Synthesis and Characterization of Multifunctional Nanovesicles Composed of POPC Lipid Molecules for Nuclear Imaging.

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

Kinetics of pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles by pH sensitive cell penetrating peptide GALA.

In order to engineer endosomal escape of drug carrying liposomes into the cytoplasm of target cells, the kinetics of bilayer poration by cell penetrating peptides needs to be well understood. To this end, we have studied pH-dependent pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles as a function of concentration of the peptide GALA. Using laser scanning confocal microscopy, we measured the rate of fluorophore transport from the suspending medium into giant unilamellar vesicles across bilayer pores induced by GALA under acidic pH conditions. We also measured the mean pore size of GALA-induced pores in large unilamellar vesicles by electron microscopy. We fitted a mathematical model of pore formation kinetics to the measured rate of fluorophore transport across the giant vesicle bilayer to estimate the rate of pore formation as a function of GALA concentration. We observed that the number of pores per vesicle and the pore density increased with increasing GALA concentration.

Membrane properties of amacrocyclic tetraether bisphosphatidylcholine lipid: Effect of a single membrane-spanning polymethylene cross-linkage between two head groups of ditetradecylphosphatidylcholine membrane.

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1'-O-octacosamethylene-2,2'-di-O-tetradecyl-bis-(sn-glycero)-3,3'-diphosphocholine (AC-(di-O-C14PC)2), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)2 monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)2 vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.

Scalable solvent-free production of liposomes.

OBJECTIVES: A major challenge faced with the manufacture of liposomes is the high volumes of organic solvents used during manufacturing. Therefore, we have implemented an organic solvent-free production method for drug-loaded liposomes and demonstrated its applicability with both aqueous core-loaded and bilayer-loaded drugs. METHODS: Liposomes were produced by high shear mixing dry powder lipids with an aqueous buffer, followed by down-sizing using a Microfluidizer processor. Liposomes were purified via tangential flow filtration and characterised in terms of size, polydispersity index, zeta potential and drug loading. KEY FINDINGS: Doxorubicin-loaded PEGylated liposomes can be manufactured using this solvent-free method with particle sizes of 100-110 nm, low polydispersity index (PDI) (<0.2) and high drug loading (97-98%). If required, liposomes can be further down-sized via microfluidic processing without impacting drug loading. Similar results were achieved with non-PEGylated liposomes. With bilayer-loaded amphotericin B liposomes, again liposomes can be prepared within a clinically appropriate size range (100-110 nm in size, low PDI) with high drug loading (98-100%). CONCLUSIONS: We apply a simple and scalable solvent-free method for the production of both aqueous core or bilayer drug-loaded liposomes.

Synthetic dimeric-drug phospholipid: a versatile liposomal platform for cancer therapy.

An amphiphilic dimeric-podophyllotoxin (PODO) phospholipid was synthesized to assemble into liposomes as a combination of prodrug and nanocarrier. The results have demonstrated that the cell membrane-like delivery system possessed an improved cellular uptake and favorable antitumor efficacy with reduced side-effects. This strategy provides a new effective platform in drug delivery for cancer chemotherapy.

Biogenic supported lipid bilayers as a tool to investigate nano-bio interfaces.

HYPOTHESIS: Extracellular Vesicles (EVs) are natural nanosized lipid vesicles involved in most intercellular communication pathways. Given their nature, they represent natural cell membrane models, with intermediate complexity between real and synthetic lipid membranes. Here we compare EVs-derived (EVSLB) and synthetic Supported Lipid Bilayers (SLBs) in the interaction with cationic superparamagnetic iron oxide nanoparticles (SPIONs). The aim is twofold: (i) exploit SPIONs as nanometric probes to investigate the features of EVSLBs as novel biogenic platforms; (ii) contribute at improving the knowledge on the behavior of SPIONs with biological interfaces. EXPERIMENTS: Quartz Crystal Microbalance, X-ray Reflectivity, Grazing-incidence Small-angle X-ray Scattering, Atomic Force Microscopy, Confocal Microscopy data on SPIONs-EVSLB were systematically compared to those on SPIONs challenging synthetic SLBs, taken as references. FINDINGS: The ensemble of experimental results highlights the much stronger interaction of SPIONs with EVSLBs with respect to synthetic SLBs. This evidence strongly supports the hypotheses on the peculiar structure of EVSLBs, with cushioned non-flat areas and extended exposed surface; in addition, it suggests that these features are relevant in the response of biogenic membranes to nano-objects. These findings contribute to the fundamental knowledge on EVSLBs, key for their development both as biomimetic membranes, or as platforms for biomedical applications.

Improved Antitumor Activity of Novel Redox-Responsive Paclitaxel-Encapsulated Liposomes Based on Disulfide Phosphatidylcholine.

The microtubule inhibitor paclitaxel (PTX) is used to treat a wide range of solid tumors. Due to the poor aqueous solubility of PTX, a continuous demand for safe, efficient PTX formulations with improved antitumor activity exists. Here, we report a novel form of redox-sensitive paclitaxel (PTX)-encapsulated liposomes based on the previously developed disulfide phosphatidylcholine (SS-PC). PTX-loaded stealth liposomes (PTX/SS-LP) composed of SS-PC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000 (DSPE-PEG2000), and cholesterol were prepared using the reverse-phase evaporation method. The characterization of the PTX/SS-LP liposomes using dynamic light scattering and transmission electron microscopy confirmed their uniform particle size and typical unilamellar vesicle structure with an average bilayer thickness of approximately 4 nm. Changes in the size and morphology as well as the rapid release of PTX triggered by the addition of dithiothreitol revealed the redox sensitivity of PTX/SS-LP. Finally, evaluations in MCF-7 and A549 cells in vitro and in BALB/c mice in vivo revealed the improved anticancer efficiency, biodistribution, and safety of PTX/SS-LP compared with those of Taxol and nonredox-sensitive PTX/LP. In conclusion, PTX/SS-LP displays a redox-responsive release of paclitaxel with improved antitumor activity and has great potential as a next-generation stealth liposomal PTX delivery system.

Disulfide phosphatidylcholines: alternative phospholipids for the preparation of functional liposomes.

Phosphatidylcholine is the main component of liposomes and other phospholipid-based nanocarriers in drug delivery. However, the functions and applications of these nanocarriers are extremely limited by conventional phospholipids. Here we report novel disulfide phosphatidylcholines (SS-PCs) and SS-PC based liposomes (SS-LPs) used as alternatives to traditional phospholipids and liposomes.

Syntheses and cytotoxicity of phosphatidylcholines containing ibuprofen or naproxen moieties.

In this study, novel phosphatidylcholines containing ibuprofen or naproxen moieties were synthesized in good yields and high purities. Under the given synthesis conditions, the attached drug moieties racemized, which resulted in the formation of phospholipid diastereomers. The comperative studies of the cytotoxicity of ibuprofen, naproxen and their phosphatidylcholine derivatives against human promyelocytic leukemia HL-60, human colon carcinoma Caco-2, and porcine epithelial intestinal IPEC-J2 cells were carried out. The results of these studies indicated that phospholipids with NSAIDs at both sn-1 and sn-2 positions (15 and 16) were more toxic than ibuprofen or naproxen themselves, whereas 2-lysophosphatidylcholines (7 and 8) were less toxic against all tested cell lines. Phospholipids with NSAIDs at sn-1 and palmitic acid at sn-2 (9 and 10) were also less toxic against Caco-2 and normal cells (IPEC-J2).

Light-Controlled Lipid Interaction and Membrane Organization in Photolipid Bilayer Vesicles.

Controlling lateral interactions between lipid molecules in a bilayer membrane to guide membrane organization and domain formation is a key factor for studying and emulating membrane functionality in synthetic biological systems. Here, we demonstrate an approach to reversibly control lipid organization, domain formation, and membrane stiffness of phospholipid bilayer membranes using the photoswitchable phospholipid azo-PC. azo-PC contains an azobenzene group in the sn2 acyl chain that undergoes reversible photoisomerization on illumination with UV-A and visible light. We demonstrate that the concentration of the photolipid molecules and also the assembly and disassembly of photolipids into lipid domains can be monitored by UV-vis spectroscopy because of a blue shift induced by photolipid aggregation.

Synthesis, Characterization, and In Vitro Cancer Cell Growth Inhibition Evaluation of Novel Phosphatidylcholines with Anisic and Veratric Acids.

Phenolic acids and its methoxy derivatives are known to induce caspase-mediated apoptosis activity and exhibit cytotoxic effect towards various cancer cell lines. However, their low stability and poor bioavailability in the human organism extensively restrict the utility of this group of compounds as anticancer and health-promoting agents. In this report, a series of eight novel phosphatidylcholines (3a-b, 5a-b, 7a-b, 8a-b) containing anisic or veratric acids (1a-b) at sn-1 and/or sn-2 positions were synthesized. The phenoylated phospholipids were obtained in good yields 28⁻66%. The structures of novel compounds were determined by their spectroscopic data. All synthesized compounds were evaluated for their antiproliferative activity towards six cancer cell lines and normal cell line Balb/3T3. Lipophilization of phenolcarboxylic acids significantly increased their anticancer properties. The asymmetrically substituted phenoylated phosphatidylcholines exhibited higher antiproliferative effect than free acids. Lysophosphatidylcholine (7b) effectively inhibited the proliferation of human leukaemia (MV4-11), breast (MCF-7), and colon (LoVo) cancer cell lines at concentrations of 9.5⁻20.7 µm and was from 19 to 38-fold more active than corresponding free veratric acid. The conjugation of anisic/veratric acids with the phosphatidylcholine have proved the anticancer potential of these phenolcarboxylic acids and showed that this type of lipophilization is an effective method for the production of active biomolecules.

High Density Lipoproteins Inhibit Oxidative Stress-Induced Prostate Cancer Cell Proliferation.

Recent evidence suggests that oxidative stress can play a role in the pathogenesis and the progression of prostate cancer (PCa). Reactive oxygen species (ROS) generation is higher in PCa cells compared to normal prostate epithelial cells and this increase is proportional to the aggressiveness of the phenotype. Since high density lipoproteins (HDL) are known to exert antioxidant activities, their ability to reduce ROS levels and the consequent impact on cell proliferation was tested in normal and PCa cell lines. HDL significantly reduced basal and H2O2-induced oxidative stress in normal, androgen receptor (AR)-positive and AR-null PCa cell lines. AR, scavenger receptor BI and ATP binding cassette G1 transporter were not involved. In addition, HDL completely blunted H2O2-induced increase of cell proliferation, through their capacity to prevent the H2O2-induced shift of cell cycle distribution from G0/G1 towards G2/M phase. Synthetic HDL, made of the two main components of plasma-derived HDL (apoA-I and phosphatidylcholine) and which are under clinical development as anti-atherosclerotic agents, retained the ability of HDL to inhibit ROS production in PCa cells. Collectively, HDL antioxidant activity limits cell proliferation induced by ROS in AR-positive and AR-null PCa cell lines, thus supporting a possible role of HDL against PCa progression.

Lipase-Catalyzed Acidolysis of Egg-Yolk Phosphatidylcholine with Citronellic Acid. New Insight into Synthesis of Isoprenoid-Phospholipids.

The development of a biotechnological method for the production of new biologically active phosphatidylcholine containing monoterpene citronellic acid (CA) was the aim of this work. Incorporation of citronellic acid (CA) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed acidolysis process was studied. Isoprenoid acid CA was used as an acyl donor and five commercially available immobilized lipases were examined as biocatalysts. The effects of organic solvent, enzyme load, reaction time and molar ratio of substrates on the incorporation of citronellic acid (CA) into the phospholipids were evaluated. Modified phospholipid fraction enriched with CA in the sn-1 position (39% of incorporation) was obtained in high 33% yield using Novozym 435 as biocatalyst. In this study a biotechnological method for production of new phospholipid biopreparation enriched with citronellic acid, which can play an important role as a nutraceutical, was applied.

Liposomes entrapping ß-cyclodextrin/ibuprofen inclusion complex: Role of the host and the guest on the bilayer integrity and microviscosity.

Multilamellar vesicles (MLVs) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared by using the dehydration-rehydration method. The ß-cyclodextrin/Ibuprofen inclusion complex (ß-CD/Ibu) was formed and solubilised into the aqueous compartments of the investigated vesicles. The resulting POPC MLVs entrapping ß-CD/Ibu complex were essentially homogeneous in shape as demonstrated by Transmission Electron Microscopy (TEM). The liposomal stability was determined at 37.0±0.1°C by following the outflux rate of 5(6)-carboxyfluorescein (CF) at pH 7.40, while the membrane microviscosity was estimated by the ratio of the fluorescence intensities of pyrene in excimer and monomer state. The results presented herein confirm that interactions between POPC and ß-CD occur and suggest that associations between POPC and Ibuprofen are also involved in the properties of the investigated liposomes.

Optimization of the Synthesis of Structured Phosphatidylcholine with Medium Chain Fatty Acid.

Structured phosphatidylcholine was successfully produced by acidolysis between phosphatidylcholine and free medium chain fatty acid, using phospholipase A1 immobilized on Duolite A568. Response surface methodology was applied to optimize the reaction system using three process parameters: molar ratio of substrates (phosphatidylcholine to free medium chain fatty acid), enzyme loading, and reaction temperature. All parameters evaluated showed linear and quadratic significant effects on the production of modified phosphatidylcholine; molar ratio of substrates contributed positively, but temperature influenced negatively. Increased enzyme loading also led to increased production of modified phosphatidylcholine but only during the first 9 hours of the acidolysis reaction. Optimal conditions obtained from the model were a ratio of phosphatidylcholine to free medium chain fatty acid of 1:15, an enzyme loading of 12%, and a temperature of 45°C. Under these conditions a production of modified phosphatidylcholine of 52.98 % were obtained after 24 h of reaction. The prediction was confirmed from the verification experiments; the production of modified phosphatidylcholine was 53.02%, the total yield of phosphatidylcholine 64.28% and the molar incorporation of medium chain fatty acid was 42.31%. The acidolysis reaction was scaled-up in a batch reactor with a similar production of modified phosphatidylcholine, total yield of phosphatidylcholine and molar incorporation of medium chain fatty acid. Purification by column chromatography of the structured phosphatidylcholine yielded 62.53% of phosphatidylcholine enriched with 42.52% of medium chain fatty acid.

Synthesis and Identification of AceDoxyPC, a Protectin-Containing Structured Phospholipid, Using Liquid Chromatography/Mass Spectrometry.

Fatty acids have many health benefits in a great variety of diseases ranging from cardiovascular to cerebral diseases. For instance, docosahexaenoic acid (DHA), which is highly enriched in brain phospholipids, plays a major role in anti-inflammatory or neuroprotective pathways. Its effects are thought to be due, in part, to its conversion into derived mediators such as protectins. 1-Lyso,2-docosahexaenoyl-glycerophosphocholine (LysoPtdCho-DHA) is one of the physiological carrier of DHA to the brain. We previously synthesized a structured phosphatidylcholine to mimic 1-lyso,2-docosahexaenoyl-glycerophosphocholine, named AceDoPC® (1-acetyl,2-docosahexaenoyl-glycerophosphocholine), that is considered as a stabilized form of the physiological LysoPtdCho-DHA and that is neuroprotective in experimental ischemic stroke. Considering these, the current study aimed at enzymatically oxygenate DHA contained within AceDoPC® to synthesize a readily structured oxidized phospholipid containing protectin DX (PDX), thereafter named AceDoxyPC (1-acetyl,2-PDX-glycerophosphocholine). Identification of this product was performed using liquid chromatography/tandem mass spectrometry. Such molecule could be used as a bioactive mediator for therapy against neurodegenerative diseases and stroke.

A Cheap and Convenient Method of Liposome Preparation Using Glass Beads as a Source of Shear Force.

Liposomes, the biocompatible lipid bilayer vesicles, have attracted immense attention due to their distinctive features such as efficient vehicle for the delivery of a wide range of therapeutic agents, adjustable formulation properties, and high drug entrapment efficiency. In this contribution, we present a simple method for the preparation of liposomes using glass beads and compared the potential of this method with conventional methods of liposome preparation. The prepared liposomes were characterized by different analytical techniques (HPLC, DLS, TEM, differential scanning calorimetry, and in vitro drug release). Our findings revealed that the particle size of liposomes is mainly dependent on the size of the glass beads and the glass bead shearing time. An average liposome size of 67.7 ± 25.5 nm was obtained using 2-mm glass beads after 24-h incubation at 200 rpm. The liposomes prepared under the optimized conditions exhibited a high encapsulation efficiency of 92.1 ± 1.7% with 31.08% drug release after 360 min at 37°C. In conclusion, the developed method is a simple and convenient process of liposome preparation of different sizes with desirable entrapment efficiency capacity.

Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles.

Giant unilamellar vesicles (GUVs) represent a versatile model system to emulate the fundamental properties and functions associated with the plasma membrane of living cells. Deformability and shape transitions of lipid vesicles are closely linked to the mechanical properties of the bilayer membrane itself and are typically difficult to control under physiological conditions. Here, we developed a protocol to form cell-sized vesicles from an azobenzene-containing phosphatidylcholine (azo-PC), which undergoes photoisomerization on irradiation with UV-A and visible light. Photoswitching within the photolipid vesicles enabled rapid and precise control of the mechanical properties of the membrane. By varying the intensity and dynamics of the optical stimulus, controlled vesicle shape changes such as budding transitions, invagination, pearling, or the formation of membrane tubes were achieved. With this system, we could mimic the morphology changes normally seen in cells, in the absence of any molecular machines associated with the cytoskeleton. Furthermore, we devised a mechanism to utilize photoswitchable lipid membranes for storing mechanical energy and then releasing it on command as locally usable work.

Syntheses and antiproliferative activities of novel phosphatidylcholines containing dehydroepiandrosterone moieties.

Dehydroepiandrosterone (DHEA) is a natural hormone with many beneficial properties including an anticancer activity. Unfortunately, DHEA is unstable in the body and exhibits cytotoxicity against healthy cells. In this study, a series of new phosphocholines containing DHEA at sn-1 and/or sn-2 positions were prepared. Succinic acid was used as a linker between the active drug and sn-glycero-3-phosphocholine. All the compounds were evaluated in vitro for their antiproliferative activities against four cell lines: Balb/3T3, HL-60, B16, and LNCaP. The results showed that phosphocholines with DHEA at sn-1 and/or sn-2 positions did not have cytotoxic effects on the normal cell line (Balb/3T3). Mixed-chain phospholipids with DHEA and fatty acid residues showed the highest activity against tumor cell lines. The most active compound, 11c, showed a moderate cytotoxic effect against the HL-60 and B16 cell lines.

Synthesis and in vitro antioxidant and antimicrobial studies of novel structured phosphatidylcholines with phenolic acids.

Novel phenoylated phosphatidylcholines were synthesized from 1,2-dipalmitoyl phosphatidylcholine/egg 1,2-diacyl phosphatidylcholine and phenolic acids such as ferulic, sinapic, vanillic and syringic acids. The structures of phenoylated phosphatidylcholines were confirmed by spectral analysis. 2-acyl-1-lyso phosphatidylcholine was synthesized from phosphatidylcholine via regioselective enzymatic hydrolysis and was reacted with hydroxyl protected phenolic acids to produce corresponding phenoylated phosphatidylcholines in 48-56% yields. Deprotection of protected phenoylated phosphatidylcholines resulted in phenoylated phosphatidylcholines in 87-94% yields. The prepared compounds were evaluated for their preliminary in vitro antimicrobial and antioxidant activities. Among the active derivatives, compound 1-(4-hydroxy-3,5-dimethoxy) cinnamoyl-2-acyl-sn-glycero-3-phosphocholine exhibited excellent antioxidant activity with EC50 value of 16.43µg/mL. Compounds 1-(4-hydroxy-3-methoxy) cinnamoyl-2-acyl-sn-glycero-3-phosphocholine and 1-(4-hydroxy-3,5-dimethoxy) cinnamoyl-2-palmitoyl-sn-glycero-3-phosphocholine exhibited good antioxidant activity with EC50 values of 36.05 and 33.35µg/mL respectively. Compound 1-(4-hydroxy-3-methoxy) cinnamoyl-2-palmitoyl-sn-glycero-3-phosphocholine exhibited good antibacterial activity against Klebsiella planticola with MIC of 15.6µg/mL and compound 1-(4-hydroxy-3-methoxy) benzoyl-2-acyl-sn-glycero-3-phosphocholine exhibited good antifungal activity against Candida albicans with MIC of 15.6µg/mL.