Prathamesh M. Kharkar

Design of Thiol- and Light-sensitive Degradable Hydrogels using Michael-type Addition Reactions.

Injectable depots that respond to exogenous and endogenous stimuli present an attractive strategy for tunable, patient-specific drug delivery. Here, the design of injectable and multimodal degradable hydrogels that respond to externally applied light and physiological stimuli, specifically aqueous and reducing microenvironments, is reported. Rapid hydrogel formation was achieved using a thiol-maleimide click reaction between multifunctional poly(ethylene glycol) macromers. Hydrogel degradation kinetics in response to externally applied cytocompatible light, reducing conditions, and hydrolysis were characterized, and degradation of the gel was controlled over multiple time scales from seconds to days. Further, tailored release of an encapsulated model cargo, fluorescent nanobeads, was demonstrated.

Dually degradable click hydrogels for controlled degradation and protein release

Click reactions have emerged as one of the most powerful paradigms in materials chemistry owing to their high regioselectivity and efficient reaction yields under mild conditions. While stability of the bonds formed by these reactions often is highly valued, their controlled cleavage is promising as an elegant approach to engineer material degradation for a number of applications, including drug delivery and tissue engineering. However, cleavage of click linkages under physiological conditions remains a major challenge in the design of degradable biomaterials. Here, we demonstrate the use of cleavable click linkages formed by Michael-type addition reactions in conjunction with hydrolytically cleavable functionalities for the degradation of injectable hydrogels by dual mechanisms for controlled protein release. Specifically, the reaction between maleimides and thiols was utilized for hydrogel formation, where thiol selection dictates the degradability of the resulting linkage under thiol-rich reducing conditions. Relevant microenvironments would include those rich in glutathione (GSH), a tripeptide that is found at elevated concentrations in carcinoma tissues. Degradation of the hydrogels was monitored with rheometry and volumetric swelling measurements. Arylthiol-based thioether succinimide linkages underwent degradation via click cleavage and thiol exchange reaction in the presence of GSH as well as ester hydrolysis, whereas alkylthiol-based thioether succinimide linkages only undergo ester hydrolysis. The resulting control over the degradation rate within a reducing microenvironment resulted in ~2.5 fold differences in the release profile of cargo molecules (fluorescently labeled bovine serum albumin as a model protein) from dually degradable hydrogels compared to non-degradable hydrogels. These unique degradable chemistries are promising for controlling the rate of degradation and local release of therapeutic cargo molecules in cancer tissue microenvironments.

Thiol–ene Click Hydrogels for Therapeutic Delivery

Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For use as a delivery vehicle, hydrophilic precursors are usually laden with bioactive moieties and then directly injected to the site of interest for in situ gel formation and controlled release dictated by precursor design. Hydrogels formed by thiol-ene click reactions are attractive for local controlled release of therapeutics owing to their rapid reaction rate and efficiency under mild aqueous conditions, enabling in situ formation of gels with tunable properties often responsive to environmental cues. Herein, we will review the wide range of applications for thiol-ene hydrogels, from the prolonged release of anti-inflammatory drugs in the spine to the release of protein-based therapeutics in response to cell-secreted enzymes, with a focus on their clinical relevance. We will also provide a brief overview of thiol-ene click chemistry and discuss the available alkene chemistries pertinent to macromolecule functionalization and hydrogel formation. These chemistries include functional groups susceptible to Michael type reactions relevant for injection and radically-mediated reactions for greater temporal control of formation at sites of interest using light. Additionally, mechanisms for the encapsulation and controlled release of therapeutic cargoes are reviewed, including i) tuning the mesh size of the hydrogel initially and temporally for cargo entrapment and release and ii) covalent tethering of the cargo with degradable linkers or affinity binding sequences to mediate release. Finally, myriad thiol-ene hydrogels and their specific applications also are discussed to give a sampling of the current and future utilization of this chemistry for delivery of therapeutics, such as small molecule drugs, peptides, and biologics.

Biomaterials for 4D stem cell culture

Stem cells reside in complex three-dimensional (3D) environments within the body that change with time, promoting various cellular functions and processes such as migration and differentiation. These complex changes in the surrounding environment dictate cell fate yet, until recently, have been challenging to mimic within cell culture systems. Hydrogel-based biomaterials are well suited to mimic aspects of these in vivo environments, owing to their high water content, soft tissue-like elasticity, and often-tunable biochemical content. Further, hydrogels can be engineered to achieve changes in matrix properties over time to better mimic dynamic native microenvironments for probing and directing stem cell function and fate. This review will focus on techniques to form hydrogel-based biomaterials and modify their properties in time during cell culture using select addition reactions, cleavage reactions, or non-covalent interactions. Recent applications of these techniques for the culture of stem cells in four dimensions (i.e., in three dimensions with changes over time) also will be discussed for studying essential stem cell processes.

Decreasing matrix modulus of PEG hydrogels induces a vascular phenotype in human cord blood stem cells

Adult and congenital cardiovascular diseases are significant health problems that are often managed using surgery. Bypass grafting is a principal therapy, but grafts fail at high rates due to hyperplasia, fibrosis, and atherosclerosis. Biocompatible, cellularized materials that attenuate these complications and encourage healthy microvascularization could reduce graft failure, but an improved understanding of biomaterial effects on human stem cells is needed to reach clinical utility. Our group investigates stem-cell-loaded biomaterials for placement along the adventitia of at-risk vessels and grafts. Here, the effects of substrate modulus on human CD34+ stem cells from umbilical cord blood were evaluated. Cells were isolated by immunomagnetic separation and encapsulated in 3, 4, and 6 weight% PEG hydrogels containing 0.032% gelatin and 0.0044% fibronectin. Gels reached moduli of 0.34, 4.5, and 9.1 kPa. Cell viability approached 100%. Cell morphologies appeared similar across gels, but proliferation was significantly lower in 6 wt% gels. Expression profiling using stem cell signaling arrays indicated enhanced self-renewal and differentiation into vascular endothelium among cells in the lower weight percent gels. Thus, modulus was associated with cell proliferation and function. Gels with moduli in the low kilopascal range may be useful in stimulating cell engraftment and microvascularization of graft adventitia.

Controlling the Release of Small, Bioactive Proteins via Dual Mechanisms with Therapeutic Potential

Injectable delivery systems that respond to biologically relevant stimuli present an attractive strategy for tailorable drug release. Here, the design and synthesis of unique polymers are reported for the creation of hydrogels that are formed in situ and degrade in response to clinically relevant endogenous and exogenous stimuli, specifically reducing microenvironments and externally applied light. Hydrogels are formed with polyethylene glycol and heparin-based polymers using a Michael-type addition reaction. The resulting hydrogels are investigated for the local controlled release of low molecular weight proteins (e.g., growth factors and cytokines), which are of interest for regulating various cellular functions and fates in vivo yet remain difficult to deliver. Incorporation of reduction-sensitive linkages and light-degradable linkages affords significant changes in the release profiles of fibroblast growth factor-2 (FGF-2) in the presence of the reducing agent glutathione or light, respectively. The bioactivity of the released FGF-2 is comparable to pristine FGF-2, indicating the ability of these hydrogels to retain the bioactivity of cargo molecules during encapsulation and release. Further, in vivo studies demonstrate degradation-mediated release of FGF-2. Overall, our studies demonstrate the potential of these unique stimuli-responsive chemistries for controlling the local release of low molecular weight proteins in response to clinically relevant stimuli.

Tuning and Predicting Mesh Size and Protein Release from Step Growth Hydrogels

Hydrogel-based depots are of growing interest for release of biopharmaceuticals; however, a priori selection of hydrogel compositions that will retain proteins of interest and provide desired release profiles remains elusive. Toward addressing this, in this work, we have established a new tool for the facile assessment of protein release from hydrogels and applied it to evaluate the effectiveness of mesh size estimations on predicting protein retention or release. Poly(ethylene glycol) (PEG)-based hydrogel depots were formed by photoinitiated step growth polymerization of four-arm PEG functionalized with norbornene (PEG-norbornene, 4% w/w to 20% w/w, Mn ∼ 5 to 20 kDa) and different dithiol cross-linkers (PEG Mn ∼ 1.5 kDa or enzymatically degradable peptide), creating well-defined, robust materials with a range of mesh sizes estimated with Flory-Rehner or rubber elasticity theory (∼5 to 15 nm). A cocktail of different model proteins was released from compositions of interest, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to facilely and quantitatively analyze temporal release profiles. Mesh size was predictive of retention of relatively large proteins and release of relatively small proteins. Proteins with diameters comparable to the mesh size, which is often the case for growth factors, were released by hindered diffusion and required experimental assessment of retention and release. With this knowledge, hydrogels were designed for the controlled release of a therapeutically relevant growth factor, PDGF-BB.