Effect of fibronectin on the binding of antithrombin III to immobilized heparin

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. \ud Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. \ud The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin

Folate‐conjugated thermo‐responsive micelles for tumor targeting

Folate‐conjugated and thermo‐responsive poly(( N ‐isopropylacrylamide)‐ co ‐ acrylamide‐ co ‐(octadecyl acrylate)‐ co ‐(folate‐(polyethylene glycol)‐(acrylic acid))) (P(NIPA‐ co ‐AAm‐ co ‐ODA‐ co ‐FPA)) micelles with mean diameter of about 60 nm and lower critical solution temperature (LCST) of about 39°C were synthesized by free radical random copolymerization. Single‐factor tests of acrylamide and octadecyl acrylate were carried out to modulate micelles' LCST and diameter, respectively. LCST, diameter, and morphology of micelles were determined by UV–vis spectrophotometer, laser particle size analyzer, and transmittance electron microscope (TEM), respectively. Fluorescein was then used as a model drug to investigate the drug loading content of micelles. Micelles with maximum amount of octadecyl acrylate (180 mg) were found to yield drug loading content of 10.48%. Near infrared dye No.10 was chosen as the tracer to monitor micelles in vivo . The targeting behaviors of micelles in folate receptor positive Bel‐7402 tumor bearing nude mice were assessed by a self‐constructed near infrared imaging system. Results showed satisfactory targeting capability of the thermo‐responsive micelles toward Bel‐7402 tumors, and targeting accumulation could last for more than 96 h, enabling P(NIPA‐ co ‐AAm‐ co ‐ODA‐ co ‐FPA) micelles to function as a diagnostic reagent as well as a targeted tumor therapy. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3134–3142, 2012.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93660/1/34230_ftp.pd

Design of chimeric GLP-1A using oligomeric bile acids to utilize transporter-mediated endocytosis for oral delivery

Background Despite the effectiveness of glucagon-like peptide-1 agonist (GLP-1A) in the treatment of diabetes, its large molecular weight and high hydrophilicity result in poor cellular permeability, thus limiting its oral bioavailability. To address this, we developed a chimeric GLP-1A that targets transporter-mediated endocytosis to enhance cellular permeability to GLP-1A by utilizing the transporters available in the intestine, particularly the apical sodium-dependent bile acid transporter (ASBT). Methods In silico molecular docking and molecular dynamics simulations were used to investigate the binding interactions of mono-, bis-, and tetra-deoxycholic acid (DOCA) (monoDOCA, bisDOCA, and tetraDOCA) with ASBT. After synthesizing the chimeric GLP-1A-conjugated oligomeric DOCAs (mD-G1A, bD-G1A, and tD-G1A) using a maleimide reaction, in vitro cellular permeability and insulinotropic effects were assessed. Furthermore, in vivo oral absorption in rats and hypoglycemic effect on diabetic db/db mice model were evaluated. Results In silico results showed that tetraDOCA had the lowest interaction energy, indicating high binding affinity to ASBT. Insulinotropic effects of GLP-1A-conjugated oligomeric DOCAs were not different from those of GLP-1A-Cys or exenatide. Moreover, bD-G1A and tD-G1A exhibited improved in vitro Caco-2 cellular permeability and showed higher in vivo bioavailability (7.58% and 8.63%) after oral administration. Regarding hypoglycemic effects on db/db mice, tD-G1A (50 μg/kg) lowered the glucose level more than bD-G1A (50 μg/kg) compared with the control (35.5% vs. 26.4%). Conclusion GLP-1A was conjugated with oligomeric DOCAs, and the resulting chimeric compound showed the potential not only for glucagon-like peptide-1 receptor agonist activity but also for oral delivery. These findings suggest that oligomeric DOCAs can be used as effective carriers for oral delivery of GLP-1A, offering a promising solution for enhancing its oral bioavailability and improving diabetes treatment.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (grant nos. 2020R1A2C1102831, 2022R1A5A8033794, 2022R1A4A3034038). This study was also supported by Regional Innovation Strategy (RIS) through the NRF funded by the Ministry of Education (MOE) (2021RIS-001

PhD

dissertationHeparin is a heterogeneous polysaccharide the prolongs the clotting time of blood by preventing the fibrin formation. Previous research has demonstrated that heparin immobilized onto a polymer substrate using grafted hydrophilic spacer improved the blood compatibility in in vitro, ex vivo, and in vivo experiments. In this current research, the previous observations were confirmed and explained by studying the binding kinetics of immobilized heparin with antithrombin III (ATIII), thrombin, and fibronectin, both under static and flow conditions. Heparin, with its high affinity for ATIII and monodispersed molecular weight (mw 6,000), was bound to polyethylene oxide (PEO, mw 3,400) which was grafted onto poly(styrene-co-p-aminostyrene). Heparin was immobilized by 1:1 binding with the PEO spacer, resulting in 16.2 % bioactivity, compared to that of free heparin. For spacer-immobilized heparin, the dominant pathway in the anticoagulant processes involved spacer-immobilized heparin initially binding to ATIII, thereby accelerating the binding of thrombin to ATIII. Directly-immobilized heparin, however, bound only to thrombin. So, the mechanism of immobilized heparin was changed by spacer. The TAT complex was then dissociated from the spacer-immobilized heparin quickly, thereby recovering the bioactivity of heparin. The effect of fibronectin on the binding of immobilized heparin with ATIII depended upon the concentration of fibronectin in bulk. Fibronectin could be displaced by ATIII on the heparin binding site at the plasma concentration. To study flow effect on the reactions of immobilized heparin, the dissociation rate of TAT complex and the mechanism of protein binding to immobilized heparin were determined by comparing the theoretical data in numerical simulation with the parallel experimental data. The dissociation rate constant of the TAT complex from spacer-immobilized heparin was gradually increased with increasing flow rate, and the binding of thrombin to heparin-ATIII complex at the surface was controlled by diffusion. Spacer-immobilized heparin reacts with ATIII and thrombin via the main pathway in low flow rates as mentioned above. Therefore, spacer-immobilized heparin surfaces can work at low flow rates as well as high flow rates

A supramolecular host-guest interaction-mediated injectable hydrogel system with enhanced stability and sustained protein release

Injectable hydrogels have been studied as drug delivery systems because of their minimal invasiveness and sustained drug release properties. Pluronic F127, consisting of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers, exhibits thermo-responsive properties and hence is injectable due to its rapid sol-gel transition. Unmodified Pluronic F127-based hydrogels, however, have limited long-term stability and controllable release of drugs entrapped within them. In this study, host-guest interactions between adamantane-conjugated Pluronic F127 (F127-Ad) and polymerized beta-cyclodextrin (CDP) were employed to develop a hydrogel-based protein delivery system. Single or multiple adamantane units were successfully introduced at the termini of Pluronic F127 with a 100% conversion yield, and the synthesized F127-Ad polymer produced a physically crosslinked micelle-packing structure when mixed with CDP. As the number of adamantanes at the terminal ends of Pluronic F127 increased, the critical gelation concentration of F127-Ad/CDP hydrogel decreased from 15 to 6% (w/v). The F127/CDP hydrogel was able to maintain its structure even with lower polymer content, and its injectability improved with a reduction of the hydrogel viscosity. The long-term stability of F127/CDP hydrogels was evaluated in vitro and in vivo, and it was demonstrated that the subcutaneously injected hydrogel did not disintegrate for up to 30 d. Throughout the drug release test using gelatin and insulin as model drugs, it was demonstrated that their release rates could be regulated via complexation between the protein drugs and the beta-cyclodextrin molecules inside the hydrogel. In conclusion, the F127-Ad/CDP hydrogel is expected to be a versatile protein delivery system with controllable durability and drug release characteristics. Statement of significance Pluronic F127 is one of the widely studied polymeric materials for thermo-sensitive injectable hydrogels due to its high biocompatibility and rapid sol-gel transition. Since the Pluronic F127-based hydrogel has some limitations in its long-term stability and mechanical property, it is inevitable to modify its structure for the application to drug delivery. In this study, mono-or multi-adamantane-conjugated Pluronic F127s were synthesized and mixed with beta-cyclodextrin polymers to form hydrogels with host-guest interaction-mediated micelle-packing structures. The host-guest interaction introduced into the hydrogel system endowed it a sustained protein drug release behavior as well as high durability in vitro and in vivo. By increasing the number of adamantane molecules at the end of the Pluronic F127, both the stability and injectability of the hydrogel could be also modulated. (C) 2021 Published by Elsevier Ltd on behalf of Acta Materialia Inc.N