Vincenzo Abbate

Polyethylene glycol conjugated polymeric nanocapsules for targeted delivery of quercetin to folate-expressing cancer cells in vitro and in vivo.

In this work we describe the formulation and characterization of chemically modified polymeric nanocapsules incorporating the anticancer drug, quercetin, for the passive and active targeting to tumors. Folic acid was conjugated to poly(lactide-co-glycolide) (PLGA) polymer to facilitate active targeting to cancer cells. Two different methods for the conjugation of PLGA to folic acid were employed utilizing polyethylene glycol (PEG) as a spacer. Characterization of the conjugates was performed using FTIR and (1)H NMR studies. The PEG and folic acid content was independent of the conjugation methodology employed. PEGylation has shown to reduce the size of the nanocapsule; moreover, zeta-potential was shown to be polymer-type dependent. Comparative studies on the cytotoxicity and cellular uptake of the different formulations by HeLa cells, in the presence and absence of excess folic acid, were carried out using MTT assay and Confocal Laser Scanning Microscopy, respectively. Both results confirmed the selective uptake and cytotoxicity of the folic acid targeted nanocapsules to the folate enriched cancer cells in a folate-dependent manner. Finally, the passive tumor accumulation and the active targeting of the nanocapsules to folate-expressing cells were confirmed upon intravenous administration in HeLa or IGROV-1 tumor-bearing mice. The developed nanocapsules provide a system for targeted delivery of a range of hydrophobic anticancer drugs in vivo.

Quantitation of hepcidin in serum using ultra‐high‐pressure liquid chromatography and a linear ion trap mass spectrometer

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.

Anabolic steroids detected in bodybuilding dietary supplements – a significant risk to public health

Twenty-four products suspected of containing anabolic steroids and sold in fitness equipment shops in the United Kingdom (UK) were analyzed for their qualitative and semi-quantitative content using full scan gas chromatography-mass spectrometry (GC-MS), accurate mass liquid chromatography-mass spectrometry (LC-MS), high pressure liquid chromatography with diode array detection (HPLC-DAD), UV-Vis, and nuclear magnetic resonance (NMR) spectroscopy. In addition, X-ray crystallography enabled the identification of one of the compounds, where reference standard was not available. Of the 24 products tested, 23 contained steroids including known anabolic agents; 16 of these contained steroids that were different to those indicated on the packaging and one product contained no steroid at all. Overall, 13 different steroids were identified; 12 of these are controlled in the UK under the Misuse of Drugs Act 1971. Several of the products contained steroids that may be considered to have considerable pharmacological activity, based on their chemical structures and the amounts present. This could unwittingly expose users to a significant risk to their health, which is of particular concern for naïve users.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

Background: Antimicrobial peptides (AMPs) have the potential to act against multiple pathogenic targets. Results: AMPs that maintain conformational flexibility are more potent against multiple pathogens and less hemolytic. Conclusion: Antimicrobial action and hemolysis proceed via differing mechanisms. Significance: The potency, selectivity, and ability of AMPs to reach intracellular pathogens can be modulated using general principles. We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Simple and mild preparation of silica-enzyme composites from silicic acid solution

Silica-enzyme composite materials including nanoparticles are formed readily from silicic acid and some hydrolase enzymes under mild conditions when the enzyme pI is greater than about 10.

Synthesis of double-clickable functionalised graphene oxide for biological applications.

Azide- and alkyne-double functionalised graphene oxide (Click2 GO) was synthesised and characterised with ATR-FTIR, TGA, and Raman spectroscopy.

Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide

Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol–ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.

Incorporation of 2,3-Diaminopropionic Acid into Linear Cationic Amphipathic Peptides Produces pH-Sensitive Vectors

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pKa of the β‐NH2 in 2,3‐diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pKa of around 6.3 is stabilized by the increased electron‐withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large‐scale conformational changes are observed in Dap‐rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene‐delivery capability on designed cationic amphipathic peptides that incorporate Dap.

68Ga-THP-PSMA: a PET imaging agent for prostate cancer offering rapid, room temperature, one-step kit-based radiolabeling

The clinical impact and accessibility of 68Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of 99mTc radiopharmaceuticals. Currently, chelating agents used in 68Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a 68Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of 68Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with 68Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing 68Ga-THP-PSMA with 68Ga-HBED-CC-PSMA. Results: Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40–60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of 68Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Conclusion: 68Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other 68Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of 68Ga radiopharmaceuticals.

Inclusion complexes between cyclic siloxanes and cyclodextrins: synthesis and characterization

Molecular inclusion complexes between cyclodextrins and cyclic siloxanes were prepared and characterized via a combination of liquid and solid state NMR, FT-IR, TGA, powder X-ray diffraction, SEM–EDS and elemental analyses. The crystalline complexes adopted the channel-type conformation. Depending from the size of both the cyclic sugar cavity and the silicon guest, various yields (between 0 and 41%) and host–guest molar ratios (between 1:1 and 4:1) were obtained. α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD) were observed to form crystalline inclusion complexes only with D3 (cyclic dimethyltrisiloxane) due to steric effects, whereas the larger γ-cyclodextrin (γ-CD) formed inclusion complexes both with D3, D4 (cyclic dimethyltetrasiloxane) and D5 (cyclic dimethylpentasiloxane). This study is believed to be the first step towards the selective removal of cyclic siloxanes impurities from commercial PDMS preparations.