Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here we show that the resulting succinimide thioether formed by a Michael type addition of a thiol to N-ethylmaleimide (NEM), generally accepted as stable, can in fact undergo retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies (1H NMR, HPLC) of NEM conjugated to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP) incubated with glutathione showed half lives of conversion from 20–80 hrs, with extents of conversion from 20–90% for MPA and N-acetylcysteine conjugates. Ring-opened the resultant succinimide thioether as well as any MP adduct did not show retro and exchange reactions. The kinetics of the retro reactions can be modulated by the Michael donor’s reactivity; therefore the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at timescales different than those currently possible via disulfide-mediated release. Such approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and sub-cellular trafficking.

Methods to assemble polymeric hydrogels on the basis of noncovalent protein-glycosaminoglycan interactions have been previously demonstrated by us and others and hold promise in the development of receptor-responsive hydrogel materials; improvements in the mechanical properties of such systems would broaden their utility. Thus, in situ crosslinkable and degradable heparin-containing hydrogels were designed for the binding and controlled release of growth factors. Specifically, maleimide-functionalized high molecular weight heparin (HMWH) was synthesized via straightforward chemical methods that permitted facile and controllable modification of carboxylates in HMWH with maleimide groups via control of catalyst and reaction conditions, as assessed via 1H NMR spectroscopy. These modified heparins were crosslinked into hydrogels via reaction with various thiol-functionalized PEGs. The gelation times and elastic moduli of the gels, as assessed through oscillatory rheometry, could be tuned by controlling the functionality of HMWH, the concentration of the hydrogel, the identity of the PEG-based crosslinker, as well as the molar ratio between maleimide and thiol groups. The capability of the hydrogels to bind to growth factors was investigated with immunochemical assays. Preliminary studies indicate the controlled release of basic fibroblast growth factor (bFGF) from these materials and suggest their broader use in the design of responsive materials.

This review presents an overview of polysaccharide-conjugated synthetic polymers and their use in tissue-engineered scaffolds and drug-delivery applications. This topic will be divided into four categories: (1) polymeric materials modified with non-mammalian polysaccharides such as alginate, chitin, and dextran; (2) polymers modified with mammalian polysaccharides such as hyaluronan, chondroitin sulfate, and heparin; (3) multi-polysaccharide-derivatized polymer conjugate systems; and (4) polymers containing polysaccharide-mimetic molecules. Each section will discuss relevant conjugation techniques, analysis, and the impact of these materials as micelles, particles, or hydrogels used in in-vitro and in-vivo biomaterial applications.

We have recently reported that retro Michael-type addition reactions can be employed for producing labile chemical linkages with tunable sensitivity to physiologically relevant reducing potentials. We reasoned that such strategies would also be useful in the design of glutathione-sensitive hydrogels for a variety of targeted delivery and tissue engineering applications. In this report, we describe hydrogels in which maleimide-functionalized low molecular weight heparin (LMWH) is crosslinked with various thiol-functionalized poly(ethylene glycol) (PEG) multi-arm star polymers. Judicious selection of the chemical identity of the thiol permits tuning of degradation via previously unstudied, but versatile chemical methods. Thiol pKa and hydrophobicity affected both the gelation and degradation of these hydrogels. Maleimide–thiol crosslinking reactions and retro Michael-type addition reactions were verified with 1H NMR during the crosslinking and degradation of hydrogels. PEGs esterified with phenylthiol derivatives, specifically 4-mercaptophenylpropionic acid or 2,2-dimethyl-3-(4-mercaptophenyl)propionic acid, induced sensitivity to glutathione as shown by a decrease in hydrogel degradation time of 4-fold and 5-fold respectively, measured via spectrophotometric quantification of LMWH. The degradation proceeded through the retro Michael-type addition of the succinimide thioether linkage, with apparent pseudo-first order reaction constants derived from oscillatory rheology experiments of 0.039 ± 0.006 h−1 and 0.031 ± 0.003 h−1. The pseudo-first order retro reaction constants were approximately an order of magnitude slower than the degradation rate constants for hydrogels crosslinked via disulfide linkages, indicating the potential use of these Michael-type addition products for reduction-mediated release and/or degradation, with increased blood stability and prolonged drug delivery timescales compared to disulfide moieties.

We study PEG–heparin hydrogels to identify compositions that lead to gel formation and measure the corresponding gelation kinetics. The material consists of a maleimide-functionalized high molecular weight heparin (HMWH) backbone covalently cross-linked with bis-thiol poly(ethylene glycol) (PEG). Using multiple particle tracking microrheology, we investigate a broad composition space, defined by the number of maleimide functional sites per HMWH (f = 3.9–11.8), the molecular weight of the PEG cross-linker (Mn = 2000, 5000, and 10 000), and the concentrations of the heparin and PEG polymers. Gelation kinetics are characterized by time–cure superposition, yielding the gel time, tc, and the critical relaxation exponent, n. Gelation times range from 5 < tc ≤ 45 min, with the fastest kinetics occurring for the highest HMWH maleimide functionalities. tc depends nonmonotonically on the PEG cross-linker molecular weight, suggesting that gelation is affected by the length of the cross-linker relative to intermolecular interactions between heparin molecules. The critical relaxation exponent decreases from n = 0.52 for PEG 2000 to n = 0.39 for PEG 10 000. Finally, 219 equilibrated samples taken over the entire composition space are identified as liquid or solid, defining the “gelation envelope”. The boundaries of this empirical gelation envelope are in good agreement with Flory–Stockmayer theory. In all, microrheological measurements enable characterization over a large parameter space and provide crucial insight into the gelation of complex, multifunctional hydrogelators used in therapeutic applications.

In light of the growing importance in understanding and controlling the physical cues presented to cells by artificial scaffolds, direct, temporally resolved measurements of the gel modulus are needed. We demonstrate that an interpolation of macro- and microrheology measurements provides a complete history of a hydrogel modulus during degradation through the reverse percolation transition. The latter is identified by microrheology, which captures the critical scaling behavior of reverse percolation, a transition of key importance in controlling cell migration, implant degradation, and tissue regeneration.

Regenerative medicine approaches offer attractive alternatives to standard vascular reconstruction; however, the biomaterials to be used must have optimal biochemical and mechanical properties. To evaluate the effects of biomaterial properties on vascular cells, heparinized poly(ethylene glycol) (PEG)-based hydrogels of three different moduli, 13.7 kPa, 5.2 kPa, and 0.3 kPa, containing fibronectin and growth factor were utilized to support the growth of three human vascular cell types. The cell types exhibited differences in attachment, proliferation, and gene expression profiles associated with the hydrogel modulus. Human vascular smooth muscle cells demonstrated preferential attachment on the highest modulus hydrogel, adventitial fibroblasts demonstrated preferential growth on the highest modulus hydrogel, and human umbilical vein endothelial cells demonstrated preferential growth on the lowest modulus hydrogel investigated. Our studies suggest that the growth of multiple vascular cell types can be supported by PEG hydrogels and that different populations can be controlled by altering the mechanical properties of biomaterials.

Hydrogels engineered for biomedical applications consist of numerous components, each of which can affect the material assembly and final mechanical properties. We present methods that rapidly generate rheological libraries to identify regimes of hydrogel assembly in a large composition parameter space. This method conserves both material and time, and leads to critical insight into assembly mechanisms and mechanics, which can then be used for further materials development and optimization.The assembly of precursor molecules and macromolecules into hydrogels, and the resulting material mechanical properties, are principal design factors in the engineering of many biologically active and therapeutic materials. 1 For instance, the gel microstructure and modulus are known to affect the viability and migration of cells 2 and the differentiation of stem cells. 3 Hydrogels intended for such biological applications are typically composed of numerous structural compounds, in addition to soluble and attached epitopes and markers, 2,4,5 each of which can influence, by design or unintentional consequence, the assembly, final material properties, and ultimate material performance. In order to facilitate the design and optimization of therapeutic hydrogel materials over a large parameter space, we apply methods of highthroughput rheological characterization using multiple particle tracking microrheology. This simple, yet powerful approach enables us to rapidly assay compositions for hydrogel formation in a relatively short period of time, while consuming only small quantities of material.In this study, the hydrogelator materials consist of maleimide-functionalized high molecular weight heparin (HMWH, MW = 15 000). 6 Covalent cross-linking occurs by the addition of dithiolized poly(ethylene glycol) (PEG, MW = 10 000).6 Heparin has the unique ability to sequester and stabilize soluble proteins, including growth factors that promote cellular proliferation and differentiation.7 Because of this, heparin hydrogels have recently been engineered to stimulate communication between cells and a surrounding gel matrix,2 behave as stimuli responsive materials,4 ,8 and serve as a three-dimensional matrix for stem cell culture. 5 In general, the high charge and conformational stiffness of many biomolecules, including heparin, may alter the anticipated gelation conditions and rheology. Thus, rapid evaluation of gelation in biomolecular gels provides a valuable tool for the synthesis of materials that mimic the chemical, structural and rheological characteristics of native biological matrices.