Protein-based polymer liquid embolics for cerebral aneurysms.

Cerebral aneurysms (CA), an abnormal bulge in the arteries that supply blood to the brain, are prone to rupture and can cause hemorrhagic stroke. Physicians can treat CA by blocking blood flow to the aneurysmal sac via clipping of the aneurysm neck via open procedure, or endovascular occlusion of the aneurysm with embolic materials to promote thrombus formation to prevent further inflow of blood into the aneurysm. Endovascular treatment options for CA still have significant limitations in terms of safety, usability in coagulopathic patients, and risks of device migration. Bioactive embolic therapies, consisting of non-toxic bioresorbable materials that encourage the growth of neointima across the aneurysm neck, are needed to improve the healing of CA. In this work, the bioinspired silk-elastinlike protein-based polymer (SELP 815K), was used to embolize aneurysms in a rabbit elastase model. SELP 815K effectively embolized the model aneurysms in vivo, achieving >90% occlusion, using commercial microcatheters. No device-associated adverse effects were observed in any of the animals, and SELP 815K showed no cytotoxicity. SELP embolization did not show any deleterious effects to local tissues, and features consistent with reendothelialization of the aneurysm neck were noted in histological examination one-month post-embolization. SELP 815K shows promise as an embolic treatment for unruptured CA. STATEMENT OF SIGNIFICANCE: Unruptured cerebral aneurysms are present in approximately 3% of the population, with a fatality rate of up to 65% upon rupture. In this work a silk-elastinlike protein polymer (SELP) is explored as a liquid embolic for occlusion of cerebral aneurysms. This embolic exists as a liquid at room temperature before rapidly forming a gel at physiological temperature. This shape filling property was used to successfully occlude cerebral aneurysms in rabbits, with stable occlusion persisting for over thirty days. SELP occlusions show evidence for reendothelialization of the aneurysm sac and provide an opportunity for delivery of bioactive agents to further improve treatments.

Translational Development of a Silk-Elastinlike Protein Polymer Embolic for Transcatheter Arterial Embolization.

Locally blocking blood flow to tumors with embolic materials is the key to transcatheter arterial embolization for treating hepatocellular carcinoma. Current microparticle agents do not deeply penetrate target tissues and are compatible with a very limited selection of therapeutic agents. Silk-elastinlike protein polymers (SELPs) combine the solubility of elastin and the strength of silk to create an easily injected liquid embolic that transition into a solid depot amenable to loading with drugs, gene therapy agents, or biologics. SELP, injected as liquid solution, penetrates the vasculature before transitioning to a solid hydrogel. The objective of this manuscript is to evaluate SELP embolization, stability, and biocompatibility at 7-, 30-, and 90-day survival intervals in a porcine model. SELP embolics selectively block blood flow in the kidneys and livers, with no off-target infarctions. As assessed with angiography, SELP renal embolization exhibits decreasing persistence for the duration of the 90-day study period. There is an increased presence of microscopic SELP emboli in the renal setting, compared to Embosphere. Histologically scored inflammatory reactions to SELP are decreased in both the renal and hepatic implantations compared to Embosphere. In conclusion, a bioresorbable SELP liquid embolic system deeply penetrates target tissue and selectively embolizes blood vessels in vivo.

Thermal Stabilization of Biologics with Photoresponsive Hydrogels.

Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded user-defined release at the point of use. The poly(ethylene glycol) (PEG)-based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (ß-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. ß-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 °C, and for 60 min at 85 °C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 °C for 24 h and 60 °C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.

SLAP deficiency decreases dsDNA autoantibody production.

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.

Modeling carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS): a trinuclear nickel complex employing deprotonated amides and bridging thiolates.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) utilizes a unique Ni-M bimetallic site in the biosynthesis of acetyl-CoA, where a square-planar Ni ion is coordinated to two thiolates and two deprotonated amides in a Cys-Gly-Cys motif. The identity of M is currently a matter of debate, although both Cu and Ni have been proposed. In an effort to model ACS's unusual active site and to provide insight into the mechanism of acetyl-CoA formation and the role of each of the metals ions, we have prepared and structurally characterized a number of Ni(II)-peptide mimic complexes. The mononuclear complexes Ni(II) N, N'-bis(2-mercaptoethyl)oxamide (1), Ni(II) N, N'-ethylenebis(2-mercaptoacetamide) (2), and Ni(II) N, N'-ethylenebis(2-mercaptopropionamide) (3) model the Ni(Cys-Gly-Cys) site and can be used as synthons for additional multinuclear complexes. Reaction of 2 with MeI resulted in the alkylation of the sulfur atoms and the formation of Ni(II) N, N'-ethylenebis(2-methylmercaptoacetamide) (4), demonstrating the nucleophilicity of the terminal alkyl thiolates. Addition of Ni(OAc)(2).4H(2)O to3 resulted in the formation of a trinuclear species (5), while 2 crystallizes as an unusual paddlewheel complex (6) in the presence of nickel acetate. The difference in reactivity between the similar complexes 2 and 3 highlights the importance of ligand design when synthesizing models of ACS. Significantly,5 maintains the key features observed in the active site of ACS, namely a square-planar Ni coordinated to two deprotonated amides and two thiolates, where the thiolates bridge to a second metal, suggesting that 5 is a reasonable structural model for this unique enzyme.