Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets.

BACKGROUND: Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes. METHODS: C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement. RESULTS: Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250µg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies. CONCLUSION: Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.

The construction of thiol-functionalized DNAsomes with small molecules response and protein release.

In this work, a poly(N-isopropylacrylamide) polymer (PNIPAAm) was prepared via the photoinduced reversible addition-fragmentation chain transfer (RAFT) polymerization using Ru(bpy)3Cl2·6H2O as photoinitiator. The design and spontaneous assembly of thiol-functionalized DNA-Thiol/PNIPAAm polymeric capsule (DNAsomes) by water-in-oil Pickering emulsion method and effective response with small molecules (Sybr green and phenanthrene) were described. The intermediate product, DNA-Thiol/PNIPAAm conjugates and DNAsomes were characterized by using 1H NMR, dynamic light scattering (DLS), SEM, TEM and UV-vis methods. The obtained results indicated that DNA-Thiol/PNIPAAm constructs assembled in a Pickering emulsion could produce DNA-based spherical DNAsomes with typically 3.3-267.7 µm in diameter. The DNAsomes showed a vesicle formation approximately 2 µm in diameter, resulting in phenanthrene molecule intercalating with DNAsomes. The phenomenon indicated that the DNA-Thiol/PNIPAAm conjugates may have potential applications in recognition polycyclic aromatic hydrocarbon molecules. The membrane of the DNAsomes could effective response toward small molecules such as Sybr green or phenanthrene, and DNAsomes has release capability of protein (BSA) under reductive agent glutathione (GSH). Our results highlight the potential of integrating aspects of supramolecular and polymer chemistry into the design and construction of DNA-polymeric capsule, guest molecule encapsulation, control delivery of drugs, recognition organic polycyclic aromatic hydrocarbon molecules and gene-directed capsule synthesis.

Xylan from Pineapple Stem Waste: a Potential Biopolymer for Colonic Targeting of Anti-inflammatory Agent Mesalamine.

We have successfully conjugated mesalamine (5-aminosalicylic acid, 5-ASA) with xylan, a biopolymer isolated from pineapple stem waste, to form xylan-5-ASA conjugate. The biopolymer was used to provide colon-targeting properties for 5-ASA, a golden standard anti-inflammatory agent commonly used for ulcerative colitis treatment. A series of data from FTIR spectroscopy, UV-Vis spectrophotometry, and HPLC confirmed the xylan-5-ASA conjugate formation. To ensure successful colon targeting properties, in vitro and in vivo drug release studies after oral administration of xylan-5-ASA conjugate to Wistar rats were performed. Xylan-5-ASA conjugate was able to retain 5-ASA release in the upper gastrointestinal tract fluid simulation but rapidly released 5-ASA in the rat colon fluid simulation. In vivo release profile shows a very low peak plasma concentration, reached at 6 h after xylan-5-ASA conjugate administration. The delayed release and the lower bioavailability of 5-ASA from xylan-5-ASA conjugate administration compared to free 5-ASA administration confirmed the successful local colon delivery of 5-ASA using xylan-5-ASA conjugate. The administration of xylan-5-ASA conjugate also exhibited greater efficacy in recovering 2,4,6-trinitrobenzene sulfonic acid-induced colon ulcer compared to free 5-ASA administration. Taken together, xylan isolated from pineapple stem waste is promising to obtain colon targeting property for 5-ASA.

Smart gated magnetic silica mesoporous particles for targeted colon drug delivery: New approaches for inflammatory bowel diseases treatment.

Magnetic mesoporous silica microparticles were loaded with safranin O (S1) and with hydrocortisone (S2) and the outer surface functionalized with a bulky azo derivative bearing urea moieties. Aqueous suspensions of both solids at pH 7.4 showed negligible payload release whereas a marked delivery was observed in the presence of sodium dithionite due to the rupture of the azo bonds. Besides, a moderate cargo release was observed at acidic pH due to the hydrolysis of the urea bonds that linked the azo derivative onto the external surface of the inorganic scaffolds. In vitro digestion models showed that S1 and S2 microparticles could be used for the controlled release of payload in the reducing colon environment (in which azoreductase enzymes are present). On the other hand, in vivo pharmacokinetic studies in rats showed that safranine O release from S1 microparticles was concentrated in colon. The performance of S2 microparticles for the treatment of colitis in rats (induced by oral administration of a 2,4,6-trinitrobenzenesulfonic acid solution) was tested. The controlled release of hydrocortisone from S2 in the colon of injured rats induced marked reduction in colon/body weight ratio and in clinical activity score. Also, histological studies showed a marked decrease in inflammation followed by intensive regeneration and almost normal mucosal structure of the individuals treated with S2. Besides, the use of a magnetic belt increased the therapeutic performances of S2 due to an enhanced retention time of the particles in the colon.

Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods.

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90°C for 30min) WPI solutions with either 20mM added NaCl (WPI+NaCl), 5mM N-ethylmaleimide (WPI+NEM) or 20mM added NaCl and 5mM NEM (WPI+NaCl+NEM). Gel electrophoresis demonstrated that the heated WPI and WPI+NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI+NEM and WPI+NaCl+NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules.

Immunomodulatory effects of Bifidobacterium bifidum 231 on trinitrobenzenesulfonic acid-induced ulcerative colitis in rats.

AIM: Ulcerative colitis and Crohn's disease are two important chronic Inflammatory bowel diseases (IBD) characterized by prominent intestinal inflammation. Probiotics are the bacteria that promote the host health by its immunomodulatory activity. The present study investigated the correlation between in vitro adhesion and immunomodulatory properties, and to assess the therapeutic potential of Bifidobacterium bifidum 231 (BIF 231), a new strain of probiotic in ulcerative colitis in rats. METHODS: In vitro adhesion assays and immunomodulatory effect of BIF 231 on interleukins (IL-1ß and IL-10) in IEC-6 cell lines were quantified by gram staining, scanning electron microscopy and q-PCR respectively. Colitis was induced by intra-rectal instillation of trinitrobenzenesulfonic acid. Colitis was evaluated by alterations in colon gross morphology, histologically and biochemically. Colonic interleukin-1ß (IL-1ß) and interleukin-10 (IL-10) mRNA and protein expression were assessed by q-PCR, ELISA and western blot. RESULTS: BIF 231 showed better adhesion and immunomodulation by up-regulating IL-10 levels in IEC-6 cell lines. In vivo studies with treatment of BIF 231 (1.4×1011 CFU/rat/day) revealed anti-inflammatory effects both macroscopically and histologically. BIF 231 lowered TBARS, nitric oxide and augmented GSH levels. BIF 231 treatment to colitic rats down regulated IL-1ß levels with concurrent increase in IL-10 levels. CONCLUSIONS: BIF 231 exerted beneficial in vitro adhesion and immunomodulatory properties which facilitated the recovery of the damaged tissue in TNBS-induced colitis.

Colon targeted oral drug delivery system based on alginate-chitosan microspheres loaded with icariin in the treatment of ulcerative colitis.

In recent years, oral colon specific drug delivery system has been paid more attention in the treatment of inflammatory bowel disease (IBD). As the special pH condition in gastrointestinal tract, the challenge for treatment of IBD was that the colon drug delivery system should endure the low pH in stomach and release drugs quickly in high pH in colon. Icariin with the poor solubility and low bioavailability limited the treatment of many diseases in clinic. In this study, the protective mechanism of alginate-chitosan microspheres loaded with icariin were investigated with trinitrobenzene sulfonic acid (TNBS)/ethanol induced colonic mucosal injury in rats. The results of drug release showed that the icariin loaded into microspheres released only 10% in simulated gastric fluid and a high amount of 65.6% released in simulated colonic fluid. The fluorescence tracer indicated high retention of targeted microspheres more than 12h in colon. The microspheres loaded with icariin could not only reduce the colonic injury by decreasing the colon mucosa damage index in rats, but also reduce the inflammatory response by reducing the production and gene expression of inflammatory mediators and cytokines in colonic mucosa. All the results indicate that targeted microspheres loaded with icariin could exert the colon-protective effects through reducing the inflammatory response, which would be developed as a potential drug controlled release system for treatment of ulcerative colitis.

Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction.

Industrial use of Novozym 435 in synthesis of structured lipids and biodiesel via alcoholysis is limited by mass transfer effects of the glycerides through immobilized enzymes and its low operational stability under operation conditions. To better understand this, differently modified Novozym 435 preparations, differing in their surface nature and in their interactions with reactants, have been compared in the alcoholysis of Camelina sativa oil. The three modifications performed have been carried out under conditions where all exposed groups of the enzyme have been modified. These modifications were: 2,4,6-trinitrobenzensulfonic acid (Novo-TNBS), ethylendiamine (Novo-EDA) and polyethylenimine (Novo-PEI). Changes in their operational performance are analyzed in terms of changes detected by scan electron microscopy in the support morphology. The hydrophobic nature of the TNBS accelerates the reaction rate; t-ButOH co-solvent swells the macroporous acrylic particles of Lewatit VP OC 1600 in all biocatalysts, except in the case of Novo-PEI. This co-solvent only increases the maximal conversions obtained at 24h using the modified biocatalysts. t-ButOH reduces enzyme inactivation by alcohol and water. In a co-solvent system, these four biocatalysts remain fully active after 14 consecutive reaction cycles of 24h, but only Novo-TNBS yields maximal conversion before cycle 5. Some deposits on biocatalyst particles could be appreciated during reuses, and TNBS derivatization diminishes the accumulation of product deposits on the catalyst surface. Most particles of commercial Novozym(®) 435 are broken after operation for 14 reaction cycles. The broken particles are fully active, but they cause problems of blockage in filtration operations and column reactors. The three derivatizations studied make the matrix particles more resistant to rupture.

Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and γ-Cyclodextrin-Enhanced Fluorescence Detection.

Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit ∼25-fold fluorescence enhancement upon the formation of inclusion complexes with γ-cyclodextrin (γ-CD). Association constants were determined as 4600 M(-1) for Ado and 1000 M(-1) for Ino by CE-LIF. The structure of the TNP-Ado:γ-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 µM for Ado and 4 µM for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence γ-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively.

Synthesis of Hapten-Protein Conjugate Vaccines with Reproducible Hapten Densities.

The ability to prepare hapten-carrier conjugates reproducibly with consistent lot-to-lot hapten densities and protein yields is a critical component of hapten vaccine development. This entails the development of appropriate coupling chemistries that do not cause protein precipitation and the development of methods to quantify hapten density. Recently, extensive efforts have been devoted to design vaccines against drugs of abuse. We describe, herein, a method for conjugation of a morphine-like hapten (MorHap) to tetanus toxoid (TT), which involves conjugation of MorHap to the surface lysines of TT through the N-hydroxysuccinimide portion of a heterobifunctional linker and the subsequent attachment of the thiol on MorHap to the maleimide portion of the cross-linker. Methods are described for the analytical quantification of the hapten density of the conjugates using modified Ellman's test, trinitrobenzenesulfonic acid (TNBS) assay, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

5-Aminosalicylic Acid Azo-Linked to Procainamide Acts as an Anticolitic Mutual Prodrug via Additive Inhibition of Nuclear Factor kappaB.

To improve the anticolitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local anesthetic, via an azo bond to prepare 5-(4-{[2-(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-ASA-azo-PA). 5-ASA-azo-PA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine, and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anticolitic effects occurred at least partly through the cooperative inhibition of NFκB activity.

New versatile approach for analysis of PEG content in conjugates and complexes with biomacromolecules based on FTIR spectroscopy.

Here we report a new approach based on FTIR-spectroscopy for determining the degree of PEGylation in biomolecules. We show that the PEG COC peak (at 1089 cm(-1)) is the main analytically valuable band in IR spectra of PEG-containing systems: it is narrow and highly intense, it is well distinguished from absorption bands of other principal functional groups of proteins and other biopolymers (carbonyl, amide, hydroxyl etc), and therefore is easily identified in the IR spectra. The proposed method is therefore "reagent-free" and allows for fast and accurate determination of the PEGylation degree of biomolecules as well as the structural characteristics of bioconjugates from a single FTIR spectrum. The measurement is not dependent on PEG polymerization degree or branching and can be applied in a wide pH range, making it a convenient replacement of laborious and unreliable methods. The developed approach was successfully used to study the range of PEG-containing covalent conjugates with chitosan and non-covalent complexes with anionic liposomes. The composition of PEG-chitosan conjugates as well as their storage stability was determined by the method based on FTIR-spectroscopy. In the case of non-covalent complexes, not only PEG content, but also the binding constants of PEG-containing ligands to the liposome membrane were evaluated with this approach. The results obtained by the FTIR method were confirmed by DLS and zeta-potential experiments where the formation of electrostatic complex was monitored by the increase in the particle radius (from 80 nm to 105 nm) and zeta-potential neutralization (from -20 mV to -12 mV). Direct comparison of the results of FTIR approach with that of TNBS or OPA titration methods shows very good agreement between the measurements, with the FTIR method showing much lower deviation.

Investigation of the colon-targeting, improvement on the side-effects and therapy on the experimental colitis in mouse of a resin microcapsule loading dexamethasone sodium phosphate.

CONTEXT: Dexamethasone is the major drug in the treatment of ulcerative colitis (UC). However, the extensive or long-time use of dexamethasone causes many toxic side-effects. Ion exchange resins react with external-ions through their own functional groups and Eudragit S occurs degradation when pH > 7. These features make them suitable for oral delivery system. OBJECTIVE: Resin microcapsule (DRM) composed by 717 anion exchange resin and Eudragit S100 was used to target dexamethasone to the colon to improve its treatment effect on UC and reduce its toxic side-effects. RESULTS: Dexamethasone sodium phosphate (DXSP) was sequentially encapsulated in 717 anion-exchange resin and Eudragit S100 to prepare the DXSP-loaded resin microcapsule (DXSP-DRM). The in vitro release study and in vivo study of pharmacokinetics and the intestinal drug residues in rat demonstrated the good colon-targeting of DXSP-DRM. Moreover, the DXSP-DRM can reduce the toxic side-effects induced by DXSP and have good therapeutic effects on colitis mouse induced by 2,4,6-trinitrobenzenesulfonic acid. DISCUSSION: Dexamethasone can be targeted to the colon by DRM, thereby enhancing its treatment effect and reducing its toxic side effects. CONCLUSION: The resin microcapsule system has good colon-targeting and can be used in the development of colon-targeted preparations.

Use of the guanidination reaction for determining reactive lysine, bioavailable lysine and gut endogenous lysine.

Determining the bioavailability of lysine in foods and feedstuffs is important since lysine is often the first limiting indispensable amino acid in diets for intensively farmed livestock (pigs and poultry) and also in many cereal-based diets consumed by humans. When foods or feedstuffs are heat processed, lysine can undergo Maillard reactions to produce nutritionally unavailable products. The guanidination reaction, the reaction of O-methylisourea with the side chain amino group of lysine that produces homoarginine, has been used to determine the unmodified lysine (reactive lysine) in processed foods and feedstuffs and also true ileal digestible reactive lysine (bioavailable lysine). The advantages of the guanidination method in comparison with other reactive lysine methods such as the fluorodinitrobenzene, trinitrobenzenesulphonic acid and dye-binding methods are that it is very specific for reactive lysine and also that the method is relatively straightforward to conduct. The specificity of the guanidination reaction for the lysine side chain amino group is particularly important, since ileal digesta will contain N-terminal groups in the form of free amino acids and peptides. The main disadvantage is that complete conversion of lysine to homoarginine is required, yet it is not straightforward to test for complete guanidination in processed foods and feedstuffs. Another disadvantage is that the guanidination reaction conditions may vary for different food types and sometimes within the same food type. Consequently, food-specific guanidination reaction conditions may be required and more work is needed to optimise the reaction conditions across different foods and feedstuffs.

In Vivo Evaluation of 5-ASA Colon-Specific Tablets Using Experimental-Induced Colitis Rat Animal Model.

Colonic drug delivery is intended not only for local treatment in inflammatory bowel disease (IBD) but also for systemic delivery of therapeutics. Intestinal myeloperoxidase (MPO) determination could be used to estimate the average level of inflammation in colon as well as to determine the efficacy of drugs to be used in the treatment of inflammatory bowel diseases or study the specificity of dosage forms to be used for colonic targeting of anti-inflammatory drugs. Colonic prodrug sulfasalazine (SASP) gets metabolized to give 5-aminosalicylic acid (5-ASA), which is the active portion of SASP. However, when given orally, 5-ASA is absorbed in upper part of gastrointestinal tract (GIT) and not made available in colon. In the present study, colon-targeted delivery of 5-ASA was achieved by formulating tablets with two natural polymers namely guar gum and pectin using compression coating method. Colonic specificity of 5-ASA tablets (prepared using guar gum and pectin as polymers) was evaluated in vitro using simulated fluids mimicking in vivo environment as well as in vivo method using chemically (2,4,6-trinitrobenzenesulfonic acid and acetic acid)-induced colitis rat model. Both colon-specific formulations of 5-ASA (guar gum and pectin) were observed to be more effective in reducing inflammation in chemically induced colitis rat models when compared to colon-specific prodrug sulfasalazine as well as conventional 5-ASA administered orally.

Visualized absorption of anti-atherosclerotic dipeptide, Trp-His, in Sprague-Dawley rats by LC-MS and MALDI-MS imaging analyses.

SCOPE: The basic dipeptide, Trp-His, was found to show an in vivo anti-atherosclerotic effect when orally administered to apo E-deficient mice. In addition, this dipeptide causes vasorelaxation in contracted rat aorta via suppression of intracellular Ca(2+) signaling cascades. In this study, we attempted to determine whether Trp-His can be absorbed after single oral administration in Sprague-Dawley (SD) rats. METHODS AND RESULTS: Trp-His and His-Trp (10 or 50 mg/kg) was orally administered to 8-week-old male SD rats. Both peptides in plasma were assayed by LC-MS/MS in combination with 2,4,6-trinitrobenzene sulfonate derivatization technique. In vitro transport experiments using Caco-2 cell monolayers were performed to evaluate the apparent permeability (Papp ). A phytic acid-aided MALDI-MS imaging (MSI) was conducted to visualize the distribution of dipeptides in the rat intestinal membrane. Trp-His was absorbed intact into SD rat blood, showing a maximal level at 1 h after administration at 10 mg/kg dose (Cmax , 28.7 ± 8.9 pmol/mL-plasma; area under the curve, 71.3 ± 18.7 pmol·h/mL-plasma). In contrast, His-Trp was surprisingly not detected, although the Papp was compatible to that of Trp-His. MSI analysis provided crucial evidence that Trp-His was visualized in the overall intestinal membrane. The Trp-His peptide was not visualized in the presence of Gly-Sar, which is a model peptide that is transported via the intestinal proton-coupled peptide transporter 1 (PepT1) transporter. The His-Trp molecular ion was not observed at the intestinal membrane. The MSI analysis illustrated that there is no absorption of His-Trp due to its unexpected hydrolysis by brush border proteases. CONCLUSION: To the best of our knowledge, this is the first study demonstrating that the vasoactive Trp-His is preferably transported across the rat intestinal membrane by PepT1 and is absorbed intact into the circulation. However, no absorption of His-Trp, a reverse sequence of absorbable Trp-His, is observed owing to hydrolysis by intestinal proteases. This suggests that the bioavailability of peptides may be determined in part by their protease resistance in the intestinal membrane.

Cysteine-functional polymers via thiol-ene conjugation.

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials.

In vivo evaluation of inflammatory bowel disease with the aid of µPET and the translocator protein 18 kDa radioligand [18F]DPA-714.

PURPOSE: The purpose of the study was to validate [18F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, as a probe to non-invasively quantify the inflammatory state in inflammatory bowel disease (IBD) animal models. PROCEDURES: Quantitative positron emission tomography (PET) imaging of intestinal inflammation was conducted with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) a glucose metabolism surrogate marker and [18F]DPA-714 a ligand of the 18 kDa TSPO, on two IBD models. The first model was induced using dextran sodium sulfate (DSS), creating global inflammation in the colon. The second model was induced by rectally administering trinitrobenzenesulfonic acid (TNBS), creating local and acute inflammation. RESULTS: The level of inflammation was analyzed using PET imaging on days 7 and 8. The analysis obtained with [18F]DPA-714, yielded a significant difference between the DSS treated (0.50 ± 0.17%ID/cc) and non-treated rats (0.35 ± 0.15%ID/cc). [18F]FDG on the other hand did not yield a significant difference. We did observe a mean glucose consumption in the colon increase from 0.40 ± 0.11 %ID/cc to 0.54 ± 0.17 %ID/cc. In the TNBS model, the uptake level of [18 F]DPA-714 increased significantly from 0.46 ± 0.23%ID/cc for the non-treated group, to 1.30 ± 0.62%ID/cc for those treated. PET signal was correlated with increased TSPO expression at cellular level. CONCLUSIONS: Results indicate that [18F]DPA-714 is suitable for studying inflammation in IBD models. [18F]DPA-714 could be a good molecular probe to non-invasively evaluate the level and localization of inflammation. Moreover, in vivo imaging using this TSPO ligand is potentially a powerful tool to stage and certainly to follow the evolution and therapeutic efficiency at molecular level within this disease family.

New method and characterization of self-assembled gelatin-oleic nanoparticles using a desolvation method via carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction.

In this study, we investigated a new method for the preparation of gelatin-oleic conjugate (GOC) as an amphiphilic biomaterial to load model anti-cancer drugs into self-assembled nanoparticles (NPs). Oleic acid (OA) was covalently bound to gelatin via carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction in water-ethanol cosolvent to form a GOC. Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance ((1)H NMR) clearly indicated the successful synthesis of GOC. The percentage of gelatin amino groups reacted with OA was up to 50% as determined using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method. Subsequently, gelatin-oleic nanoparticles (GONs) were prepared using a desolvation method with glutaraldehyde or genipin used as a crosslinker for comparison. Irinotecan hydrochloride (IRT) was used as a model drug to load into GONs using incubation or an in-process adding method for comparison. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) data showed that the sizes of GONs and IRT-loaded GONs (IRT-GONs) were below 250 nm. The zeta potentials of the GONs and irinotecan-loaded IRT-GONs were below -20 mV, which was found to be stable in suspension against the aggregation process. The incubation method was more suitable for drug loading because it did not affect the process of GON formation and thus did not increase their size much compared to the change in size with the in-process adding method. The lipophilic property of the oleic moiety in the GOC increased the affinity between GOC molecules, thus reducing the amount of crosslinking agents needed to stabilize GONs compared to gelatin nanoparticles (GNs). As novel approaches for the synthesis of protein-fatty acid complexes, chemical reaction has been suggested for the synthesis of GOC. The above results show that GOC synthesized via new method is a promising biomaterial based upon preparation of nanoparticles.

Comparison of noncontrast MRI magnetization transfer and T2 -Weighted signal intensity ratios for detection of bowel wall fibrosis in a Crohn's disease animal model.

PURPOSE: To compare the abilities of magnetization transfer magnetic resonance imaging (MT-MRI) and T2 -weighted signal intensity (T2 WSI) ratios to detect intestinal fibrosis in a Crohn's disease animal model. MATERIALS AND METHODS: Ten rats ("Group 1") received one trinitrobenzenesulfonic acid enema to induce acute colonic inflammation, while 10 additional animals ("Group 2") received multiple enemas to induce colonic inflammation and fibrosis. Gradient recalled-echo MT-MRI (5 and 10 kHz off-resonance) and T2 -weighted spin-echo imaging were performed 2 days after the last enema. MT ratios (MTR) and T2 WSI ratios were calculated in the area of greatest colonic thickening. Bowel wall MTR, bowel wall MTR normalized to paraspinous muscle MTR ("normalized MTR"), and T2 WSI ratios were compared between animal groups using Student's t-test. RESULTS: At 10 kHz off-resonance, mean bowel wall MTR for Group 1 was 24.8 ± 3.1% vs. 30.3 ± 3.2% for Group 2 (P = 0.001). Mean normalized MTR was 0.45 ± 0.05 for Group 1 and 0.58 ± 0.08 for Group 2 (P = 0.0003). At 5 kHz off-resonance, mean bowel wall MTR for Group 1 was 34.7 ± 5.2% vs. 40.3 ± 3.6% for Group 2 (P = 0.015). Mean normalized MTR was 0.53 ± 0.08 for Group 1 and 0.64 ± 0.07 for Group 2 (P = 0.003). Mean T2 WSI ratio was 5.32 ± 0.98 for Group 1 and 3.01 ± 0.66 for group 2 (P < 0.0001). Mean T2 WSI ratio/MTR (10 kHz off-resonance) was 12.06 ± 2.70 for Group 1 and 5.22 ± 1.29 for Group 2 (P < 0.0001), with an ROC c-statistic of 0.98. CONCLUSION: MTR and T2 WSI ratios detect bowel wall fibrosis in a Crohn's disease animal model.