Effect of cationic surfactants on enhancement of firefly bioluminescence in the presence of liposomes

Firefly bioluminescence (BL) was greatly affected by cationic surfactants coexisting with liposomes containing phosphatidylcholine and cholesterol. In this study, the effects of the type and concentration of cationic surfactants on BL were studied in the presence of the liposomes. Three types of cationic surfactant: benzalkonium chloride (BAC), n-dodecyltrimethylammonium bromide (DTAB), and benzethonium chloride (BZC), were used. As a common effect in these surfactants, BL intensity was increased and then drastically decreased with increasing surfactant concentration. This can be explained by the formation of cationic liposomes as BL enhancers at low concentration of the surfactant, and by the transformation into cationic (mixed) micelles as inhibitors at high concentration. The maximal BL intensity and the concentration for the maximal BL were dependent on the type of the surfactants. To explain the differences in these parameters in the enhanced BL, we determined the distribution coefficient, K, of the surfactants to the liposomal membrane. The result indicated that the surfactant with higher K value gives the maximal BL intensity at lower concentration.     

Enhanced delivery of retinoic acid to skin by cationic liposomes

We studied the delivery of retinoic acid to skin by using cationic liposomes consisting of double-chained cationic surfactant, phosphatidylcholine (PC) and retinoic acid in excised guinea pig dorsal skin. Egg yolk PC liposomes contaning retinoic acid at a molar ratio of 4 : 1 increased the delivery of retinoic acid about two-fold, compared with its addition as an isopropyl myristate solution. Cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) further enhanced the incorporation dependent on the DOTAP content. Liposomes consisting of DOTAP, egg yolk PC, and retinoic acid at a molar ratio of 2 : 2 : 1 induced a 3.7-fold increase in the skin incorporation compared with the egg yolk PC liposomes without DOTAP. Significant difference was not observed when either dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) was used instead of egg yolk PC as well as when dimethyldipalmitylammonium was used instead of DOTAP. These results suggest the potential of the use of the cationic liposomes for the intradermal delivery of lipophilic drugs like retinoic acid.     

快速检测细菌总数的便携式生物荧光传感器

细菌数与细菌中三磷酸腺苷(ATP)含量成比例,可通过生物荧光法检测细菌中ATP含量,间接获得细菌总数.研制了一种便携式生物荧光传感器,可依次进行细菌ATP的提取和检测,最终实现对细菌总数的快速检测.以十六烷基三甲基溴化铵(CTMAB)作为细菌ATP提取剂,当CTMAB浓度为5 mmol/L、提取时间2 min、中和剂浓度为5 mmol/L时,ATP提取效率最高.在优化的实验条件下,传感器响应光强与细菌总数线性相关,线性范围58～5.89×10.7 cfu/mL,相关系数达0.979.传感器检测结果与传统方法吻合,线性相关系数为0.873,满足现场使用要求.从加样到ATP提取到最后获得检测结果所需总时间小于5 min.所研制的传感器具有操作简单、易于携带、成本低,适用于食品卫生质量控制、环境检测等领域的细菌总数现场检测.     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH

A multi-function biosensor for the determination of either ATP or NADH using a single bioluminescence-based fibre-optic probe is described. This was made possible by co-immobilizing the firefly luciferase from Photinus pyralis for ATP analysis with the bacterial/oxidoreductase system from Vibrio harveyi for NADH analysis, on the same preactivated polyamide membrane. Compatible analytical conditions with regard to the activity and stability of each bioluminescent system were selected, enabling them to attain their highest performances. It was possible to perform continuous-flow measurements of ATP and NADH over a wide (log-log) linear calibration range with a relative standard deviation of 4.0–4.5% and detection limits of 0.25 pmol ATP and 5 pmol NADH.     

Measuring the simultaneous effects of hypoxia and deformation on ATP release from erythrocytes

It is known that adenosine triphosphate (ATP) is released from red blood cells (RBCs) due to various forms of stimulation such as deformation, pharmacological stimuli, and hypoxia. To date, these various stimuli have been investigated individually. Here, we have combined a microflow system capable of initiating deformation-induced release of ATP from the RBCs at various levels of hypoxia as measured by percent oxygen saturation in the RBC sample. When values of ATP released from deformation and hypoxia are compared to values of ATP release due to hypoxia alone, the relationship between the two stimuli can be deduced. Measurement of RBC-derived ATP with the well-known chemiluminescence assay employing luciferin/luciferase indicates that RBCs deoxygenated for 4 min released 1.84 +/- 0.075 microM ATP. The largest decrease in oxygen saturation was found to be between 0 s (66.3% O(2) saturation) and 15 s (22.3% O(2) saturation). RBCs deoxygenated to a 22.3% O(2) saturation released 0.374 +/- 0.011 microM ATP when pumped through the microflow system. This value is an increase from 0.281 +/- 0.007 microM ATP in the presence of flow alone. The ATP release after exposure to hypoxia at 22.3% O(2) saturation was 0.381 +/- 0.014 microM ATP, a value statistically equivalent to that of hypoxia and flow combined. These data suggest that, at an oxygen saturation point of around 25.0% or above, deformation contributes to ATP release from the RBC; however, beyond this saturation point, the ATP release is largely due to hypoxia.     

Transformation of actoHMM assembly confined in cell-sized liposome

To construct a simple model of a cellular system equipped with motor proteins, cell-sized giant liposomes encapsulating various amounts of actoHMM, the complexes of actin filaments (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), with a depletion reagent to mimic the crowding effect of inside of living cell, were prepared. We adapted the methodology of the spontaneous transfer of water-in-oil (W/O) droplets through a phospholipid monolayer into the bulk aqueous phase and successfully prepared stable giant liposomes encapsulating the solution with a physiological salt concentration containing the desired concentrations of actoHMM, which had been almost impossible to obtain using currently adapted methodologies such as natural swelling and electro-formation on an electrode. We then examined the effect of ATP on the cytoskeleton components confined in those cell-sized liposomes, because ATP is known to drive the sliding motion for actoHMM. We added α-hemolysin, a bacterial membrane pore-forming toxin, to the bathing solution and obtained liposomes with the protein pores embedded on the bilayer membrane to allow the transfer of ATP inside the liposomes. We show that, by the ATP supply, the actoHMM bundles inside the liposomes exhibit specific changes in spatial distribution, caused by the active sliding between F-actin and HMM. Interestingly, all F-actins localized around the inner periphery of liposomes smaller than a critical size, whereas in the bulk solution and also in larger liposomes, the actin bundles formed aster-like structures under the same conditions.     

Cationic Polymerization Behavior of Some Vinyl Ketone Cyclodimers

Cationic polymerizations of 2,3-dihydropyran derivatives, methyl isopropenyl ketone cyclodimer (MIPKD), chloromethyl vinyl ketone cyclodimer (CMVKD), and methyl isopropenyl ketone-chloromethyl vinyl ketone cyclocodimer, D(MIPK-CMVK), were investigated in the presence of various Lewis acids at 0°C. These monomers were found to undergo cationic polymerization to give low molecular weight polymer which consisted mainly of a vinylene structure. However, the ring-opening polymerization was found to occur to a minor extent in cationic polymerizations of MIPKD and D(MIPK-CMVK) cyclodimer.     

荧光素酶法测定活性污泥中的ATP

选择了４种不同的提取剂，对曝气池活性污泥的腺苷三磷酸进行提取，并用荧光素酶法进行了测定。结果表明，不同提取剂所提取的ＡＴＰ量有很大不同；同一提取剂对不同水样ＡＴＰ的提取效果也不同。     

Extracellular ATP is generated by ATP synthase complex in adipocyte lipid rafts

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 microM) synthesis from the added ADP and P(i) in the adipocyte media suggests the involvement of the surface ATP synthase complex for the extracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.     

Silicone-stabilized liposomes

The present work is focused on stabilization of liposomes by covering their surface with a thin silicone layer. The appropriate silicone monomer was obtained by the hydrosilylation of vinylmethyldimethoxysilane with 1,3,5,7-tetramethylcyclotetrasiloxane. The surface potential of egg yolk phosphatidylcholine vesicles was modified by incorporation of a cationic double-tailed surfactant, dimethyldioctadecylammonium bromide (DODAB), yielding cationic liposomes. The silicone material was deposited on the cationic liposomes in base-catalyzed polycondensation/polymerization processes of the monomer at the liposomal surface. In order to initialize the processes pH of the liposomal dispersion was adjusted to the values of 8.5 or 10.2. The formed structures were characterized using dynamic light scattering (DLS) and zeta potential measurements. The DLS measurements show that the size of covered liposomes decrease during the reaction and the zeta potential turned to negative value, as can be expected. The morphology of the structures was evaluated using transmission cryo-electron microscopy (cryo-TEM). The cryo-TEM micrographs revealed the presence of the covered liposomes of sizes lower than the initial liposomes, which is in line with DLS measurements. However, some disintegration of the liposomes occurred during the covering procedure, especially at high pH value. Using the surfactant lysis and calcein-release study it was shown that silicone-covered liposomes are stable.     

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 micro m in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5'-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length.An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.     

Structure and order of DODAB bilayers modulated by dicationic gemini surfactants

Cationic liposomes have been extensively studied from the experimental and theoretical standpoints, motivated both by their fundamental interest and by potential applications in drug delivery and gene therapy. However, a detailed understanding of the nature of interactions within mixed bilayers containing cationic gemini surfactants is still lacking. This work focuses on the structural and dynamic properties of DODAB membranes in the presence of dicationic gemini surfactants. A thermodynamic characterization of the phase transitions in the mixed systems has been carried out by differential scanning calorimetry, while insight into the molecular interactions in the bilayer has been provided by molecular dynamics. For this purpose, variations in the gemini spacer and tail length, as well as in the respective molar fraction, have been included in both experimental and simulation studies. The results indicate that the influence of cationic gemini surfactants upon the thermotropic behavior and degree of order of DODAB structures is controlled by a complex interplay between charge density, conformation and hydrophobic effects, for which a detailed rationale is provided.     

Adsorption of bacteria and polycations on model surfaces of cellulose, hemicellulose and wood extractives studied by QCM-D

Quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) were used as the tools to study the adsorption of bacteria onto surfaces of silica and polystyrene coated with materials related to papermaking. Cationic polyelectrolytes used as fixatives and retention aids in paper industry were found to promote irreversible adsorption of the ubiquitous white water bacterium, Pseudoxanthomonas taiwanensis, onto model surfaces of cellulose (pH 8). The high charged low molecular weight polyelectrolyte, poly(diallyldimethyl) ammonium chloride (pDADMAC) adsorbed to silica surface as a flat and rigid layer, whereas the low charged cationic polyacryl amide (C-PAM) of high molecular weight adsorbed as a thick and loose layer. AFM images showed that the polyelectrolytes accumulated as layers around each bacterial cell. In the presence of wood hemicellulose (O-acetyl-galactoglucomannan) the bacteria adsorbed massively, as large, tightly packed rafts (up to 0.05mm in size) onto the polystyrene crystal surface coated with wood extractives (pH 4.7). AFM and FESEM micrographs also showed large naked areas (with no bacteria) in between the bacterial rafts on the crystal surface. In this case, QCM-D only incompletely responded to the massiveness of the bacterial adsorption. The results indicate that cationic polymers can be used to increase the retention of bacteria from the process water onto the fibre web and that, depending on the balance between hemicelluloses and wood extractives and pH of the process waters, bacteria can be drawn from process waters onto surfaces.     

Adsorption and desorption behaviors of cationic liposome-DNA complexes upon lipofection in inside and outside biomembrane models using a dynamic quasi-elastic laser scattering method.

The dynamic behaviors of cationic liposome-DNA complexes in inside and outside biomembrane models upon lipofection were investigated using the time-resolved quasi-elastic laser scattering (QELS) method. Inside and outside biomembrane models with similar phospholipid compositions to those in living cells were formed at a tetradecane/phosphate buffered saline (TD/PBS) interface. Cationic liposome-DNA complexes were injected into the buffer subphase, and their adsorption/desorption behaviors at the biomembrane models were monitored through changes in the interfacial tension. We found that the adsorption rate of the complexes increased 2.6 times more in the outside model than in the inside one. The adsorption rate of DNA alone did not show a remarkable difference from one side to the other; however, the adsorption rate of the cationic liposome alone showed a similar tendency to that of the liposome-DNA complex. These results indicated that the difference in lipid composition induced a different dynamic behavior of exogenous biomolecules and that the cationic liposomes played an important role in the faster incorporation of DNA into cells upon lipofection.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.     

Cationic liposomes in mixed didodecyldimethylammonium bromide and dioctadecyldimethylammonium bromide aqueous dispersions studied by differential scanning calorimetry, Nile Red fluorescence, and turbidity

The thermotropic phase behavior of cationic liposomes in mixtures of two of the most investigated liposome-forming double-chain lipids, dioctadecyldimethylammonium bromide (DODAB) and didodecyldimethylammonium bromide (DDAB), was investigated by differential scanning calorimetry (DSC), turbidity, and Nile Red fluorescence. The dispersions were investigated at 1.0 mM total surfactant concentration and varying DODAB and DDAB concentrations. The gel to liquid-crystalline phase transition temperatures (Tm) of neat DDAB and DODAB in aqueous dispersions are around 16 and 43 degrees C, respectively, and we aim to investigate the Tm behavior for mixtures of these cationic lipids. Overall, DDAB reduces the Tm of DODAB, the transition temperature depending on the DDAB content, but the Tm of DDAB is roughly independent of the DODAB concentration. Both DSC and fluorescence measurements show that, within the mixture, at room temperature (ca. 22 degrees C), the DDAB-rich liposomes are in the liquid-crystalline state, whereas the DODAB-rich liposomes are in the gel state. DSC results point to a higher affinity of DDAB for DODAB liposomes than the reverse, resulting in two populations of mixed DDAB/DODAB liposomes with distinctive phase behavior. Fluorescence measurements also show that the presence of a small amount of DODAB in DDAB-rich liposomes causes a pronounced effect in Nile Red emission, due to the increase in liposome size, as inferred from turbidity results.     

Polyadenylic acid binding on cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine

In this work unilamellar liposomes doped with a novel non-ionic 5′-Uridine-head nucleolipid, Lauroyl Uridine (LU), were prepared and characterized for their ability to interact with the polynucleotide polyadenylic acid (poly-A). Vesicles, were made up of the cationic lipid DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), the zwitterionic lipid DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine), and the novel amphiphile Lauroyl Uridine. The influence of the non-ionic nucleolipid on essential liposomes properties, such as the structure and net charge was first investigated by a comparative analysis performed on the different lipoplex preparations by means of ζ-potential and size measurements. Both structure and net charge of liposomes were shown to be not modified by the presence of the non-ionic nucleolipid.The role of the synthetic lipid inserted as anchor in the liposome bilayer in the condensation process between vesicles and the polynucleotide poly-A was then analyzed by UV–vis, Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The data presented comparative UV–vis analyses that evidenced the occurrence of staking interactions in the poly-A only in LU containing lipoplexes. CD and NMR studies indicated the presence of H-bonding interaction between Lauroyl Uridine containing vesicles and the polynucleotide poly-A. The results presented in this work support a role for Lauroyl Uridine in A-U molecular recognition, thus, suggesting that cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine could represent a model system to study molecular interactions among single stranded polynucleotides and lipid anchor bearing the complementary bases.     

Multicomponent cationic lipid-DNA complex formation: role of lipid mixing

Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3'[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dioleoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed.