Oligonucleotide-polymer conjugates: effect of the method of synthesis on their structure and performance in diagnostic assays

Oligonucleotide-polymer conjugates have been described to improve the sensitivity of an enzyme-linked oligosorbent assay diagnostic test. To understand the influence of their structure and conformation in solution on the efficiency of the test during the capture step, two different ways of synthesizing these conjugates were compared. The first consisted of coupling 5' amino modified oligonucleotides to poly(maleic anhydride-alt-methylvinyl ether) and poly(maleic anhydride-alt-ethylene). The second resulted from direct synthesis of oligonucleotides from poly(maleic anhydride-alt-ethylene) previously grafted onto a controlled pore glass support. The different conjugates were analyzed by size-exclusion chromatography and viscometry. The former method for conjugate synthesis produced aggregates, which was not the case for the latter. These conjugates were then used in the capture phase of a hybridization assay using a HBV DNA target, on a bioMérieux VIDAS instrument. Different parameters were studied, such as the purity of the conjugate solution and the number of oligonucleotides per polymer chain. The amount of conjugate coated on the solid-phase receptacle surface at the time of the capture phase was evaluated by radioactive labeling. Finally, it was demonstrated that conjugates produced an amplification factor of 50 versus the capture oligonucleotide probe used as the reference. The detection limit reached 10(8) copies/mL.     

Salivary β-glucosidase as a direct factor influencing the occurrence of halitosis

β-Glucosidases are enzymes present in all living organisms, playing a pivotal role in diverse biological processes. These enzymes cleave β-glycosidic bonds between carbohydrates, or between a carbohydrate and a non-carbohydrate moiety, which may result in the liberation of volatile aglycones. Released compounds execute diverse physiological roles, while the industry takes advantage of exogenously added β-glucosidases for aroma enrichment during food and beverage production. β-Glucosidase enzymatic activity has been reported in human saliva and given the fact that these enzymes are involved in aroma release, we investigated here the correlation between β-glucosidase activity in human saliva and the occurrence of halitosis. Measurement of salivary enzyme activity of 48 volunteers was performed using p-nitrophenyl-β-d-glucopyranoside as substrate. Each volunteer was clinically evaluated by a dental surgeon and clinical and laboratorial data were statistically analyzed. Gas-chromatography of saliva headspace allowed the analysis of the direct role of exogenous β-glucosidase on aromatic /volatile profile of saliva samples. The data demonstrated a positive correlation between halitosis and enzymatic activity, suggesting that the enzyme exerts a direct role in the occurrence of bad breath. Gas-chromatography analysis demonstrated that exogenously added enzyme led to the alteration of volatile organic content, confirming a direct contribution of β-glucosidase activity on saliva volatile compounds release. Although halitosis is a multifactorial condition, the complete understanding of all governing factors may allow the development of more effective treatment strategies. Such studies may pave the way to the use of β-glucosidase inhibitors for halitosis clinical management.     

Aromatic aldols and 1,5-diketones as optimized fragrance photocages

Aromatic aldols and 1,5-diketones with abstractable γ-hydrogen atoms are highly photoactive cage molecules for the release of fragrance carbonyl compounds (aldehydes and Michael ketones, respectively). Aldols 3a-d are easily accessible by Mukaiyama addition and are cleaved to form the substrates with high quantum yields under solar radiation. By tuning the properties of the chromophores, a series of δ-damascone cages 5 were developed that can be used for selective and fast (5a,e) or slow (5b,d) release of fragrances under air and solar irradiation. The intermediates of the Norrish II process were observed by laser transient absorption spectroscopy.     

Controlled Release of Volatile Secondary and Tertiary Alcohols by Neighboring Group Participation: Stepwise Cyclization and Re‐Opening of 2,2′‐Bis(carbamoyl)dibenzoates at Neutral pH

Bioactive, volatile, secondary and tertiary fragrance alcohols are efficiently released by intramolecular neighboring‐group‐assisted hydrolysis of 2,2′‐bis(carbamoyl)dibenzoates at neutral pH. The stepwise cyclization of 2,2′‐[(methylimino)bis(propane‐3,1‐diylcarbamoyl)]dibenzoates is followed by the re‐opening of the intermediately formed diphthalimide and proceeds in an overall four‐step consecutive reaction sequence. Kinetic rate constants for all four reaction steps could be determined pairwise by reversed‐phase HPLC. At neutral pH, secondary alcohols were released by one order of magnitude faster than the tertiary alcohols, and the rate constants for the re‐opening of the diphthalimides were found to be in the same order of magnitude as the release of the tertiary alcohols. Dynamic headspace analysis on a dry cotton surface finally confirmed the efficient release of tertiary alcohols under mild reaction conditions generally encountered for applications in functional perfumery.     

Potential semiochemicals in urine from free ranging wolverines (Gulo gulo Pallas, 1780)

Urine deposition has been observed as an important scent-marking behaviour among wolverines (Gulo gulo, Mustelinae, Mustelidae). Solid phase microextraction (SPME) of headspace volatiles of the urine from free ranging wolverines were examined by gas chromatography–mass spectrometry (GC–MS). Urine samples were collected directly from the bladder of live-trapped animals or from frozen samples deposited in snow. Nineteen potential semiochemicals were identified in the headspace from 22 urine samples. The composition of these volatile compounds varied by type and amount with each sample, but a number of chemicals were regularly found in many samples. The most commonly found constituents were the ketones; 2-heptanone, 4-heptanone and 4-nonanone; and the terpenes: α-pinene, β-pinene, limonene, linalool and geraniol. Mammalian urinary discharge of ingested α-pinene, β-pinene, limonene and other hydrocarbon terpenes is unusual, as these compounds are usually oxidized before excretion. The source of the hydrocarbon monoterpenes likely includes conifer needles, as they have been found in wolverine scat.     

Inflammation responsive logic gate nanoparticles for the delivery of proteins

Oxidative stress and reduced pH are important stimuli targets for intracellular delivery and for delivery to diseased tissue. However, there is a dearth of materials able to deliver bioactive agents selectively under these conditions. We employed our recently developed dual response strategy to build a polymeric nanoparticle that degrades upon exposure to two stimuli in tandem. Our polythioether ketal based nanoparticles undergo two chemical transformations; the first is the oxidation of the thioether groups along the polymer backbone of the nanoparticles upon exposure to reactive oxygen species (ROS). This transformation switches the polymeric backbone from hydrophobic to hydrophilic and thus allows, in mildly acidic environments, the rapid acid-catalyzed degradation of the ketal groups also along the polymer backbone. Dynamic light scattering and payload release studies showed full particle degradation only in conditions that combined both oxidative stress and acidity, and these conditions led to higher release of encapsulated protein within 24 h. Nanoparticles in neutral pH and under oxidative conditions showed small molecule release and swelling of otherwise intact nanparticles. Notably, cellular studies show absence of toxicity and efficient uptake of nanoparticles by macrophages followed by cytoplasmic release of ovalbumin. Future work will apply this system to inflammatory diseases.     

Design,synthesis and ex-vivo release studies of colon-specific polyphosphazene–anticancer drug conjugates

Colon-specific azo based polyphosphazene–anticancer drug conjugates (11–18) have been synthesized and evaluated by ex-vivo release studies. The prepared polyphosphazene drug conjugates (11–18) are stable in acidic (pH = 1.2) buffer which showed that these polymer drug conjugates are protected from acidic environment which is the primary requirement of colon specific targeted drug delivery. The ex-vivo release profiles of polyphosphazene drug conjugates (11–18) have been performed in the presence as well as in the absence of rat cecal content. The results showed that more than 89% of parent drugs (methotrexate and gemcitabine) are released from polymeric backbone of polyphosphazene drug conjugates (14 and 18) having n-butanol (lipophilic moiety). The in-vitro cytotoxicity assay has also been performed which clearly indicated that these polymeric drug conjugates are active against human colorectal cancer cell lines (HT-29 and COLO 320 DM). The drug release kinetic study demonstrated that Higuchi’s equation is found to be best fitted equation which showed that release of drug from polymeric backbone as square root of time dependent process based on non-fickian diffusion. Therefore, the synthesized polyphosphazene azo based drug conjugates of methotrexate and gemcitabine are the potential candidates for colon targeted drug delivery system with minimal undesirable side effects.     

Stabilized hemiacetal complexes as precursors for the controlled release of bioactive volatile alcohols

Hemiacetals of pyridine-2-carbaldehyde derivatives and volatile alcohols can be stabilized in organic solution in the presence of protons or different metal cations. Despite the inherent instability of hemiacetals in H(2) O, stabilizing them with zinc(II) triflate and adding them to a cationic surfactant formulation resulted in the slow release of the alcohol from cotton surfaces being treated with the hemiacetal complex. Stabilized hemiacetals might thus be suitable delivery systems of bioactive volatiles by rapid hydrolysis in H(2) O-based media.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

转Bt基因棉花及其受体品种主要挥发性物质的测定

应用顶空进样气质联机系统（Headspace-GC-MS)测定了转Bt基因棉花及其对照亲本主要挥发性物质。结果表明棉花营养器官和繁殖器官的主要挥发性物质的差异,棉花植株现蕾期前,叶片中挥发性物质以α-蒎烯为主,而现蕾后,蕾、花和铃中主要以β-月桂烯为主。研究初步发现,外源Bt杀虫蛋白表达对棉花自身主要挥发性物质的合成不会造成不利影响。     

Polysaccharide-poly(ethylene glycol) star copolymer as a scaffold for the production of bioactive hydrogels

The production of polysaccharide-derivatized surfaces, polymers, and biomaterials has been shown to be a useful strategy for mediating the biological properties of materials, owing to the importance of polysaccharides for the sequestration and protection of bioactive proteins in vivo. We have therefore sought to combine the benefits of polysaccharide derivatization of polymers with unique opportunities to use these polymers for the production of bioactive, noncovalently assembled hydrogels. Accordingly, we report the synthesis of a heparin-modified poly(ethylene glycol) (PEG) star copolymer that can be used in the assembly of bioactive hydrogel networks via multiple strategies and that is also competent for the delivery of bioactive growth factors. A heparin-decorated polymer, synthesized by the reaction of thiol end-terminated four-arm star PEG (M(n) = 10 000) with maleimide functionalized low molecular weight heparin (LMWH, M(r) = 3000), has been characterized via (1)H NMR spectroscopy and size-exclusion chromatography; results indicate attachment of the LMWH with at least 73% efficiency. Both covalently and noncovalently assembled hydrogels can be produced from the PEG-LMWH conjugate. Viscoelastic noncovalently assembled hydrogels have been formed on the basis of the interaction of the PEG-LMWH with a PEG polymer bearing multiple heparin-binding peptide motifs. The binding and release of therapeutically important proteins from the assembled hydrogels have also been demonstrated via immunochemical assays, which demonstrate the slow release of basic fibroblast growth factor (bFGF) as a function of matrix erosion. The combination of these results suggests the opportunities for producing polymer-polysaccharide conjugates that can assemble into novel hydrogel networks on the basis of peptide-saccharide interactions and for employing these materials in delivery applications.     

转Bt基因棉挥发性气味的化学成分及其对棉铃虫的电生理活性

报道了对转Bt基因棉“GK-12”及其常规棉亲本“泗棉3号”挥发性化学物质的分析结果,以及棉铃虫对Bt棉挥发性化学物质的电生理反应,以期为抗虫棉的生态学和安全性评价提供化学生态学的证据。结果表明,在7～8个真叶期,转Bt基因棉的α-蒎烯和β-蒎烯的相对含量比常规棉高许多,倍半萜烯C和一个含量很低的化合物（该化合物对棉铃虫有电生理活性）是常规棉所没有的。转基因Bt棉的挥发性物质中有9个化合物可引起棉铃虫的触角电位反应。     

Flavor release of diacetyl and 2-heptanone from cream style dressings in three mouth model systems

The release of volatile compounds from a cream style dressing, which consisted of a thickening agent dispersed in the water phase of an oil in water (o/w) type of emulsion, was studied by the purge-and-trap (PT), dynamic head space mastication (DHM) and dynamic headspace (DH) model systems for diacetyl and 2-heptanone as two volatile compounds. Big differences were detected in the quantity of volatiles released by the three models for both diacetyl and 2-heptanone: PT released the most, followed by DHM and DH. Nitrogen gas bubbling in PT and plunger up-and-down motion in DHM mimic mouth movements and promoted volatile release more than DH. The quantity of volatiles released depended on the nitrogen gas flow rate and isolation period with both the PT and the DHM model. Static headspace measurements indicated that no interaction occurred between the volatiles and the dispersion thickening agent, nor between the volatiles and protein of saliva.     

Thioether Profragrances: Parameters Influencing the Performance of Precursor‐Based Fragrance Delivery in Functional Perfumery

A series of thioether profragrances was prepared by reaction of different sulfanylalkanoates with δ‐damascone and tested for their release efficiencies in a fabric‐softener and an all‐purpose cleaner application. Dynamic headspace analysis on dry cotton and on a ceramic plate revealed that the performance of the different precursors depended on the structure, but also on the particular conditions encountered in different applications. Moreover, profragrances derived from other α,β‐unsaturated fragrance aldehydes and ketones were synthesized analogously and evaluated using the same test protocol. Thioethers were found to be suitable precursors to release the corresponding fragrances, but neither the quantity of profragrance deposited from an aqueous environment onto the target surface, nor the amount of fragrance released after deposition could be linearly correlated to the hydrophilicity or hydrophobicity of the compounds. Different sets of compounds were found to be the best performers for different types of applications. Only one of the compounds evaluated in the present work, namely the thiolactic acid derivative of δ‐damascone, efficiently released the corresponding fragrance in both of the tested applications. Profragrance development for functional perfumery thus remains a partially empirical endeavour. More knowledge (and control) of the various application conditions are required for an efficient profragrance design.     

A spectrophotometric method for the estimation of polymer-supported sulfhydryl groups

A sensitive and simple method is described for the quantitative determination of free sulfhydryl (-SH) groups on polymer supports. The method includes the reaction of 4,4'-dimethoxytrityloxy-S-(2-thio-5-nitropyridyl)-2-mercapto ethane (DTNPME) with polymer-supported sulfhydryl groups. After removal of excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the polymer support into the solution. The dimethoxytrityl cation (lambda max = 498 nm, epsilon 498 = 70,000/M) is then quantified spectrophotometrically. A comparative study of the reagent DTNPME with 2,2'-dithiobis(5-nitropyridine) is also described.     

Targeted delivery of atorvastatin via asialoglycoprotein receptor (ASGPR)

Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.     

A novel application of thermo-responsive polymer to affinity precipitation of polysaccharide

The lectin receptors concanavalin A (ConA) and wheat germ lectin (WGL) were successfully conjugated to a thermo-responsive hydrogel polymer (poly-N-isopropylacrylamide) (PNIPAAm) as an affinity ligand. The coupling efficiencies of the ConA and WGL to PNIPAAm were 5.1% and 44%, respectively. These lectin receptor-polymer conjugates were then tested for their efficiency in purification of various polysaccharides or polysaccharide-containing compounds such as beta-glucan. Results indicated that these conjugates separated various polysaccharides from a complex mixture. The use of a thermo-responsive polymer in low-temperature purification of potentially heat-labile glycoproteins is advantageous. Additionally, other affinity ligands could be coupled to the polymer for separation of the respective bioactive molecules.     

Reversible Blocking of Amino Groups of Octreotide for the Inhibition of Formation of Acylated Peptide Impurities in Poly(Lactide-co-Glycolide) Delivery Systems

The purpose of this study was to develop a novel method to inhibit the formation of acylated peptide impurities in poly(d,l-lactide-co-glycolide) (PLGA) formulations by reversely blocking the amino groups of octreotide with maleic anhydride (MA). Two mono-MA conjugates with different modification sites (N terminus and Lys residue) and di-MA conjugate of octreotide were prepared and isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). The polymer interaction of peptides and the formation of acylated peptides were monitored by RP-HPLC. The stability of MA-octreotide conjugates in PLGA films was studied in 0.1 M phosphate buffer (pH 7.4) at 37°C. The conjugation of MA to octreotide substantially inhibited the interaction of peptide with PLGA polymer and the subsequent formation of acylated peptide impurities. The MA-octreotides were successfully converted to intact octreotide as pH drops by PLGA hydrolysis. In PLGA films, MA-octreotide also showed complete inhibition of peptide acylation. In conclusion, MA conjugation provides a viable approach for stabilizing peptides in PLGA delivery systems.     

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.     

Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.