An analysis of interactions between three structurally diverse anthocyanidins, as well as their glucosides, and model biological membranes, albumin, and plasmid DNA.

The aim of the study is to investigate the differences in the interaction of three structurally diverse anthocyanidins, namely peonidin, petunidin, and delphinidin, as well as their glucosides with model biological membranes, human albumin, and plasmid DNA in order to look into their structure-activity relationships. Fluorimetric studies, as well as ATR-FTIR analyses, were jointly used in order to determine the changes observed in both the hydrophilic and hydrophobic layers of cell-mimic membranes (MM) which reflected the membrane lipid composition of tumour cells and red blood cell membranes (RBCM). Our results showed that anthocyanins and anthocyanidins can cause an increase in the packing order of the polar heads of lipids, as well as interact with their deeper layers by reducing the fluidity of lipid chains. The results presented here indicate that all compounds tested here possessed the ability to bind to human serum albumin (HSA) and the presence of a glucose molecule within the structures formed by anthocyanidin reduces their ability to bind to proteins. Using fluorescence correlation spectroscopy, it was demonstrated that the compounds tested here were capable of forming stable complexes with plasmid DNA and, particularly, strong DNA conformational changes were observed in the presence of petunidin and corresponding glucoside, as well as delphinidin. The results we obtained can be useful in comprehending the anthocyanins therapeutic action as molecular antioxidants and provide a valuable insight into their mechanism of action.

Antitumor and antioxidant activities of purple potato ethanolic extract and its interaction with liposomes, albumin and plasmid DNA.

The aim of the study was to broadly determine the biological activities of purple potato ethanolic extract of the Blue Congo variety (BCE). The antioxidant activity of BCE was determined in relation to liposome membranes, and peroxidation was induced by UVB and AAPH. To clarify the antioxidant activity of BCE, we investigated its interactions with hydrophilic and hydrophobic regions of a membrane using fluorimetric and FTIR methods. Next, we investigated the cytotoxicity and pro-apoptotic activities of BCE in two human colon cancer cell lines (HT-29 and Caco-2) and in normal cells (IPEC-J2). In addition, the ability to inhibit enzymes that are involved in pro-inflammatory reactions was examined. Furthermore, BCE interactions with serum albumin and plasmid DNA were investigated using steady state fluorescence spectroscopy and a single molecule fluorescence technique (TCSPC-FCS). We proved that BCE effectively protects lipid membranes against the process of peroxidation and successfully inhibits the cyclooxygenase and lipoxygenase enzymes. Furthermore, it interacts with the hydrophilic and hydrophobic parts of lipid membranes as well as with albumin and plasmid DNA. It was observed that BCE is more cytotoxic against colon cancer cell lines than normal IPEC-J2 cells; it also induces apoptosis in cancer cell lines, but does not induce cell death in normal cells.

Interaction of Newly Platinum(II) with Tris(2-carboxyethyl)phosphine Complex with DNA and Model Lipid Membrane.

Structural properties of plasmid DNA and model lipid membrane treated with newly synthesized platinum(II) complex cis-[PtCl2{P(CH2CH2COOH)3}2] (cis-DTCEP for short) were studied and compared with effects of anticancer drug cisplatin, cis-[Pt(NH3)2Cl2] (cis-DDP for short). Time Correlated Single Photon Counting Fluorescence Correlation Spectroscopy (TCSPC-FCS) was employed to study interactions between those platinum complexes and DNA. The TCSPC-FCS results suggest that bonding of cis-DTCEP derivative to DNA leads to plasmid strain realignment towards much more compact structure than in the case of cis-DDP. Application of both differential scanning calorimetry and infrared spectroscopy to platinum complexes/DPPC showed that cis-DTCEP slightly increases the phospholipid's main phase transition temperature resulting in decreased fluidity of the model membrane. The newly investigated compound-similarly to cis-DDP-interacts mainly with the DPPC head group however not only by the means of electrostatic forces: this compound probably enters into hydrophilic region of the lipid bilayer and forms hydrogen bonds with COO groups of glycerol and PO2- group of DPPC.

New cytotoxic butyltin complexes with 2-sulfobenzoic acid: Molecular interaction with lipid bilayers and DNA as well as in vitro anticancer activity.

New butyltin complexes with 2-sulfobenzoic acid: [Sn(C4H9)2{O3SC6H4COO-2}(H2O)]·(C2H5OH) (DBTsbz), [Sn(C4H9)3{O3SC6H4COOH-2}] (TBTsbz) and [Sn2(C4H9)6{µ-O3SC6H4COO-2}] (DTBTsbz) are very effective cytotoxic agents against tumor cells. The molecular interaction of these complexes with lipid membranes and DNA has been investigated. The IR spectra and changes of (1)H, (13)C chemical shifts suggest that SO3 and COO groups of 2-sulfobenzoato ligand interact with O atom of glycerin fragment of DPPC. Moreover, the compounds form Sn-OP bonds with phosphate groups of DPPC, which was shown by the lower frequency shift of the νs(PO2(-)) and νas(PO2(-)) band, by change of (31)P NMR signals and by DFT calculation. Another possibility is the interaction of the phosphate group of DPPC owing to formation of hydrogen bond O-H…O-P between water molecule coordinated to Sn and oxygen atom from the phosphate group. Using TCSPC-FCS we characterized DNA supramolecular assemblies' formation upon increasing TBTsbz, DTBTsbz and DBTsbz concentration. Diffusion time, lifetime and particle number changes are altered systematically with increasing Ccomp/CDNAbp ratio in following effectiveness order DBTsbz > TBTsbz > DTBTsbz. From those parameters we can conclude that all these compounds lead to a change of DNA winding, strand but not to DNA compaction. Investigated compounds show very high cytotoxic activity against cancer cell lines. All compounds exhibit efficient in vitro antitumor activity toward Jurkat (T-cell leukemia), CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line) and D-17 (canine osteosarcoma). The DBTsbz is more effective then carboplatin against canine osteosarcoma.

New gluconamide-type cationic surfactants: Interactions with DNA and lipid membranes.

New linear cationic surfactants - 2-(alkyldimethylammonio)ethylgluconamide bromides, denoted as CnGAB, n=10, 12, 14 and 16 - were synthesized from natural resources and characterized with respect to their potential as gene-delivery agents in gene therapy applications. Interactions with plasmid DNA and with model membranes were studied both experimentally and theoretically. The compounds with n=12, 14 and 16 show exponentially increasing ability to fully condense DNA. C16GAB condenses DNA at 1:1 surfactant to nucleotide molar ratio. Furthermore, CnGABs interact with model membrane, slightly lowering the temperature of the main phase transition Tm of the DPPC bilayer. C10GAB is found to interact only at the membrane surface. C16GAB reduces Tm less than C12GAB and C14GAB, and forms domains in the bilayer at the surfactant/DPPC molar ratio of 0.1 and higher. The results suggest that C16GAB can be a promising candidate for building gene-delivery carrier systems.

Protonation of lipids impacts the supramolecular and biological properties of their self-assembly.

We assessed in this work how a chemical structure difference could influence a supramolecular organization and then its biological properties. In our case study, we considered two amphiphilic lipidic gene vectors. The chemical difference was situated on their hydrophilic part which was either a pure neutral thiourea head or a mixture of three thiourea function derivatives, thiourea, iminothiol, and charged iminothiol. This small difference was obtained thanks to the last chemical deprotection conditions of the polar head hydroxyl groups. Light, neutron, and X-ray scattering techniques have been used to investigate the spatial structure of the liposomes and lipoplexes formed by the lipids. The chemical structure difference impacts the supramolecular assemblies of the lipids and with DNA as shown by fluorescence correlation spectroscopy (FCS), X-ray, and neutron scattering. Hence the structures formed were found to be highly different in terms of liposomes to DNA ratio and size and polydispersity of the aggregates. Finally, the transfection and internalization results proved that the differences in the structure of the lipid aggregates fully affect the biological properties of the lipopolythiourea compounds. The lipid containing three functions is a better gene transfection agent than the lipid which only contains one thiourea moiety. As a conclusion, we showed that the conditions of the last chemical step can influence the lipidic supramolecular structure which in turn strongly impacts their biological properties.

Lipopolythiourea/DNA interaction: a biophysical study.

Lipopolythioureas (LPT) are original non cationic systems representing an alternative to cationic lipids. Their high transfection efficiency prompted us to investigate further their biophysical properties, and in particular how thiourea lipids interact with DNA. The interaction of lipopolythiourea with DNA was investigated by fluorescence correlation microscopy (FCS). Influence of the lipid length and nature of the thiourea head on the thiourea/DNA interaction were studied. FCS revealed a strong interaction between lipopolythiourea and DNA, occurring at 1 equivalent of a thiourea lipid by a DNA phosphate group, and leading to a condensed plasmid state. From previous in vitro experiments, we could conclude that the lipid leading to the more condensed state of DNA was also the more efficient to transfect cells.

On mechanism of intermediate-sized circular DNA compaction mediated by spermine: contribution of fluorescence lifetime correlation spectroscopy.

The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Thus, it is in the scope of research interest. It was shown, that spermine-induced compaction of large DNA molecules occurs in a discrete "all-or-non" regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (approximately 10 kbp), so far, it was stated that the mechanism of folding is continuous. Here, we show, that neither a standard benchmark technique-dynamic light scattering, nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach-fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen during the titration with spermine and based on that, it reveals the discrete mechanism of the process. Furthermore, we show that it allows for observation of the equilibrium state transition dynamics.

Equilibrium dynamics of spermine-induced plasmid DNA condensation revealed by fluorescence lifetime correlation spectroscopy.

The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto- and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.

Lipopolyamine-mediated single nanoparticle formation of calf thymus DNA analyzed by fluorescence correlation spectroscopy.

PURPOSE: The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N4,N9-dioleoylspermine and N1-cholesteryl spermine carbamate. METHODS: Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. RESULTS: N4,N9-dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 x 10(-12) m2/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio = 1.0-1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N1-cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5-2.5) with D = 1.3 x 10(-12) m2/s and PN value of 5.2. N4,N9-dioleoylspermine is a more efficient DNA-condensing agent than N1-cholesteryl spermine carbamate. CONCLUSIONS: FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

DNA-spermine and DNA-lipid aggregate formation visualized by fluorescence correlation spectroscopy.

BACKGROUND: Fluorescence correlation spectroscopy (FCS) can be used for the determination of diffusion coefficients of single molecules. Since diffusion coefficients are correlated with size and shape of the labeled species, FCS provides information on conformational changes in plasmids aggregates. METHODS: A 10-kbp plasmid stained with PicoGreen was condensed by spermine or liposomes formulated from cationic lipid and egg phosphatidylcholine. RESULTS: The diffusion coefficient of DNA increases from 1.0 x 10(-12) m2/s to 3.2 x 10(-12) m2/s by the addition of spermine, whereas the addition of cationic liposomes leads to complexes characterized by diffusion coefficients with values ranging from 1.7 to 1.9 x 10(-12) m2/s. CONCLUSIONS: FCS experiments allow determining the diffusion coefficients of DNA-containing aggregates which provide information regarding the topology and homogeneity of the aggregate.

Propidium iodide and PicoGreen as dyes for the DNA fluorescence correlation spectroscopy measurements.

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe-nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

Fluorescence correlation spectroscopy (FCS) as a tool to study DNA condensation with hexadecyltrimethylammonium bromide (HTAB).

The presented data show that the FCS technique can be used to detect the DNA condensation process induced with the cationic compound hexadecyltrimethylammonium bromide (HTAB). We have shown that HTAB induces plasmid condensation upon interaction with it. Condensation can be considered to be complete when the diffusion constant reaches its maximum. The HTAB induced increase in diffusion time does not correlate well with the changes observed when count rate and particle number are considered. This observation contradicts data published for another cationic agent, spermine. This apparent discrepancy proves that the mechanisms of interaction between these compounds and DNA are different. Consequently, the different characters of the plots of count rate, diffusion time, and particle number versus condensing agent concentration can be a source of additional information about the nature of cationic compound-DNA interaction.

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy.

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHbetaAPr-1-neo (10 kbp; contour length 3.4 microm) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 microM (spermine/DNAphosphate=0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.0x10(-12) m2/s to 6.0x10(-12) m2/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dye's dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

The application of fluorescence correlation spectroscopy in detecting DNA condensation.

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).