Calorimetric study of the interaction of binary DMTAP/DOTAP cationic liposomes with plasmid DNA

Cationic liposomes have been suggested as possible agents for nonviral gene transfer. The interaction of plasmid DNA (pDNA) with dispersions of stable unilamellar cationic liposomes based on the binary lipid system 1,2-dimyristoyl-3-trimethyl-ammonium-propane (DMTAP):1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) has been studied by using isothermal titration calorimetry (ITC), high-precision differential scanning calorimetry (DSC), dynamic light scattering (DLS), and circular dichroism (CD). Systematic calorimetric and DLS exploration of the DMTAP:DOTAP binary system reveals that single-bilayer liposomes are stable at the 4:1 molar ratio, exhibiting the main lipid-phase transition temperature at ~25.3°C, and a total enthalpy change δH = 8.5 ± 0.4 kcal/mol. The interaction of pDNA with unilamellar DMTAP:DOTAP vesicles was investigated by ITC experiments, which clearly distinguished endothermic binding between the phosphate and the ammonium groups from exothermic processes, driven by slow kinetics, corresponding to interliposomal, DNA-triggered aggregation that leads to the formation of large multilamellar liposome/pDNA assemblies. Lipid-added-to-pDNA and pDNA-added-to-lipid experiments have been carried out in order to systematically explore the interaction mechanisms. Complex ITC profiles are revealed, which may be linked to packing rearrangements of the pDNA molecules bound at the outer liposomal surface, possibly due to binding to more than one liposome or due to p-DNA-enhanced heterogeneity in the local lipid concentration. DNA-mediated aggregation effects are detected at high [ammonium]/[phosphate] molar ratios in the case of lipid-added-to-pDNA interactions and at relatively low [phosphate]/[ammonium] molar ratios in the case of pDNA-added-to-lipid.     

Liposomal solubilization of new 3-hydroxy-quinolinone derivatives with promising anticancer activity: a screening method to identify maximum incorporation capacity

Four new 3-hydroxy-quinolinone derivatives with promising anticancer activity could be solubilized using liposomes as vehicle to an extent that allows their in vitro and in vivo testing without use of toxic solvent(s). A screening method to identify the maximum incorporation capacity of hydrophobic drugs within liposomes was successfully applied. The compounds and lipid(s) were dissolved in methanol, and the solvent was removed by rotary evaporation. The film was resuspended with phosphate buffer (pH 7.4), and the dispersion was sonicated to reduce vesicle size. Ultracentrifugation was used to separate liposome-associated drug from free (i.e., precipitated) drug, and the amount of drug incorporated within the liposomes was quantified using high-performance liquid chromatography. All four compounds were found to be significantly incorporated within soy phosphatidylcholine (SPC) liposomes, resulting in a 200–500-fold increase in apparent solubility. Drug-to-lipid ratios in the range of 2–5 µg/mg were obtained. Interestingly, the four quinolinone derivatives have shown different association tendencies with liposomes, probably due to the physicochemical properties of the different group bonded in position 2 of the quinolinone ring. None of the alternative lipids/lipid blends tested incorporated as much drug as SPC. Photon correlation spectroscopy analyses indicated that use of ultrasounds produced an efficient reduction in liposome size. The present approach appears suitable for incorporation capacity studies of any lipophilic drug in liposomes.     

Increased circulatory half-life of liposomes after conjunction with dextran

Dextran was covalently coupled to neutral unilamellar liposomes. Dextran conjugated liposomes were cleared from the circulation at a much slower rate than unconjugated liposomes. The uptake of dextran conjugated liposomes by liver and spleen was also decreased. The amount of dextran on the surface of liposomes was found to be a determining factor for their stability in circulation. Dextran conjugated liposomes therefore may be a more effective way of controlled drug release     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.     

Binding of antibodies in liposomes to extracellular matrix antigens

We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (less than 2%). The binding of antibody-containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome-antibody complex with the antigen was estimated at 1-10 X 10(-9) M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.     

DNA strand-breaks induced by the topoisomerase I inhibitor camptothecin in unstimulated human white blood cells

Camptothecin (CPT) and actinomicyn-induced strand-breaks, repair and apoptosis in unstimulated human blood cells were studied using the DNA comet assay, and electrophoresis of low molecular weight DNA extracts. On the one hand, incubation of G0 leukocytes for 1 h with CPT induced DNA strand-breaks that were observed using the single cell gel electrophoresis technique. On the other hand, internucleosomal DNA fragments were not observed, suggesting that apoptosis had not occurred. DNA-strand-breaks caused by CPT were repaired 24 h after treatment; the migration of DNA fragments was assessed by a reduction in the number of comets. These data strongly suggest that the unexpected clastogenic effect of this topoisomerase I inhibitor is not due to the collision of the cleavage complex with the replication fork, since replication does not occur in G0. In our opinion, this effect could be due instead to the topoisomerase I enzyme being able to bind DNA in the absence of replication, probably in a way that is not strictly related to the progression of the cell cycle. Thus, CPT does not provoke apoptosis in quiescent leukocytes.     

定量测定储粮害虫胃毒作用的方法——混药薄片法

本实验制定一种定量测定储粮害虫胃毒作用的方法,命名为混药薄片法。通过试验验证饲料薄片对烟草甲Lasioderma serricorne(Fabricius)的幼虫成活率、发育历期、虫体重量、产卵量等生长发育无影响。在排除烯虫酯对烟草甲的触杀作用的前提下,将混有烯虫酯溶液的混药薄片作为饲料饲养烟草甲3龄幼虫,得到烯虫酯对烟草甲3龄幼虫的准确致死剂量为(0.46±0.03)μg。     

Lipophilic prodrug conjugates allow facile and rapid synthesis of high-loading capacity liposomes without the need for post-assembly purification

Abstract

Dihydropyridopyrazoles are simplified synthetic analogues of podophyllotoxin that can effectively mimic its molecular scaffold and act as potent mitotic spindle poisons in dividing cancer cells. However, despite nanomolar potencies and ease of synthetic preparation, further clinical development of these promising anticancer agents is hampered due to their poor aqueous solubility. In this article, we developed a prodrug strategy that enables incorporation of dihydropyridopyrazoles into liposome bilayers to overcome the solubility issues. The active drug was covalently connected to either myristic or palmitic acid anchor via carboxylesterase hydrolyzable linkage. The resulting prodrugs were self-assembled into liposome bilayers from hydrated lipid films using ultrasound without the need for post-assembly purification. The average particle size of the prodrug-loaded liposomes was about 90?nm. The prodrug incorporation was verified by differential scanning calorimetry, spectrophotometry and gel filtration reaching maximum at 0.3 and 0.35 prodrug/lipid molar ratios for myristic and palmitic conjugates, respectively. However, the ratio of 0.2 was used in the particle size and biological activity experiments to maintain long-term stability of the prodrug-loaded liposomes against phase separation during storage. Antiproliferative activity was tested against HeLa and Jurkat cancer cell lines in vitro showing that the liposomal prodrug retained antitubulin activity of the parent drug and induced apoptosis-mediated cancer cell death. Overall, the established data provide a powerful platform for further clinical development of dihydropyridopyrazoles using liposomes as the drug delivery system.     

Effect of incorporation of the adjuvant Quil A on structure and immune stimulatory capacity of liposomes

Liposomes have been widely used as drug delivery systems for many years. However, they are of limited use as delivery systems for subunit vaccines due to their low immunogenicity. Here we examine the effect of incorporating the adjuvant Quil A into liposomes on the type of particles produced, on the ability of the different particles to incorporate antigen and on the ability of the different particles to stimulate murine bone-marrow-derived dendritic cells (DC) and lymphocytes. The incorporation of increasing amounts of Quil A, from 20% to 70% of the total lipid into liposomes, reduces the size of the particles that form in aqueous dispersion and decreases antigen incorporation and uptake by DC. Interestingly, the particles with 20% Quil A were more toxic to cells in culture than the particles containing 70% Quil A, and the 20% particles were also more immunostimulatory.     

The Effects of pH and Intraliposomal Buffer Strength on the Rate of Liposome Content Release and Intracellular Drug Delivery

Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (~pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H⁺] increases 250-fold from 4 × 10^–8 M (pH 7.4, outside the cell) to 10^–5 M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.     

Synthesis of phospholipid-conjugated bile salts and interaction of bile salt-coated liposomes with cultured hepatocytes

To examine the possibility of targeting liposomes to hepatocytes via bile salts, the bile salt lithocholyltaurine was covalently linked to a phospholipid. The isomeric compounds disodium 3alpha-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy)-5beta-cholan-24-oyl-2'-aminoethansulfonate and disodium 3beta-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy-5beta-cholan-24-oyl-2'-aminoethansulfonate (DSPE-3beta-LCT) were synthesized and incorporated into liposomal membranes. Confocal laser scanning microscopy studies showed that bile salt-bearing liposomes (BSLs) attach to the surface of rat hepatocytes in culture. Studies with radioactively labeled liposomes revealed that the bile salt linked via the 3beta-conformation resulted in a higher attachment efficiency than that with the 3alpha-derivative. In the presence of BSLs corresponding to 2 mM liposomal phosphatidylcholine, uptake of 50 microM cholyltaurine (CT) into hepatocytes was reduced by approximately 40% by the 3beta-derivative and by approximately 17% by the 3alpha-derivative. When added simultaneously with the liposomes, CT up to 75 microM inhibited the binding of DSPE-3beta-LCT-bearing liposomes. By contrast, increasing concentrations reversed this inhibition and resulted in an increased bile salt-mediated binding. The same was true when CT was added 10 min before the liposomes were added. The attachment of BSLs to the surface of hepatocytes opens up promising possibilities for hepatocyte-specific drug delivery. More generally, not only substrates for cellular endocytosing receptors but also substrates for cellular carrier proteins should be suitable ligands for the cell-specific targeting of nanoscale particles such as liposomes.     

Designing of 'intelligent' liposomes for efficient delivery of drugs

The liposome- vesicles made by a double phospholipidic layers which may encapsulate aqueous solutions- have been introduced as drug delivery vehicles due to their structural flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a large variety of both hydrophilic and hydrophobic compounds. With time the liposome formulations have been perfected so as to serve certain purposes and this lead to the design of "intelligent" liposomes which can stand specifically induced modifications of the bilayers or can be surfaced with different ligands that guide them to the specific target sites. We present here a brief overview of the current strategies in the design of liposomes as drug delivery carriers and the medical applications of liposomes in humans.     

Observation on the incorporation cone in the rat

At the time of in vivo sperm–egg fusion in the rat, a small region of the oolemma under the head of the fertilizing sperm is observed to be free of microvilli. The microvilli-free region increases in area, and by one hour after sperm–egg contact extends over an area 20–30 μ in circumference and bulges out to form an “incorporation cone” visible by light microscopy. The microvilli-free incorporation cone reaches its maximum size at about two hours after sperm–egg interaction. It soon becomes smaller and has disappeared three to four hours after sperm–oocyte fusion. The cone cytoplasm is characterized by a 0.1 μ zone of thin filaments below the plasma membrane. Cytochalasin-B, 2.5 μg/ml, prevents formation of the cone or destroys the intact cone. It is suggested that micro filaments may be involved in the formation of the incorporation cone.     

接种时期对丛枝菌根喜树幼苗喜树碱含量的影响

喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种, 因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注。通过温室盆栽接种试验, 观察了喜树幼苗不同生长时期接种蜜色无梗囊霉(Acaulospora mellea)和根内球囊霉(Glomus intraradices)对喜树幼苗喜树碱积累的影响。结果表明接种两种丛枝菌根真菌均促进了喜树幼苗喜树碱的积累, 表现为喜树碱产量(单株幼苗所含的喜树碱量, 喜树碱含量与幼苗生物量的乘积)的显著提高。进一步分析发现, 接种丛枝菌根真菌导致幼喜树苗喜树碱产量的提高, 早期(幼苗出土20天)接种主要是源于喜树碱含量的提高, 特别是叶片喜树碱含量的提高, 而晚期(幼苗出土60天)接种则主要是源于幼苗生物量的增加。     

Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity

A microprocedure for the preparation of Na,K-ATPase-containing liposomes with a minimal starting material (200 microgram) of purified Na,K-ATPase is presented. Phosphatidylcholine is added gradually to cholate-solubilized Na,K-ATPase of various concentrations and the lipid-induced decrease in enzyme activity is monitored. After removal of the detergent by dialysis, the transport parameters of the resulting Na,K-ATPase-liposomes are established by a microassay. By relating the transport properties to the Na,K-ATPase activity preset before dialysis, a procedure is developed which allows to prepare standardized Na,K-ATPase-liposomes with predictable transport properties.     

光强对喜树幼苗叶片次生代谢产物喜树碱的影响

喜树碱是我国特有树种——喜树中所含的重要次生代谢产物 ,在人工控制条件下观察了光强对喜树幼苗叶片喜树碱含量的影响。喜树幼苗叶片的喜树碱含量随着遮荫程度的增加 (光照强度降低 )而增加 ,但严重遮荫的 (光强为全光照的 2 0 % )在处理后期 (75 d)喜树碱含量降低。叶片的喜树碱产量 (喜树碱含量与叶片生物量乘积 )在处理初期 (30 d)随光强减弱而缓慢地略有增加 ,处理后期 (45 d以后 )随光强的减弱而有明显增加 ,但光强低于全光照的 6 0 %以后喜树碱产量迅速下降。喜树碱的增加可能是喜树幼苗通过次生代谢过程对不良环境 (遮荫 )的一种适应性反应     

Binding of cationic liposomes to apoptotic cells

One of the most prominent hallmarks of apoptotic cells is the altered characteristics of their plasma membrane, with its blebbing and exposure of the anionic phospholipid, phosphatidylserine (PS), in the outer leaflet of the lipid bilayer. The latter feature provides the basis of distinguishing apoptotic cells from most normal cells due to staining with fluorescently labeled annexin V, binding specifically to PS. In this article, we report on the binding to apoptotic leukemic T cells (Jurkat cell line, treated with different apoptotic inducers) of cationic liposomes (CLs) composed of the cationic gemini surfactant SS-1 ((2S,3S)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide), the fluorescent lipid analog DOPRho (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Control cells showed negligible and irregular binding patterns of CLs, whereas apoptotic cells revealed a strongly augmented staining of their plasma membrane. Morphological observations and comparison with standard procedures for detecting apoptotic cells further demonstrated the binding of CLs to be intense for cells undergoing apoptosis. In addition, some apoptotic cells with higher caspase-3 activity also revealed more pronounced staining by CLs. Our data suggest that the binding of CLs to apoptotic cells is mediated through an electrostatic interaction between the positively charged head group of SS-1 and the translocated anionic phospholipid PS in the plasma membrane. Because the fluorescent lipid tracer can be freely selected, this approach provides convenient and versatile means for the fluorescence detection of apoptotic cells.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.