Intracellular nitric oxide delivery from stable NO-polymeric nanoparticle carriers.

The encapsulation of S-nitrosoglutathione into polymeric nanoparticles substantially improves NO stability in aqueous media without affecting the efficacy of intracellular delivery. The combination of nano-NO delivery and chemotherapy has been found to enhance antitumour activity of chemotherapeutics, as demonstrated using preliminary in vitro experiments with neuroblastoma cells.

Selective Postmodification of Copolymer Backbones Bearing Different Activated Esters with Disparate Reactivities.

In this communication, we report an easy method for introducing functional groups into polymer structures by successively reacting two different activated ester functionalities (pentafluorophenyl (PFP) ester and azlactone (AZ)) with different functional amine compounds. By exploiting the difference in reactivity of the two activated esters (PFP and AZ) toward different amino compounds, we demonstrate, for the first time, a selective modification of the different activated ester groups, thereby introducing functional groups to the polymer backbone in a controlled manner. Statistical and block copolymers of vinyl dimethyl azlactone (VDM) and pentafluorophenyl acrylate (PFPA), i.e.,(p(VDM-stat-PFPA)) and (p(VDM-block-PFPA)), were prepared using reversible addition-fragmentation transfer (RAFT) polymerization and subsequently modified using a library of amino compounds, yielding macromolecules with bespoke functionality. In additional work, the functional macromolecules were self-assembled into nanoparticles.

Probing bacterial-toxin inhibition with synthetic glycopolymers prepared by tandem post-polymerization modification: role of linker length and carbohydrate density.

Probing the depths: A tandem post-polymerization modification strategy was used to systematically probe the multivalent inhibition of a bacterial toxin as a function of linker length (see scheme), carbohydrate density, and glycopolymer chain length. Guided by structural-biology information, the binding-pocket depth of the toxin was probed and used as a means to specifically improve inhibition of the toxin by the glycopolymer.

Direct peptide bioconjugation/PEGylation at tyrosine with linear and branched polymeric diazonium salts.

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG(2000) to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca(2+)] plasma levels by mPEG(2000)-sCT conjugate in rat animal models.

Highly efficient disulfide bridging polymers for bioconjugates from radical-compatible dithiophenol maleimides.

The direct synthesis of dithiophenol maleimide functional polymers by living radical polymerisation is described without the need for protecting group chemistry. The synthesised polymers have been successfully employed as disulfide bridging agents for salmon calcitonin when used in equimolar quantities, negating the requirement for complex purification strategies, traditionally associated with peptide bioconjugation.

Polymeric dibromomaleimides as extremely efficient disulfide bridging bioconjugation and pegylation agents.

A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while maintaining the disulfide bridge and thus peptide conformation. The insertion is initiated by disulfide reduction using a water-soluble phosphine, tris(2-carboxyethyl)phosphine (TCEP) which allows for subsequent substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary structure of the peptide is efficiently maintained.

Dibromomaleimide End Functional Polymers by RAFT Polymerization Without the Need of Protecting Groups.

Polymers bearing the dibromomaleimide (DBM) group as a functional chain end have been synthesized by RAFT polymerization. A DBM functional chain transfer agent (CTA) was utilized to afford well-defined PtBA, PMA, and PTEGA, without the requirement of protecting group chemistry. It was found that RAFT polymerization of NIPAM and styrene with this CTA was severely retarded/inhibited which is ascribed to their relatively low propagation rate constants compared to acrylates. This observation is accounted for by a reversible trapping of propagating radicals by the DBM group in RAFT polymerizations using a monomer with low kp. However, further attempts to synthesize DBM-terminated PtBA and PMA by ATRP using an analogous initiator were unsuccessful, and broad PDI were observed. Furthermore, highly efficient postpolymerization functionalization of DBM-terminated PMA produced by RAFT, with the model compound thiophenol was also demonstrated.

A facile route to end-functionalised polymers synthesised by SET-LRP via a one-pot reduction/thiol-ene Michael-type addition.

We report the facile synthesis of well defined, disulfide containing polymers via SET-LRP. A one-pot reduction/conjugation reaction enables post polymerisation modification with functional (meth)acrylates and acrylamides.

Phosphine-mediated one-pot thiol-ene "click" approach to polymer-protein conjugates.

We employ water-soluble organic phosphines as key reagents in a one-pot synthetic protocol where a (poly)peptide disulfide bridge is first reduced followed by subsequent reaction of the two thiols in situ with poly(monomethoxy ethylene glycol)(meth)acrylates (p(mPEG)(M)A); we use salmon calcitonin (sCT) as a disulfide bridge-containing peptide, which contains a disulfide bridge-Cys1-Cys7-that can be reduced to give two sulfhydryl units available for thiol functionalisation; bioactivity is retained.

Pleistocene glaciation events shape genetic structure across the range of the American lobster, Homarus americanus.

A north/south discontinuity along the northeastern coast of North America in the genetic structure of the American lobster (Homarus americanus) was detected using a suite of 13 microsatellite loci assessed using spatial analyses. Population genetic data laid over existing data on physiographic changes and sea-surface temperatures were used to reconstruct the Pleistocene distribution of this species. A postglacial northern-edge colonization model best explains the relative genetic homogeneity of the northern region compared to the southern region centred in the Gulf of Maine. Population genetic analyses identified significant structure (range of standardized theta 0-0.02) but no significant evidence for isolation by distance. The novel application of spatial genetic analyses to a marine species allowed us to interpret these results by providing a greater insight into the evolutionary factors responsible for shaping the genetic structure of this species throughout is natural range.

Isolation and characterization of tetranucleotide microsatellites from Atlantic haddock (Melanogrammus aeglefinus).

Five tetranucleotide microsatellite loci developed from Atlantic haddock (Melanogrammus aeglefinus) are presented. Loci were isolated using a modified magnetic bead-hybridization selection procedure that enriched for tetranucleotide microsatellites. Loci were polymorphic (3-99 alleles per locus; mean, 33.2) and exhibited high levels of observed heterozygosity (0.07-0.99; mean, 0.73) in a sample of 70 haddock collected from the Scotian Shelf in the Northwest Atlantic Ocean. Primer sets for 5 tetranucleotide microsatellites, originally developed from cod (Gmo34), were also tested in Atlantic haddock; one pair yielded readily detectable product and was variable in the population assayed (29 alleles; heterozygosity, 0.96). These loci are suitable for kinship analyses in aquaculture-related applications, and are potentially useful for resolving population structure in the wild.