Transferrin and tocopheryl-polyethylene glycol-succinate dual ligands decorated, cisplatin loaded nano-sized system for the treatment of lung cancer.

Nanocarriers decorated with different ligands were used to achieve lung cancer treatment. Surface decoration of nanoparticulate system will assist in targeting the drug to specific tumor cells and tissues. The aim of this research was to develop a dual ligands decorated nanocarriers (NCs), which could increase the cell uptake and anti-tumor efficiency. Two different ligands: Transferrin (Tf) and D-α-tocopheryl polyethylene glycol succinate (TPGS) containing ligands were synthesized. Dual ligands decorated nanocarriers (DL-NCs) was constructed. The in vitro cytotoxicity, in vivo biodistribution, and in vivo antitumor efficacy of the DL-NCs were evaluated. DL-NCs can efficiently deliver cisplatin (CDDP) into lung cancer cells in vitro and reduced xenograft tumor size in vivo. The encapsulation of CDDP in the DL-NCs significantly improved the cytotoxicity and antitumor efficacy. DL-NCs held great potential for achieving an optimal therapeutic effect in the treatment of lung cancer.

Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation.

Lipids have fundamental roles in the structure, energetics, and signaling of cells and organisms. The recent discovery of fatty acid esters of hydroxy fatty acids (FAHFAs), lipids with potent antidiabetic and anti-inflammatory activities, indicates that our understanding of the composition of lipidome and the function of lipids is incomplete. The ability to synthesize and test FAHFAs was critical in elucidating the roles of these lipids, but these studies were performed with racemic mixtures, and the role of stereochemistry remains unexplored. Here, we synthesized the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-9-PAHSA). Access to highly enantioenriched PAHSAs enabled the development of a liquid chromatography-mass spectrometry (LC-MS) method to separate and quantify R- and S-9-PAHSA, and this approach identified R-9-PAHSA as the predominant stereoisomer that accumulates in adipose tissues from transgenic mice where FAHFAs were first discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL), a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.

5-Fluorouracil-lipid conjugate: potential candidate for drug delivery through encapsulation in hydrophobic polyester-based nanoparticles.

The encapsulation of 5-fluorouracil (5-FU) in hydrophobic polymeric materials is made feasible by a lipid-based prodrug approach. A lipid-5-FU conjugate of 5-FU with palmitic acid was synthesized in two-step process. A synthesized dipalmitoyl derivative (5-FUDIPAL) was characterized using Fourier transform infrared spectroscopy and (1)H-nuclear magnetic resonance. The 5-FUDIPAL was encapsulated in polyester-based polymers by the double emulsion-solvent evaporation method. The nanoparticles were characterized by scanning electron microscopy, transmission electron microscopy and dynamic light scattering. The thermal stability was assessed by differential scanning calorimetry data. In vitro release kinetics measurements of the drug from nanoparticles showed the controlled release pattern over a period of time. Cytotoxicity measurements by MTT assay confirmed that dipalmitoyl derivative in nano formulation successfully inhibited the cell growth. Thus the combined physical and biological evaluation of the different polyester-based nanoparticle containing the modified drug showed a facile approach to delivering 5-FU to the tumour site with enhanced efficacy.

Nanoscale lithography mediated by surface self-assembly of 16-[3,5-bis(mercaptomethyl)phenoxy]hexadecanoic acid on Au(111) investigated by scanning probe microscopy.

The solution-phase self-assembly of bidentate 16-[3,5-bis(mercapto-methyl)phenoxy]hexadecanoic acid (BMPHA) on Au(111) was studied using nano-fabrication protocols with scanning probe nanolithography and immersion particle lithography. Molecularly thin films of BMPHA prepared by surface self-assembly have potential application as spatially selective layers in sensor designs. Either monolayer or bilayer films of BMPHA can be formed under ambient conditions, depending on the parameters of concentration and immersion intervals. Experiments with scanning probe-based lithography (nanoshaving and nanografting) were applied to measure the thickness of BMPHA films. The thickness of a monolayer and bilayer film of BMPHA on Au(111) were measured in situ with atomic force microscopy using n-octadecanethiol as an internal reference. Scanning probe-based nanofabrication provides a way to insert nanopatterns of a reference molecule of known dimensions within a matrix film of unknown thickness to enable a direct comparison of heights and surface morphology. Immersion particle lithography was used to prepare a periodic arrangement of nanoholes within films of BMPHA. The nanoholes could be backfilled by immersion in a SAM solution to produce nanodots of n-octadecanethiol surrounded by a film of BMPHA. Test platforms prepared by immersion particle lithography enables control of the dimensions of surface sites to construct supramolecular assemblies.

Organic synthesis of maize starch-based polymer using Rhizopus oryzae lipase, scale up, and its characterization.

The industrial utilization of native starches is limited because of their inherit nature, with characteristics such as water insolubility and their tendency to form unstable pastes and gels. In this investigation, a lipase produced from Rhizopus oryzae was used for modification of maize starch with palmitic acid at a reaction temperature of 45°C for 18 hr in the presence of dimethyl sulfoxide (DMSO). The synthesis of maize starch palmitate was confirmed by Fourier-transform infrared (FT-IR) and (1)H-nuclear magnetic resonance (NMR) spectra with a higher degree of substitution (DS) of 1.68. Thermal gravimetric analysis (TGA) showed that the maize starch palmitate is more stable even up to 496°C as compared to unmodified maize starch (231.4°C). Maize starch palmitate possesses high degree of substitution and thermal properties and thus can be widely used in food and pharmaceutical industry.

[Synthesis and properties of 5-fluorouracil derivatives].

A series of N-acyl derivatives of 5-fluorouracil (5-FU) bearing residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and 1-adamantanecarbonic acids was synthesized. Relative rates of hydrolysis of derivatives mentioned under physiological conditions, at pH 7.2 and 37 degrees C, have shown that stability of these compounds increases with reducing of spatial accessibility of amide group at N1 in 5-FU. These substances incorporate easily into lipid bilayer; their liposomal preparations showed substantial cytostatic activity on HBL (human breast lymphoma) cells and are of interest as potential antitumor preparations. Also, a fluorescent analog, 1-[8-(3-perylenyl)octanoyl]-5-fluorouracil, destined for studies of the 5-FU derivatives behavior in cells and tissues was prepared.

Automated production of [11C]acetate and [11C]palmitate using a modified GE Tracerlab FX(C-Pro).

As researchers explore new applications for positron emission tomography radiopharmaceuticals, the demand for effective and readily available radiopharmaceuticals continues to increase. The syntheses of two such radiopharmaceuticals, [(11)C]acetate and [(11)C]palmitate, can be automated on the GE Tracerlab FX(C-Pro) by utilizing Grignard reactions. Radiochemical purities of the [(11)C]acetate and the [(11)C]palmitate products were high (>98% and >99.9%, respectively) with average non-corrected yields of 18% (n = 3) and 10% (n = 5), respectively. These data comprise the validation trials for site qualification of clinical production of both radiopharmaceuticals.

A stereo-controlled synthesis of 2,4-dimethyl-4-hydroxy-16-phenylhexadecanoic acid 1,4-lactone and its PPAR activities.

A novel class of natural PPAR agonists, 2,4-dimethyl-4-hydroxy-16-phenylhexadecanoic acid 1,4-lactone (1), were discovered in marine natural product libraries. The synthesis of 1 was accomplished starting from vinylmethyl ketone. Ring formation of the α,γ dialkyl γ-lactone was achieved via the stereo-controlled reaction of a ketyl radical anion with a chiral methacrylate. In the PPAR agonistic assay, the most potent of the four stereoisomers had EC(50) values of 12 µM for mPPARα, 9 µM for mPPARδ and >100 µM for mPPARγ.

Non-radioactive detection of palmitoylated mitochondrial proteins using an azido-palmitate analogue.

While palmitoylation is typically thought of as a cytosolic process resulting in membrane attachment of the palmitoylated proteins, numerous mitochondrial proteins have been shown to be palmitoylated following in vitro labeling of mitochondria with radioactive or bioorthogonal analogues of fatty acids. The fatty acylation of two liver mitochondrial enzymes, methylmalonyl semialdehyde dehydrogenase and carbamoyl phosphate synthetase 1, has been studied in great detail. In both cases palmitoylation of an active site cysteine residue occurred spontaneously and resulted in inhibition of enzymatic activity, thus, suggesting that palmitoylation may be a direct means to regulate the activity of metabolic enzymes within the mitochondria. The progress of investigators working on protein fatty acylation has long been impeded by the long exposure time required to detect the incorporation of [(3)H]-fatty acids into protein by fluorography (often 1-3 months or more). Significant reduction in exposure times has been achieved by the use of [(125)I]-iodofatty acids but these analogues are also hazardous and not commercially available. Herein, we describe a sensitive chemical labeling method for the detection of palmitoylated mitochondrial proteins. The method uses azido-fatty acid analogues that can be attached to proteins and reacted with tagged phosphines via a modified Staudinger ligation. Recently, we used this labeling method, combined with mass spectrometry analysis of the labeled proteins, to identify 21 palmitoylated proteins from rat liver mitochondria.

The use of 3-aminophthalimide as a pro-chemiluminescent label in chemiluminescence and fluorescence-based cellular assays.

We present a labeling system for direct chemiluminescence-based cellular bioassays using the stable pro-chemiluminescent, luminol precursor, 3-aminophthalimide (API). API-coupled reporter molecules are detected chemiluminometrically after treatment with hydrazine, which converts the API label to luminol. API derivatives containing a variety of functional groups are readily synthesized, allowing for ease of coupling via the imide nitrogen to a host of reporter molecules. The fluorescent nature of APIs further allows for dual fluorescence and chemiluminescence studies. To highlight the utility of this label, we show that API-labeled insulin can be successfully utilized in cellular binding and transport assays and that an API-coupled mitochondrial probe (API-triphenylphosphonium(+)) can be used to both fluorescently and chemiluminometrically investigate mitochondrial function. We also assess the use of API as a polysaccharide and nucleic acid label, and we show that API-labeled palmitic acid undergoes cellular transport and lipid metabolism.

Design, synthesis, and evaluation of an isotopic labeling strategy for studying fatty acid-protein binding by NMR.

Here we describe the synthesis and evaluation of a new isotopic labeling strategy for fatty acids to be used as probes for studying ligand binding by NMR. We synthesized palmitic acid with carbons C-3 through C-16 perdeuterated, C-1 and C-2 with 13C atoms and hydrogens at C-2. Our strategy began with commercially available perdeuterated myristic acid and built up to palmitic acid using a Horner-Wadsworth-Emmons reaction. To evaluate the power of this isotopic enrichment strategy, we evaluated ligand binding to a prototypical member of the intracellular lipid binding protein family, FABP2. This small 15 kDa protein is well known to bind fatty acids with high affinity, and we used this system to illustrate the spectral filtering abilities of our described labeling strategy. Herein we show how having two vicinal 13C-enriched carbons, two hydrogens at the alpha-position, and a perdeuterated aliphatic tail allows the efficient use of multidimensional NMR experiments to effectively filter all background resonances from the protein and facilitate the study of ligand binding.

Synthesis and characterization of a dipalmitoylated lipopeptide derived from paralogous lipoproteins of Mycoplasma pneumoniae.

Genomic analysis of Mycoplasma pneumoniae revealed the existence of a large number of putative lipoprotein genes compared with the numbers in other bacteria. However, the pathogenic roles of M. pneumoniae lipoproteins are still obscure. In this study, we synthesized a lipopeptide (designated M. pneumoniae paralogous lipoprotein 1 [MPPL-1]) in which an S-dipalmitoylglyceryl cysteine was coupled to a peptide with a consensus sequence of a putative paralogous lipoprotein group characteristic of M. pneumoniae. The cytokine-inducing activity of MPPL-1 in human monocytic cells was much weaker (approximately 700-fold weaker) than that of the known mycoplasmal S-dipalmitoylated lipopeptide FSL-1 or MALP-2. MPPL-1 required Toll-like receptor (TLR2) to activate NF-kappaB-dependent gene transcription in HEK293 cells, although a 1,000-fold-larger amount of MPPL-1 was needed to exert activity similar to that of FSL-1 in the cells. TLR2-mediated recognition of MPPL-1 was synergistically upregulated by TLR6 but not by TLR1 or TLR10, although the activity was still weak. In addition, MPPL-1 did not antagonize FSL-1 recognition in human monocytic cells and TLR2/TLR6-expressing HEK293 cells. Thus, these results suggest that there is preferential selective recognition of diacylated lipopeptides due to the magnitude of an affinity with TLR2 and TLR6 and the roles of increased paralogous lipoprotein genes of M. pneumoniae in evasion of TLR2 recognition.

A near-field microscopy study of submicron domain structure in a model lung surfactant monolayer.

The submicron domain structure of coexisting liquid condensed (LC) and liquid expanded (LE) phases in monolayers composed of palmitic acid and 20 wt% of a lung surfactant protein B fragment has been investigated. Near-field microscopy was used to simultaneously measure topography and fluorescence images of monolayers that were prepared at a surface pressure of 15 mN/m and a temperature of 22 degrees C. The use of a fluorescently tagged peptide allowed for unambiguous determination of the peptide location in the two-component system. The LC and LE phases in the monolayers are measured on the submicron length scale. A 6-11 A height difference between the LC and LE phases was evident in the height images. Gradual transitions between the LC and LE domains were observed across a 1.3 microm length scale in the near-field fluorescence images, but were significantly sharper in the simultaneously collected topography images and in the separately measured AFM images. These results may reflect the occurrence of peptide encroachment into the LC domains.

Identification and total synthesis of novel fatty acids from the Siphonarid limpet Siphonaria denticulata.

The novel fatty acids 17-methyl-6(Z)-octadecenoic acid and 17-methyl-7(Z)-octadecenoic acid were identified for the first time in nature in the mollusk Siphonaria denticulata from Queensland, Australia. The principal fatty acids in the limpet were hexadecanoic acid, octadecanoic acid, and (Z)-9-octadecenoic acid, while the most interesting series of monounsaturated fatty acids was a family of five nonadecenoic acids with double bonds at either Delta(7), Delta(9), Delta(11), Delta(12), or Delta(13). The novel compounds were characterized using a combination of GC-MS and chemical transformations, such as dimethyl disulfide derivatization. The first total syntheses for the two novel methyl-branched nonadecenoic acids are also described, and these were accomplished in four to five steps and in high yields.

Synthesis and preliminary evaluation of (18)F-labeled 4-thia palmitate as a PET tracer of myocardial fatty acid oxidation.

Interest remains strong for the development of a noninvasive technique for assessment of regional fatty acid oxidation rate in the myocardium. (18)F-labeled 4-thia palmitate (FTP, 16-[(18)F]fluoro-4-thia-hexadecanoic acid) has been synthesized and preliminarily evaluated as a metabolically trapped probe of myocardial fatty acid oxidation for positron emission tomography (PET). The radiotracer is synthesized by Kryptofix 2.2.2/K(2)CO(3) assisted nucleophilic radiofluorination of an iodo-ester precursor, followed by alkaline hydrolysis and by purification by reverse phase high performance liquid chromatography. Biodistribution studies in rats showed high uptake and long retention of FTP in heart, liver, and kidneys consistent with relatively high fatty acid oxidation rates in these tissues. Inhibition of carnitine palmitoyl-transferase-I caused an 80% reduction in myocardial uptake, suggesting the dependence of trapping on the transport of tracer into the mitochondrion. Experiments with perfused rat hearts showed that the estimates of the fractional metabolic trapping rate (FR) of FTP tracked inhibition of oxidation rate of palmitate with hypoxia, whereas the FR of the 6-thia analog 17-[(18)F]fluoro-6-thia-heptadecanoic acid was insensitive to hypoxia. In vivo defluorination of FTP in the rat was evidenced by bone uptake of radioactivity. A PET imaging study with FTP in normal swine showed excellent myocardial images, prolonged myocardial retention, and no bone uptake of radioactivity up to 3 h, the last finding suggesting a species dependence for defluorination of the omega-labeled fatty acid. The results support further investigation of FTP as a potential PET tracer for assessing regional fatty acid oxidation rate in the human myocardium.

P450BM-3: absolute configuration of the primary metabolites of palmitic acid.

P450BM-3, a catalytically self-sufficient, soluble bacterial P450, contains on the same polypeptide a heme domain and a reductase domain. P450BM-3 catalyzes the oxidation of short- and long-chain, saturated and unsaturated fatty acids. The three-dimensional structure of the heme domain both in the absence and in the presence of fatty acid substrates has been determined; however, the fatty acid in the substrate-bound form is not adequately close to the heme iron to permit a prediction regarding the stereoselectivity of oxidation. In the case of long-chain fatty acids, the products can also serve as substrate and be metabolized several times. In the current study, we have determined the absolute configuration of the three primary products of palmitic acid hydroxylation (15-, 14-, and 13-OH palmitic acid). While the 15- and 14-hydroxy compounds are produced in a highly stereoselective manner (98% R, 2% S), the 13-hydroxy is a mixture of 72% R and 28% S. We have also examined the binding of these three hydroxy acids to P450BM-3 and shown that only two of them (14-OH and 13-OH palmitic acid) can bind to and be further metabolized by P450BM-3. The results indicate that in contrast to the flexibility of palmitoleic acid bound to the oxidized enzyme, palmitic acid is rigidly bound in the active site during catalytic turnover.