Stability Of Conjugates Research Articles

Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

Chitosan‐hydroxycinnamic acid conjugates: Synthesis, photostability and phytotoxicity to seed germination of barley

AbstractChitosan‐caffeic and chitosan‐ferulic acid conjugates are synthesized by carbodiimide method and characterized by UV, FTIR, and NMR spectroscopy. There are two obtained forms of conjugates: freshly synthesized and reconstituted freeze‐dried. A comparative study of the stability of various conjugate forms over time and under UV irradiation has been carried out. It has been shown that the absorption intensity of an aqueous solution of the freshly synthesized chitosan‐caffeic acid conjugate decrease in ~2 times during 20 weeks of storage, while the maximum rate of intensity decrease is observed during the first 40 days. The absorption intensity of the chitosan‐ferulic acid conjugate decrease by 6–7% during the entire storage period. It has been found that freeze‐dried powders of chitosan‐caffeic and chitosan‐ferulic acid conjugates are stable for at least 1 year. It has been established that freeze‐dried chitosan‐caffeic and chitosan‐ferulic acid conjugates are more stable under UV irradiation (in 4–8 and 1.4–1.9 times, respectively) compared to freshly synthesized ones. A comparative study of the influence of conjugates on barley seedlings (Hordeum vulgare L.) growth has been carried out.

The essential synchronization backbone problem.

Network optimization strategies for the process of synchronization have generally focused on the re-wiring or re-weighting of links in order to (1) expand the range of coupling strengths that achieve synchronization, (2) expand the basin of attraction for the synchronization manifold, or (3) lower the average time to synchronization. A new optimization goal is proposed in seeking the minimum subset of the edge set of the original network that enables the same essential ability to synchronize in that the synchronization manifolds have conjugate stability. We call this type of minimal spanning subgraph an essential synchronization backbone of the original system, and we present two algorithms: one is a strategy for an exhaustive search for a true solution, while the other is a method of approximation for this combinatorial problem. The solution spaces that result from different choices of dynamical systems and coupling schemes vary with the level of a hierarchical structure present and also the number of interwoven central cycles. Applications can include the important problem in civil engineering of power grid hardening, where new link creation may be costly, and the defense of certain key links to the functional process may be prioritized.

Abstract 1873: GlycoConnect™ ADCs based on topoisomerase 1 inhibitor exatecan (SYNtecan E™) show excellent in vivo efficacy and tolerability

Abstract Camptothecins form a powerful class of chemotherapy drugs (e.g. irinotecan, topotecan, belotecan), based on their ability to inhibit topoisomerase I. Some camptothecins, such as DXd and SN-38, have also proven powerful payloads for antibody-drug conjugates (ADCs), as exemplified by the recent market approval of Enhertu® and Trodelvy®, respectively. We have reported earlier that the native glycan of monoclonal antibodies is a priviliged conjugation site for ADCs (GlycoConnect™)1, while the highly polar spacer technology (HydraSpace™)2 enables the conjugation of any cytotoxic payload, providing ADCs with significantly expanded therapeutic index (TI) versus mainstream ADC technologies. We here show that exatecan (DX-8951f), a clinically validated and potent campothecin, is readily combined with HydraSpace™ technology to provide linker-payload (SYNtecan E™). Homogeneous and stable GlycoConnect™ADCs were generated from trastuzumab and SYNtecan E™ and compared head-to-head with DXd or SN-38payload, as well as deruxtecan, the linker-payload applied in Enhertu®. Complete tumor regression was observed in a mouse xenograft study (BT-474) after a single dose, while safety studies in mouse corroborated the high tolerability of SYNtecan E™ ADCs. Taken together, these studies demonstrate the potential of SYNtecan E™ for application in stable and site-specific ADCs with excellent therapeutic index. 1 van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of NativemAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. 2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Floris van Delft. GlycoConnect™ ADCs based on topoisomerase 1 inhibitor exatecan (SYNtecan E™) show excellent in vivo efficacy and tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1873.

Site-Specific Quantitation of Drug Conjugations on Antibody-Drug Conjugates (ADCs) Using a Protease-Assisted Drug Deconjugation and Linker-like Labeling (PADDLL) Method.

Antibody-drug conjugates (ADCs) are biopharmaceuticals for the targeted delivery of antitumor agents. ADCs can be highly heterogeneous with various drug-to-antibody ratio (DAR) species, conjugation sites, and occupancy levels. The conjugation site can modulate the ADC stability and efficacy and therefore can be considered to be a critical quality attribute (CQA) during development. Traditional mass spectrometry (MS)-based peptide mapping methods cannot accurately quantify site-specific conjugations due to a significant ionization discrepancy between unconjugated native peptides and conjugated peptides. Here, we developed a novel protease-assisted drug deconjugation and linker-like labeling (PADDLL) method to quantify the levels of linker payload at specific conjugation sites. We utilized optimized papain digestion to deconjugate the drug payload and labeled unoccupied conjugation sites with a linker-like structure to provide comparable ionization efficiency for MS-based quantitation. This method was successfully applied on two cysteine-linked, protease-cleavable ADCs, and the method demonstrated good linearity and reliability, reaching a limit of quantitation of below 1%. The calculated DARs were comparable with the results from intact mass analysis. The lot-to-lot variation in conjugation distribution and inferior conjugation stability at HC Cys225 to other interchain cysteines were observed. This method provides a valuable tool for ADC design and product development. To the best of our knowledge, this is the first analytical method developed to accurately quantify site-specific linker-drug payload conjugations for ADCs.

Trph-222, a Novel Anti-CD22 Antibody Drug Conjugate (ADC), Has Signficant Anti-Tumor Activity in NHL Xenografts and Is Well Tolerated in Non-Human Primates

CD22 is a B-cell specific transmembrane glycoprotein belonging to the SIGLEC family of lectins. Its expression is restricted to B lineage cells from the early immature/pre-B-cell stage through the mature naïve stage, and is downregulated on the surface of plasma cells. CD22 is highly expressed on a wide range of malignancies arising from mature B-cells, including NHL (non-Hodgkin's lymphoma), DLBCL (diffuse large B cell lymphoma), MCL (mantle cell lymphoma), MZL, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and small lymphocytic leukemia. The restricted lineage of CD22 and rapid internalization upon antibody binding make CD22 an appealing target for an antibody-drug conjugate (ADC) therapeutic for B cell malignancies. Prior attempts at targeting CD22 using ADC technology have been limited clinically in NHL due to an unacceptable or poor therapeutic index. Subsequently the ADC field has focused on more stable and site specific conjugation of payloads to increase the therapeutic index of ADCs.TRPH-222 is an ADC comprised of an anti-CD22 antibody site-specifically modified at one site in each heavy chain to express formylglycine (FG), allowing for site-specific conjugation of a maytansinoid payload, a protease-insensitive spacer, and a functional group for coupling to an aldehyde on the antibody FG residues. This SMARTAG™ site specific non-cleavable conjugation enforces a maximum drug to antibody ratio (DAR) of 2.0.The aim of this study was to characterize the anti-tumor activity of TRPH-222 in mouse xenografts and tolerability in non-human primates (NHP).In the WSU-DLCL2 xenograft model, once weekly intravenous (IV) dosing of 1, 3 or 10mg/kg or TRPH-222 reduced tumor volume by 51%, 100% and 100% after 28 days. A once every three-week dose of 10mg/kg reduced tumor volume by 100% from pre-dosing measurements. In contrast, rituximab (30mg/kg, weekly) resulted in 55% tumor growth inhibition. In both SU-DHL-2 (DLBCL) and Granta-519 (MCL) xenograft models, weekly dosing at 10mg/kg resulted in tumor stasis.The tolerability and PK of TRPH-222 were evaluated in cynomolgus monkeys. Animals were dosed IV once every 3 weeks (the anticipated clinical dosing schedule) for either 2 (5, 12.5, or 25 mg/kg) or 3 (50 mg/kg) doses. TRPH-222 was well tolerated at all doses evaluated and all animals survived to study termination, with no TRPH-222-related effects on clinical observations or body weight. Aspartate aminotransferase (AST) activity was mildly increased in animals at ≥12.5 mg/kg/dose, while minimal increases in alanine aminotransferase (ALT) activity were noted at 50 mg/kg/dose. Minor microscopic findings of Kupffer cell hypertrophy/hyperplasia were noted in the livers of monkeys treated at 50 mg/kg IV; this finding was not considered adverse based on minimal severity and lack of degeneration/necrosis. Preliminary PK data demonstrate the stability of TRPH-222, as the ratio of the area under the curve (AUC) from day 0-42 for the ADC was ~76-88% that of the antibody backbone suggesting that the ADC has a long half-life and that the linker is stable in vivo.In conclusion, the studies reported here demonstrate that TRPH-222 has significant anti-tumor activity in a range of B-cell lymphoma models and that very high doses of TRPH-222 are well tolerated in monkeys. IND-enabling safety studies are ongoing to support clinical evaluation of TRPH-222 in 2018. These data support the argument that TRPH-222 will have a large therapeutic window due to the stability of conjugation of the maytansinoid payload to the anti-CD22 antibody; doses tolerated in the monkey study described above correspond to human equivalent doses significantly higher than for most ADCs approved or currently under clinical evaluation. To our knowledge, this is the highest dose of a maytansine containing ADC that has been tolerated in monkeys and raises the potential for a best in class therapy for NHL in addition to a best in class ADC technology. DisclosuresMaclaren:Triphase Accelerator: Employment. Levin:Triphase Accelerator: Employment. Lowman:Triphase Accelerator: Employment. Trikha:Triphase Accelerator: Employment; Encycle Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Diethynyl Phosphinates for Cysteine-Selective Protein Labeling and Disulfide Rebridging.

Diethynyl phosphinates were developed as bisfunctional electrophiles for the site‐selective modification of peptides, proteins and antibodies. One of their electron‐deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra‐ or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery. Finally, this reagent class was used to generate functional homogeneously rebridged antibodies that remain specific for their target. Their modular synthesis, thiol selectivity and conjugate stability make diethynyl phosphinates ideal candidates for protein conjugation for biological and pharmaceutical applications.

Structural properties of native and conjugated black soldier fly (Hermetia illucens) larvae protein via Maillard reaction and classification by SIMCA

Black soldier fly (Hermetia illucens) has received considerable interest as an alternative protein source. Aqueous solutions of black soldier fly larvae (BSFL) protein and glucose (2:1 w.w−1, pH 9) were heated at 50, 70 and 90 °C, for 2–10 h at 2 h intervals, respectively. The zeta-potential (ζ) of BSFL-Glu conjugates heat-treated at 70 °C ranged from -10.25 to -25.25 mV while the native BSFL protein ranged from -12.84 to -16.70 mV. The ζ-potential analysis revealed that the glycation reaction modified the surface charge density of the BSFL protein as a function of reaction time and temperature. In addition, an increase in thermal stability of the BSFL-Glu conjugates was observed by means of Thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the most apparent structural changes in the BSFL protein were in the amide I and amide II region. Well-separated clusters permitting differentiation between native BSFL and BSFL-Glu conjugates were observed by using principal component analysis (PCA) on FT-IR spectra. At 50, 70 and 90 °C the first two principal components (PC1 and PC2) showed an accumulated total variance of 91, 96 and 95%, respectively. A classification efficiency of 91% was obtained when using soft independent modelling of class analogy (SIMCA). Infrared spectroscopy combined with SIMCA is a powerful tool to monitor the formation of edible insect protein–sugar conjugates by Maillard reaction. As a result, combining FT-IR spectroscopy with multivariate techniques (PCA and SIMCA) exhibited a strong potential to differentiate between native and glycated protein samples from black soldier fly larvae.

Tailored Mobility in a Zeolite Imidazolate Framework (ZIF) Antibody Conjugate*.

Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral‐flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme‐linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC‐MS/MS provided evidence that a covalent link was established between these metal‐organic frameworks and lysine residues in IgG antibodies.

BSA-drug-ZnO-PEI conjugates interaction with glycans of gp60 endothelial cell receptor protein for targeted drug delivery: a comprehensive spectroscopic study

The zinc oxide (ZnO) nanoparticles (NPs) have several biomedical applications such as drug delivery, bio-imaging, and biomedical research. ZnO NPs were remedied with polyethyleneimine (PEI) and modified with bovine serum albumin (BSA). Two anticancer drugs - Cisplatin (CIS) and Gemcitabine (GEM) were used in conjugation with BSA. BSA-ZnO-PEI (conjugate 1), BSA-CIS-ZnO-PEI (conjugate 2), and BSA-GEM-ZnO-PEI (conjugate 3) can be used for targeted drug delivery via glycans - N-acetylneuraminic acid (NANA), L-fucose (FUC), N-acetyl glucosamine (NAG), D-mannose (MAN), and D-galactose (GAL), of albumin binding membrane receptor protein (gp60). Considerable interaction and the strong binding of conjugate 2 and conjugate 3 with NANA were observed by UV-visible absorption and fluorescence spectra. The electrostatic stability of conjugate 2 and conjugate 3 with NANA was considerably increased in comparison to conjugate 1 as evident with zeta potential values. The fluorescence quenching data (Ksv and kq) and binding parameters (K and n) of BSA-CIS, BSA-GEM, conjugate 2, and conjugate 3 with NANA and FUC attributes to the strong binding. Amide I and amide III bands of the Raman signal suggested insignificant loss in alpha-helical and beta-sheet content of conjugate 2 and conjugate 3 with NANA and FUC. Therefore, the present study is going to assist in the comprehensive development of conjugates for targeted drug delivery based on the differential glycation pattern of gp60 protein. Communicated by Ramaswamy H. Sarma

Stability and bioaccessibility improvement of capsorubin using bovine serum albumin-dextran-gallic acid and sodium alginate

This study attempted to prepare ternary conjugate emulsion from bovine serum albumin (BSA), dextran (DEX) and gallic acid (GA) to improve the stability of conjugate emulsion and the bioaccessibility of capsorubin. The release of capsorubin was further delayed by sodium alginate capsules in the intestinal phase. First, protein formed new functional groups and covalent bonds was analyzed by Fourier transform infrared (FTIR) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the stability of the ternary conjugate showed distinct pH correlation and the higher stability near the isoelectric point. Finally, the bioaccessibility of capsorubin embedded in sodium alginate emulsion was higher than that of ternary conjugate emulsion (65% and 34%).

Synthesis and characterization of Chitosan-Catechol conjugates: Development and in vitro, in silico and in vivo evaluation of mucoadhesive pellets of lafutidine

Present study was aimed to synthesize and characterize Chitosan-Catechol conjugates and to design and develop mucoadhesive pellets loaded with lafutidine. SEM images indicated the presence of fibrous structures responsible for enhanced mucoadhesive potential of Chitosan-Catechol conjugates. Thermodynamic stability and amorphous nature of conjugates was confirmed by DSC and XRD studies respectively. Rheological studies were used to evaluate polymer mucin interactions wherein strong interactions between Chitosan-Catechol conjugate and mucin was observed in comparison to pristine chitosan and mucin. The mucoadhesion potential of Chitosan-Catechol (Cht-C) versus Chitosan (Cht) was assessed in silico using molecular mechanics simulations and the results obtained were compared with the in vitro and ex vivo results. Cht-C/mucin demonstrated much higher energy stabilization (∆E ≈ −65 kcal/mol) as compared to Cht/mucin molecular complex. Lafutidine-loaded pellets were prepared from Chitosan (LPC) and Chitosan-Catechol conjugates (LPCC) and were evaluated for various physical properties viz. flow, circularity, roundness, friability, drug content, particle size and percent mucoadhesion. In vitro drug release studies on LPC and LPCC pellets were performed for computing t50%, t90% and mean dissolution time. The values of release exponent from Korsmeyer-Peppas model was reported to be 0.443 and 0.759 for LPC and LPCC pellets suggesting Fickian and non-Fickian mechanism representing drug release, respectively. In vivo results depicted significant controlled release and enhanced residence of the drug after being released from the chitosan-catechol coated pellets. Chitosan-Catechol conjugates were found to be a promising biooadhesive polymer for the development of various mucoadhesive formulations.

Innovative Bioconjugation Technology for Antibody-Drug Conjugates: Proof of Concept in a CD30-Positive Lymphoma Mouse Model.

To overcome stability and heterogeneity issues of antibody-drug conjugates (ADCs) produced with existing bioconjugation technologies incorporating a maleimide motif, we developed McSAF Inside, a new technology based on a trifunctionalized di(bromomethyl)pyridine scaffold. Our solution allows the conjugation of a linker-payload to previously reduced interchain cysteines of a native antibody, resulting in disulfide rebridging. This leads to highly stable and homogeneous ADCs with control over the drug-to-antibody ratio (DAR) and the linker-payload position. Using our technology, we synthesized an ADC, MF-BTX-MMAE, built from anti-CD30 antibody cAC10 (brentuximab), and compared it to Adcetris, the first line treatment against CD30-positive lymphoma, in a CD30-positive lymphoma model. MF-BTX-MMAE displayed improved DAR homogeneity, with a solid batch-to-batch reproducibility, as well as enhanced stability in thermal stress conditions or in the presence of a free thiol-containing protein, such as human serum albumin (HSA). MF-BTX-MMAE showed antigen-binding, in vitro cytotoxicity, in vivo efficacy, and tolerability similar to Adcetris. Therefore, in accordance with current regulatory expectations for the development of new ADCs, McSAF Inside technology gives access to relevant ADCs with improved characteristics and stability.

Enhanced heat stability and antioxidant activity of myofibrillar protein-dextran conjugate by the covalent adduction of polyphenols

In the present study, three types of polyphenols, namely, (−)-epigallocatechin-3-gallate (EGCG), catechin (C), and gallic acid (GA), were grafted to myofibrillar protein (MP)-dextran (DX) conjugate through a free radical-mediated adduction method. The analysis of secondary structure showed that conjugation of polyphenols induced a decrease in contents of α-helix structures. The surface hydrophobicity of MP-DX conjugate was increased after polyphenols were covalently adducted, while that of the free amino, thiol groups, and tyrosine residues were decreased, especially with the addition of EGCG (p < 0.05). Analysis of rheological properties showed that covalently linking of polyphenols decreased the thermal gelling capacity by inhibiting myosin-head aggregation and myosin tails interaction. Moreover, polyphenol adduction was able to remarkably improve the thermal stability and antioxidant activity of MP-DX conjugate. The findings regarding enhanced functionalities evidence potential of applying the ternary adduct as a novel antioxidant.

An All-in-One Tannic Acid-Containing Hydrogel Adhesive with High Toughness, Notch Insensitivity, Self-Healability, Tailorable Topography, and Strong, Instant, and On-Demand Underwater Adhesion.

Hydrogels that are mechanically tough and capable of strong underwater adhesion can lead to a paradigm shift in the design of adhesives for a variety of biomedical applications. We hereby innovatively develop a facile but efficient strategy to prepare hydrogel adhesives with strong and instant underwater adhesion, on-demand detachment, high toughness, notch-insensitivity, self-healability, low swelling index, and tailorable surface topography. Specifically, a polymerization lyophilization conjugation fabrication method was proposed to introduce tannic acid (TA) into the covalent network consisting of polyethylene glycol diacrylate (PEGDA) of substantially high molecular weight. The presence of TA facilitated wet adhesion to various substrates by forming collectively strong noncovalent bonds and offering hydrophobicity to allow water repellence and also provided a reversible cross-link within the binary network to improve the mechanical performance of the gels. The long-chain PEGDA enhanced the efficacy and stability of TA conjugation and contributed to gel mechanics and adhesion by allowing chain diffusion and entanglement formation. Moreover, PEGDA/TA hydrogels were demonstrated to be biocompatible and capable of accelerating wound healing in a skin wound animal model as compared to commercial tissue adhesives and can be applied for the treatment of both epidermal and intracorporeal wounds. Our study provides new, critical insight into the design principle of all-in-one hydrogels with outstanding mechanical and adhesive properties and can potentially enhance the efficacy of hydrogel adhesives for wound healing.

SYNTHESIS AND PROPERTIES OF NANOCELLULOSE-DYE CONJUGATES

Synthesis of conjugates of cellulose nanocrystals (CNC) with chlortriazine reactive dyes (RD) has been developed. The obtained CNC-RD conjugates were characterized using a complex of modern methods: UV-Vis, IR and 13C solid state NMR spectroscopy, thermogravimetric and elemental analysis, particle size and zeta potential analysis, scanning electron and polarization optical microscopy. IR spectra of CNC-RD films show absorption bands typical of cellulose. The formation of a covalent cellulose-dye bond is confirmed by the presence of characteristic absorption bands of the resulting ether bond in the region of 1000-1300 cm-1. Based on the analysis of solid-state 13C NMR spectra, it was concluded that the degree of crystallinity of the CNC-RD conjugates does not change compared to the CNC, i.e. the RD covalently bind to the surface of the CNC particles and do not affect the crystal structure of the CNC. Using a simple model of CNC with square cross-section, and based on the elemental analysis data, the amount of covalently bound dye on the surface of CNC particles was estimated. The results obtained allow us to conclude that covalent bound of RD occurs selectively at one of the ends of the cellulose nanocrystal (the so-called reducing end of the cellulose polymer chain). Successful CNC surface modification is confirmed by UV-Vis spectroscopy. It was found that aqueous suspensions of CNC-RD conjugates have indicator properties. The sizes and charges of the particles of CNC-RD conjugates in dilute aqueous suspensions were determined. High colloidal stability of the CNC-RD aqueous suspensions was confirmed. It has been shown that CNC-RD colloidal suspensions possess liquid crystalline properties, which is clearly demonstrated by their iridescent color in polarized light with stirring. CNC films modified with RD also possess birefringence properties and exhibit an iridescent color in polarized light. The morphology of CNC-RD particles was studied using scanning electron microscopy. The thermal stability of the CNC-RD conjugates was studied.

Highly efficient synthesis and characterization of starch aldehyde-catechin conjugate with potent antioxidant activity

In this study, cassava starch aldehyde was functionalized with catechin through acid catalyzed condensation reaction. The structural characterization, stability and antioxidant activity of starch aldehyde-catechin conjugates were investigated. Thin layer chromatography revealed the conjugates did not contain free catechin. UV–vis spectra of the conjugates exhibited an absorption band at 280 nm, attributing to the B-ring of catechin moiety. Fourier-transform infrared and proton nuclear magnetic resonance spectroscopy demonstrated the conjugation occurred between the H–6/H–8 of catechin A-ring and the aldehyde groups of starch aldehyde. X-ray diffraction pattern indicated that the conjugates had an amorphous structure. Scanning electron microscopy showed the conjugates were fragmentary slices with rough surfaces. Notably, the conjugates were more stable than catechin in phosphate buffered saline (pH 7.4). In addition, the conjugates could not be digested in simulated saliva, gastric and small intestinal juices. The reducing power and free radical scavenging activity of starch aldehyde were remarkably elevated by conjugating with catechin. Meanwhile, the conjugates were non-cytotoxic to RAW264.7 mouse macrophage cells and possessed higher resistant starch contents than starch. Our results suggest starch aldehyde-catechin conjugates can be used as antioxidants in food industry.

Влияние конструкции сопряжения водобойной части сооружения с нижним бьефом на кинематику водного потока

Purpose: to determine the impact of the conjugation construction of the apron section structure with the downstream on the water flow kinematics with the use of the performed field and laboratory studies data and their processing. The relevance of this issue lies in the fact that in case of unsatisfactory conjugation of the apron with the discharge canal slopes, negative phenomena are formed in it in the form of faulty currents, whirlpools at the canal slopes, etc., affecting the stability of the apron conjugation, creating a threat to the stability of the structure itself. Materials and Methods: the materials were the data of field studies carried out on a number of hydraulic structures of irrigation systems in Stavropol Territory, as well as on models in laboratory conditions with the identification of the impact of conjugation structures of the apron with the discharge canal slopes on the water flow distribution in tailrace. The measurement of velocities in natural conditions was carried out by observing the integrating floats movement in the discharge canals. On the model, the velocities were measured using a miniflowmeter, and the trajectories were traced using luminous floats. Results: graphs of the dependence of the sought factors on the known parameters were built on the basis of the information obtained, and using the methods of mathematical statistics, the coefficient of the conjugation of the apron structure with the canal slopes in tailrace was obtained. The result of the research was an empirical dependence for determining the flow velocity along its dynamic axis, which is the scientific novelty of the research performed. Conclusions: the type of conjugation of the apron of the structure with the distribution canal slopes has a great influence on the hydraulic flow regime on the apron and in the discharge canal. Each type of conjugation creates a sudden expansion of the flow on the apron, which affects the value of the average flow velocity along the dynamic axis, on which the deformation of the discharge canal depends.

Sonochemical effects on formation and emulsifying properties of zein-gum Arabic complexes

Zein is a widely used plant protein, but its application is limited by its poor water solubility under neutral conditions. In this work, we present an approach to develop a water-soluble zein-gum arabic (GA) complex and to explore its application in stabilizing lemon oil-in-water emulsions. As a simple, inexpensive and rapid method, sonication was used to accelerate the glycation reaction between zein and GA. The grafting degree of zein and GA was highest when the sonication power was 400 W and the treatment time was 25 min. SDS-PAGE and 1H NMR confirmed the formation of covalent bonds between zein and GA. The water-dispersibility, antioxidant activity, and thermal stability of zein-GA conjugates were better than those of native zein. The conjugates were used to form stable high internal phase emulsions (HIPEs) containing 80% v/v oil, which had relatively small mean droplet diameter (<10 μm). The HIPEs were semi-solids that exhibited strong shear-thinning behavior. After encapsulation in the HIPEs, the chemical degradation of lemon oil when exposed to UV-light and heat was significantly reduced. This study shows that the functional performance of zein can be extended by covalently linking it to GA using a combination of sonication and heat.

(Invited) Magnetic Relaxation Nanoparticle Biosensor for Dosimetric, Longitudinal Biochemical Monitoring In Vivo

Introduction: Harnessing the diversity of sensing platforms for applications in complex environments has been an ongoing challenge in biosensing. Most techniques exhibit limitations ranging from biocompatibility, sensitivity, and signal transduction through opaque media, to longevity under non-laboratory conditions. Through multidisciplinary advances in nanoscience and medical devices, we have developed an implant for the measurement of biomarkers using a magnetic particle assay platform1,2. Exposure to target induces a switch from a dispersed to an assembled state with a corresponding change in magnetic relaxation properties allowing for robust contrast. Current applications focus on diagnostics in chronic cardiac disease and spikes in biomarker presence frequently missed by serial sampling. We address design parameters for sensing functionality, tunability, and degradation to lay the groundwork for multi-month implantation in vivo. Materials and Methods: Particle core materials provided magnetic contrast while biological functionality was tuned by surface ligands for affinity toward multivalent targets (Figure 1a). Polyclonal antibodies were conjugated to superparamagnetic iron oxide nanoparticles (20-50 nm) by maleimide-thiol chemistry. Proton Magnetic Relaxation measurements were acquired on a custom-made, single-sided, inhomogeneous field relaxometer (0.43 Tesla, 25°C, NMR MOUSE) fitted with a programmable robotic scanning stage3. A Carr-Purcell-Meiboom-Gill pulse sequence was used: TE = 0.035 ms, 5454 echoes, 15 scans, TR = 6 s. Relaxation times were determined by single exponential fit to echo peak intensities with a custom MATLAB script. Delrin diffusion devices were filled with particle colloidal suspension and sealed with 30 nm pore polycarbonate semi-permeable membranes via double-sided, pressure-sensitive adhesive (Figure 1b). Devices were exposed to target while sensor response was read every 90-120 minutes for up to 48 hrs. Degradation was measured by quantitating loss of ligand from the surface (ELISA) and changes in sensor performance after elevated temperature storage (37°C) for up to 12 weeks. Results and Discussion: Ligand valency was tuned over an order of magnitude (4-40 Antibodies/Particle) by varying bioconjugation strategies and reagents. Conjugation stability under standard storage conditions indicated a lifetime >5 months by isolating free from bound protein by centrifugation. A maximum 1% of conjugated ligand was released, minimally decreasing valency and sensor functionality. Assay performance showed prozone agglutination behavior with quantitative exposure ranging from 50-2000 ng/mL of target and contrast in <2 hrs. Device dosimeter performance was validated for a cardiac Myoglobin model. Saturation was tuned from 4 to 12 μg/mL*hrs corresponding with 8 to >24 hrs of continuous biomarker elevation at 0-500 ng/mL. 4 hr equilibration and irreversible dosimeter signal stability post-exposure for >12 hrs was also observed (Figure 1c). Biochemical and aggregation performance degradation parameters were evaluated. Over 6.5 weeks of elevated temperature storage, 1.8% of the conjugated antibody was unbound. Sensor longitudinal performance on exposure to target over 12 weeks of incubation showed a monotonically decreasing response magnitude and rate (Figure 1d). Exponential fit to sensor response decay curve predicts a 29 week “switch” (on/off) sensor lifetime for an infarction model (500 ng/mL Myo). These results indicate our sensor can be derivatized for a cardiac biomarker with usable sensitivity and saturation dosimeter response corresponding to elevations expected in a Myocardial Infarction (MI). Device performance can be tuned by matching sensor characteristics with the physiological range of target sensitivity and saturation desired. By tuning bioconjugation strategies we enhanced the performance of the biosensor by an order of magnitude over prior studies. Sensor lifetime and nanostructure degradation studies have shown the limiting performance factor is not the loss of ligand from the surface of the particles but rather the loss of binding activity as seen in elevated temperature experiments. Conclusions: This implant has the potential to broaden diagnostics in personalized medicine by addressing the hurdles of longevity and robust sensor signal stability in complex environments. In situ diagnostics offer continuous sentineling of critical biomarkers, providing a deeper understanding of local biology in dynamic, heterogeneous systems. This monitoring provides valuable timescale data to clinicians with insight into their patients’ lab results for early intervention and data collection throughout treatment. This implant has the capacity to bridge the gap between lab and clinical grade sensors, significantly improving interventional medicine by leveraging the robust and tunable nature of magnetic relaxation particle assays.