Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Synthesis and fluorine-18 radiolabeling of a phospholipid as a PET imaging agent for prostate cancer.

INTRODUCTION: Altered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. METHODS: Lipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. RESULTS: [18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/µmol (2300 ± 93 mCi/µmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET.

Integrin-targeted AmpRGD sunitinib liposomes as integrated antiangiogenic tools.

We report here the preparation, physico-chemical characterization, and biological evaluation of a new liposome formulation as a tool for tumor angiogenesis inhibition. Liposomes are loaded with sunitinib, a tyrosine kinase inhibitor, and decorated with cyclo-aminoprolineRGD units (cAmpRGD), efficient and selective ligands for integrin αVß3. The RGD units play multiple roles since they target the nanovehicles at the integrin αVß3-overexpressing cells (e.g. activated endothelial cells), favor their active cell internalization, providing drug accumulation in the cytoplasm, and likely take part in the angiogenesis inhibition by interfering in the αVß3-VEGFR2 cross-talk. Both in vitro and in vivo studies show a better efficacy of this integrated antiangiogenic tool with respect to the free sunitinib and untargeted sunitinib-loaded liposomes. This system could allow a lower administration of the drug and, by increasing the vector specificity, reduce side-effects in a prolonged antiangiogenic therapy.

Isoprenoid-phospholipid conjugates as potential therapeutic agents: Synthesis, characterization and antiproliferative studies.

The aim of this research was to extend application field of isoprenoid compounds by their introduction into phospholipid structure as the transport vehicle. The series of novel isoprenoid phospholipids were synthesized in high yields (24-97%), their structures were fully characterized and its anticancer activity was investigated in vitro towards several cell lines of different origin. Most of synthesized compounds showed a significantly higher antiproliferative effect on tested cell lines than free terpene acids. The most active phosphatidylcholine analogue, containing 2,3-dihydro-3-vinylfarnesoic acids instead of fatty acids in both sn-1 and sn-2 position, inhibits the proliferation of colon cancer cells at 13.6 µM.

A reflection of the lasting contributions from Dr. Robert Bittman to sterol trafficking, sphingolipid and phospholipid research.

With the passing of Dr. Robert Bittman from pancreatic cancer on the 1st October 2014, the lipid research field lost one of the most influential and significant personalities. Robert Bittman's genius was in chemical design and his contribution to the lipid research field was truly immense. The reagents and chemicals he designed and synthesised allowed interrogation of the role of lipids in constituting complex biophysical membranes, sterol transfer and in cellular communication networks. Here we provide a review of these works which serve as a lasting memory to his life.

Peptidophospholipids: synthesis, phospholipase A2 catalyzed hydrolysis, and application to development of phospholipid prodrugs.

New phospholipid analogues incorporating sn-2-peptide substituents have been prepared to probe the fundamental structural requirements for phospholipase A2 catalyzed hydrolysis of PLA2-directed synthetic substrates. Two structurally different antiviral oligopeptides with C-terminal glycine were introduced separately at the sn-2-carboxylic ester position of phospholipids to assess the role of the α-methylene group adjacent to the ester carbonyl in allowing hydrolytic cleavage by the enzyme. The oligopeptide-carrying phospholipid derivatives were readily incorporated into mixed micelles consisting of natural phospholipid (dipalmitoyl phosphatidylcholine, DPPC) and Triton X-100 as surfactant. Hydrolytic cleavage of the synthetic peptidophospholipids by the phospholipase A2 occurred slower, but within the same order of magnitude as the natural substrate alone. The results provide useful information toward better understanding the mechanism of action of the enzyme, and to improve the design and synthesis of phospholipid prodrugs targeted at secretory PLA2 enzymes.

SiRNA-phospholipid conjugates for gene and drug delivery in cancer treatment.

Due to low charge density and stiff backbone structure, small interfering RNA (siRNA) has inherently poor binding ability to cationic polymers and lipid carriers, which results in low siRNA loading efficiency and limits siRNA success in clinical application. Here, siRNA-phospholipids conjugates are developed, which integrate the characteristics of the two phospholipids to self-assemble via hydrophilic siRNA and hydrophobic phospholipid tails to overcome the siRNA's stiff backbone structures and enhance the siRNA loading efficiency. In this study, the thiol-modified sense and antisense siRNA are chemically conjugated with phospholipids to form sense and antisense siRNA-phospholipid, and then these sense or antisense siRNA-phospholipids with equal amounts are annealed to generate siRNA-phospholipids. The siRNA-phospholipids can serve dual functions as agents that can silence gene expression and as a component of nanoparticles to embed hydrophobic anticancer drugs to cure tumor. siRNA-phospholipids together with cationic lipids and DSPE-PEG2000 fuse around PLGA to form siRNA-phospholipids enveloped nanoparticles (siRNA-PCNPs), which can deliver siRNAs and hydrophobic anticancer drugs into tumor. In animal models, intravenously injected siRNA-PCNPs embedded DOX (siPlk1-PCNPs/DOX) is highly effective in inhibiting tumor growth. The results indicate that the siRNA-PCNPs can be potentially applied as a safe and efficient gene and anticancer drug delivery carrier.

Antioxidant phospholipid calix[4]arene mimics as micellular delivery systems.

Amphiphilic calix[4]arenes were designed as phospholipid mimics by incorporating PO3H2 or NMe3(+) head groups. Using PC12 cells and three stressors (H2O2, menadione and glutamate), we established safe calix[4]arene levels that are able not only to deliver antioxidant payloads of curcumin, but intriguingly also have inherent antioxidant properties. The calix[4]arenes appear to be potent synthetic antioxidants that could be used as nano-carriers for drug delivery.

Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids.

The design of the drug delivery depends upon different parameters. One of the most noticeable factors in design of the drug delivery is drug-release profile which determines the site of action, the concentration of the drug at the time of administration, the period of time that the drug must remain at a therapeutic concentration. To get a better understanding of drug release, large unilamellar liposomes containing calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; calcein was chosen as a model of hydrophilic drug. The calcein permeability across liposomal membrane (with different compositions) was evaluated on the basis of the first-order kinetic by spectrofluorometer. Also, the effects of liposome composition/fluidity as well as the incubation temperature/pH were investigated. Furthermore, we simulated the digestion condition in the gastrointestinal tract in humans, to mimic human gastro-duodenal digestion to monitor calcein release during the course of the digestion process. In vitro digestion model ''pH stat'' was used to systematically examine the influence of pH/enzyme on phospholipid liposomes digestion under simulated gastro-duodenal digestion. The results revealed that calcein permeates across liposomal membrane without membrane disruption. The release rate of calcein from the liposomes depends on the number and fluidity of bilayers and its mechanical/physical properties such as permeability, bending elasticity. Chemo-structural properties of drugs like as partition coefficient (Log P), H-bonding, polar surface area (PSA) are also determinative parameter in release behavior. Finally, stimulated emission depletion (STED) microscopy was used to study calcein translocation through liposomal bilayers.

Synthesis and surface properties of polyurethane end-capped with hybrid hydrocarbon/fluorocarbon double-chain phospholipid.

To improve hemocompatibility of biomedical polyurethanes (PUs), a series of new fluorinated phospholipid end-capped polyurethanes (FPCPUs) as blending PU additives were designed and synthesized using diphenyl methane diisocyanate and 1,4-butanediol as hard segment, poly(tetramethylene glycol), polypropylene glycol, polycarbonate diols, and polyethylene glycol as soft segments, respectively, aminofunctionalized hybrid hydrocarbon/fluorocarbon double-chain phospholipid as end-capper. The bulk structures and surface properties of the obtained FPCPUs were fully characterized by (1)H NMR, Fourier transform infrared, gel permeation chromatography, X-ray photoelectron spectroscopy, differential scanning calorimetry, atomic force microscopy, and water contact angle measurement. It was found that the phosphatidylcholine groups could enrich on the surfaces and subsurfaces with the help of the fluorocarbon chains and self-assemble into mimic biomembrane on these polymer surfaces. These surfaces could effectively suppress fibrinogen adsorption, as evaluated by enzyme-linked immunosorbent assay method. Our work indicates that the FPCPUs should be one of the most potential modified additives for enhancing hemocompatibility of traditional medical PUs.

Magnetic resonance contrast media sensing in vivo molecular imaging agents: an overview.

Metabolic imaging is commonly performed by nuclear medicine facilities such as PET or SPECT, etc. The production and biomedical applications of bio-molecular sensing in vivo MRI metabolic contrast agents has recently become of great universal research interest, which follows its great success as a potential cost effective, less radioactive, nuclear medicine alternative. Temperature, redox potential, enzyme activity, free radial/metal ion responsive and/or pH sensitive molecular metabolic MR contrast agents are among the famous instances exemplified, which basically promote MR image contrast enhancement ability to distinguish molecular metabolic/gene expression features. Overall, these MRI contrast agents provide a framework to achieve a greater degree of accuracy from MRI as a low cost, more available facility, non radioactive radiation producing and highly sensitive biomedical tool to propound as a new suggesting opponent for PET nuclear medicine imaging. In the present review, the design, development, examination and future of the above agents will be discussed in detail.

Synthetic glycosidated phospholipids induce apoptosis through activation of FADD, caspase-8 and the mitochondrial death pathway.

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD--/-- and caspase-8--/-- cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas--/-- Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.

Synthesis of novel phospholipids that bind phenylalkanols and hydroquinone via phospholipase D-catalyzed transphosphatidylation.

Phenylalkanols such as tyrosol and hydroxytyrosol (h-tyrosol), which possess antioxidant and anticancer properties, were phosphatidylated by phospholipase D (PLD)-catalyzed transphosphatidylation. After a 24-hour reaction of phosphatidylcholine (PC) and tyrosol with PLD, a new product was detected by TLC and identified to phosphatidyl-tyrosol by high-resolution MS and NMR analyses. The optimum reaction conditions were as follows: soyPC 50µmol, tyrosol 500µmol, ethyl acetate 1.6ml, PLD 1.6U, 0.2m sodium acetate buffer (pH 5.6) 0.8ml, 37°C for 24 hours. Under the optimum reaction conditions, the yields of phosphatidyl-tyrosol, hydroquinone (HQ), 2-(4-aminophenyl)ethanol (4APE), h-tyrosol and 2-phenylethanol (PEA) were 87±3.7, 13±1.3, 90±2.3, 64±5.5 and 85±1.0mol%, respectively. Furthermore, from the results of transphosphatidylation of soyPC with several phenylethanols and phenylpropanols, we established the following details about the reaction specificity of transphosphatidylation by PLD from Streptomyces sp.: (1) para-amino and para-hydroxyl groups in the benzene ring of PEA derivatives do not affect the transphosphatidylation by PLD, whereas meta-hydroxyl group slightly inhibits the transphosphatidylation. (2) Meta- and ortho-methyl groups in the benzene ring of PEA derivatives also slightly inhibit the transphosphatidylation. (3) Secondary and tertiary alcohols and hydroquinone are difficult to transphosphatidylate by PLD.

Static and dynamic characterization of nanodiscs with apolipoprotein A-I and its model peptide.

The class A amphipathic α-helical peptide 18A is known to form discoidal phospholipid complexes (nanodiscs) similar to that formed by apolipoprotein A-I (apoA-I). To reveal the structural differences in nanodiscs formed with this protein and peptide, we prepared nanodiscs with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and applied fluorescence techniques to these nanoparticles. Fluorescence resonance energy transfer between 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) in nanodiscs revealed that lipid exchange with 18A nanodiscs is mediated by collisions between nanodiscs. The fluorescence lifetime of dansyl phosphatidylethanolamine and excimer fluorescence of 1,2-bis(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine showed that the degree of hydration of the membrane surface and lateral pressure of acyl chains in 18A nanodiscs are independent of the disc size, suggesting that 18A nanodiscs form planar lipid bilayers irrespective of their size, which differs from apoA-I nanodiscs, whose bilayer deforms to a saddle surface with decreasing size. These results suggest that the flexible structure of a chain of helices in apoA-I is crucial for the formation of saddle surfaces in nanodiscs.

Formulation development of parenteral phospholipid-based microemulsion of etoposide.

The aim of the present study was to investigate the potential of a phospholipid-based microemulsion formulation for parenteral delivery of anticancer drug, etoposide. The microemulsion area was identified by constructing pseudoternary phase diagrams. The prepared microemulsions were subjected to different thermodynamic stability tests. The microemulsion formulations that passed thermodynamic stability tests were characterized for optical birefringence, droplet size, viscosity measurement, and pH measurements. To assess the safety of the formulations for parenteral delivery, the formulation was subjected to compatibility studies with various intravenous infusions and in vitro erythrocyte toxicity study. The developed formulation was found to be robust and safe for parenteral delivery.

Disterolphospholipids: nonexchangeable lipids and their application to liposomal drug delivery.

Extreme makeover of cholesterol: Cholesterol exchange is a major reason for the instability of liposomes in blood. The formation of a covalent hybrid between cholesterol and glycerophosphocholine preserves the bilayer-stabilizing effect of free cholesterol but prevents its transfer from the bilayer. Thus, disterolphospholipids (e.g. 1) are valuable new components for liposome formulation.

PK-PD modeling of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine and the enhanced antitumor effect of its phospholipid derivatives in long-circulating liposomes.

The efficacy of an antitumor nucleoside, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl)cytosine (3'-ethynylcytidine, ECyd), was analyzed in vitro and in vivo. The in vivo antitumor effect of ECyd encapsulated into long-circulating liposomes was also examined. Based on pharmacokinetic (PK) and pharmacodynamic (PD) analyses, a model that quantitatively explains the in vivo effects of ECyd was proposed, using the concept of minimum effective concentration. The model suggests that ECyd followed a time-dependent mechanism of action in vivo, and that availability of ECyd in tumor tissue was highly important. To improve the availability of ECyd, its phospholipid derivatives were synthesized and encapsulated into long-circulating liposomes, which increased the antitumor effect. These results indicate that it is very important to design carriers of antitumor drugs based on PK-PD modeling.

Synthetic surfactants: where are we? Evidence from randomized, controlled clinical trials.

The benefits of exogenous synthetic or animal-derived surfactants for prevention or treatment of respiratory distress syndrome (RDS) are well established. Data from head-to-head trials comparing animal-derived surfactants primarily with the synthetic surfactant colfosceril suggest that the major clinical advantages afforded by the presence of surfactant protein (SP)-B and SP-C in animal-derived preparations relate to faster onset of action, a reduction in the incidence of RDS when used prophylactically, and a lower incidence of air leaks and RDS-related deaths. However, no benefits in terms of overall mortality or BPD have been shown in these head-to-head comparisons. Findings from trials of a new-generation synthetic surfactant containing a peptide that mimics SP-B, as well as their follow-up study suggest that its administration improves short-term clinical outcomes compared with colfosceril and results in survival through 1 year of age, which is at least comparable, if not perhaps superior, to that seen with animal-derived surfactants.

Synthesis and transfection activity of new cationic phosphoramidate lipids: high efficiency of an imidazolium derivative.

In an effort to enhance the gene-transfer efficiencies of cationic lipids and to decrease their toxicities, a series of new phosphoramidate lipids with chemical similarity to cell membrane phospholipids was synthesised. These lipids contained various cationic headgroups, such as arginine methyl ester, lysine methyl ester, homoarginine methyl ester, ethylenediamine, diaminopropane, guanidinium and imidazolium. Their transfection abilities, either alone or with the co-lipid DOPE, were evaluated in HEK293-T7 cells. We found that imidazolium lipophosphoramidate 7 a/DOPE lipoplexes gave the most efficient transfection with low toxicity (15 %). The luciferase activity was 100 times higher than that obtained with DOTAP/DOPE lipoplexes. The size, zeta potential, pDNA-liposome interactions and cellular uptakes of the lipoplexes were determined. No definitive correlation between the zeta potential values and the transfection efficiencies could be established, but the uptake of lipoplexes by the cells was correlated with their final transfection efficiencies. Our results show that imidazolium phosphoramidate lipids constitute a potential new class of cationic lipids for gene transfer.