In Situ‐Forming Cross‐linking Hydrogel Systems: Chemistry and Biomedical Applications

With the development of chemical synthetic strategies and available building blocks, in situ‐forming hydrogels have attracted significant attention in the biomedical fields over the past decade. Due to their distinct properties of easy management and minimal invasiveness via simple aqueous injections at target sites, in situ‐forming hydrogels have found a broad spectrum of biomedical applications including tissue engineering, drug delivery, gene delivery, 3D bioprinting, wound healing, antimicrobial research, and cancer research. The objective of this chapter is to provide a comprehensive review of updated research methods in chemical synthesis of in situ‐forming cross‐linking hydrogel systems and their diverse applications in the biomedical fields. This chapter concludes with perspectives on the future development of in situ‐forming hydrogels to facilitate this multidisciplinary field

Further characterization of the binding of heparin to granulocyte colony-stimulating factor: Importance of sulfate groups

Heparin mediates fundamental biological mechanisms through interaction with proteins. Previously, we have shown that standard heparin binds to granulocyte colony-stimulating factor (G-CSF) with an affinity of 4.8 x 10(5) M-1. To further study the structural features in heparin that are responsible for this interaction, we studied the bindings of G-CSF and N-desulfated and 2,3-O-desulfated heparin by CZE. Results showed that the N-desulfated heparin had a similar affinity for G-CSF ((5.4 +/- 0.9) x 10(5) M-1), but the 2,3-O-desulfated heparin had a 1000-fold lower affinity ((3.4 +/- 1.2) x 10(2) M-1) in comparison to standard heparin. The results showed that 2,3-O-sulfate groups are more important than N-sulfate groups in heparin-G-CSF interaction

Studying drug-plasma protein interactions by two-injector microchip electrophoresis frontal analysis

We developed a simple, rapid, and sensitive two-injector microchip electrophoresis frontal analysis (MCE-FA) method for studying drug-plasma protein interactions. In this method, large volumes of a reference sample and drug-plasma protein mixture were simultaneously introduced into the respective sections of the microchannel through the separated injectors and then electrophoresed. Since the reference sample did not meet with the interacting species during migration, it could be used as an external standard. The interaction between heparin and HSA was quantitatively characterized as a model system. The binding constant was found to be (1.53 +/- 0.01) X 10(4) M-1

Sensing Active Heparin by Counting Aggregated Quantum Dots at Single-Particle Level

Developing highly sensitive and highly selective assays for monitoring heparin levels in blood is required during and after surgery. In previous studies, electrostatic interactions are exploited to recognize heparin and changes in light signal intensity are used to sense heparin. In the present study, we developed a quantum dot (QD) aggregation-based detection strategy to quantify heparin. When cationic micelles and fluorescence QDs modified with anti-thrombin III (AT III) are added into heparin sample solution, the AT III-QDs, which specifically bind with heparin, aggregate around the micelles. The aggregated QDs are recorded by spectral imaging fluorescence microscopy and differentiated from single QDs based on the asynchronous process of blue shift and photobleaching. The ratio of aggregated QD spots to all counted QD spots is linearly related to the amount of heparin in the range of 4.65 × 10 ^–4 U/mL to 0.023 U/mL. The limit of detection is 9.3 × 10 ^–5 U/mL (∼0.1 nM), and the recovery of the spiked heparin at 0.00465 U/mL (∼5 nM) in 0.1% human plasma is acceptable

Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins: OPPORTUNITIES FOR POTENT, SELECTIVE MODULATION OF ANTITHROMBIN FUNCTION*

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891–31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80–87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis