding model proposed for this elementary step shows that for short AT bridges, hole transfer between two G bases proceeds via quantum mechanical tunneling. By contrast, hopping over long bridges requires thermal activation. The condition for crossover between tunneling and thermal activation near room temperature is specified and applies to the analysis of experimental data. We show that thermal activation dominates, if the bridge between two G bases contains more than three AT pairs. Our theoretical findings predict that the replacement of AT base pairs by GC pairs increases the efficiency of hole transport only in the case of short base pair sequences. For long sequences, however, the opposite effect is expected.     

Salicylaldiminato derivatives of cyclotriveratrylene: flexible strategy for new rim-metalated CTV complexes

The amino-derivatized cyclotriveratrylene analogue, triaminotrimethoxytribenzocyclononene [CTV(NH2)3(OMe)3], 1, is readily converted into triply substituted imine compounds [CTV(sal)3(OMe)3], 2, in high yield by treatment of the acid salt of 1 with a variety of substituted salicylaldehydes. Cleavage of the protecting methoxy group generates the tristridentate chelate CTV(sal)3(OH)3, 3, which is readily converted into new rim-metalated species CTV(sal)3(ONiL)3, 4a (a, L = pyrrolidine; b, L = 1-n-butyl-imidazole). Taken together, these results illustrate the remarkable synthetic flexibility that is possible for the CTV-based metal complexes by alteration of the metal, the salicylaldehyde component of the CTV ligand, or the ancillary ligands coordinated to the metal.     

'N-in-one' strategy for metabolite identification using a liquid chromatography/hybrid triple quadrupole linear ion trap instrument using multiple dependent product ion scans triggered with full mass scan

This paper describes a new strategy that utilizes the fast trap mode scan of the hybrid triple quadrupole linear ion trap (QqQ(LIT)) for the identification of drug metabolites. The strategy uses information-dependent acquisition (IDA) where the enhanced mass scan (EMS), the trap mode full scan, was used as the survey scan to trigger multiple dependent enhanced product ion scans (EPI), the trap mode product ion scans. The single data file collected with this approach not only includes full scan data (the survey), but also product ion spectra rich in structural information. By extracting characteristic product ions from the dependent EPI chromatograms, we can provide nearly complete information for in vitro metabolites that otherwise would have to be obtained by multiple precursor ion scan (prec) and constant neutral loss (NL) analysis. This approach effectively overcomes the disadvantages of traditional prec and NL scans, namely the slow quadrupole scan speed, and possible mass shift. Using nefazodone (NEF) as the model compound, we demonstrated the effectiveness of this strategy by identifying 22 phase I metabolites in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. In addition to the metabolites reported previously in the literature, seven new metabolites were identified and their chemical structures are proposed. The oxidative dechlorination biotransformation was also discovered which was not reported in previous literature for NEF. The strategy was further evaluated and worked well for the fast discovery setting when a ballistic gradient elution was used, as well as for a simulated in vivo setting when the incubated sample (phase I metabolites) was spiked to control human plasma extract and control human urine.