Zheng Qu

Cell surface engineering with polyelectrolyte multilayer thin films.

Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.

Polymeric Materials for Tissue Engineering of Arterial Substitutes

Cardiovascular disease is the leading cause of mortality in the United States. The limited availability of healthy autologous vessels for bypass grafting procedures has led to the fabrication of prosthetic vascular conduits. Synthetic polymeric materials, while providing the appropriate mechanical strength, lack the compliance and biocompatibility that bioresorbable and naturally occurring protein polymers offer. Vascular tissue engineering approaches have emerged in order to meet the challenges of designing a vascular graft with long-term patency. In vitro culture techniques that have been explored with vascular cell seeding of polymeric scaffolds and the use of bioactive polymers for in situ arterial regeneration have yielded promising results. This review describes the development of polymeric materials in various tissue engineering strategies for the improvement in the mechanical and biological performance of an arterial substitute.

Thrombomodulin Improves Early Outcomes After Intraportal Islet Transplantation

Primary islet nonfunction due to an instant blood mediated inflammatory reaction (IBMIR) leads to an increase in donor islet mass required to achieve euglycemia. In the presence of thrombin, thrombomodulin generates activated protein C (APC), which limits procoagulant and proinflammatory responses. In this study, we postulated that liposomal formulations of thrombomodulin (lipo‐TM), due to its propensity for preferential uptake in the liver, would enhance intraportal engraftment of allogeneic islets by inhibiting the IBMIR. Diabetic C57BL/6J mice underwent intraportal transplantation with B10.BR murine islets. In the absence of treatment, conversion to euglycemia was observed among 29% of mice receiving 250 allo‐islets. In contrast, a single infusion of lipo‐TM led to euglycemia in 83% of recipients (p = 0.0019). Fibrin deposition (p < 0.0001), neutrophil infiltration (p < 0.0001), as well as expression TNF‐α and IL‐β (p < 0.03) were significantly reduced. Significantly, thrombotic responses mediated by human islets in contact with human blood were also reduced by this approach. Lipo‐TM improves the engraftment of allogeneic islets through a reduction in local thrombotic and inflammatory processes. As an enzyme‐based pharmacotherapeutic, this strategy offers the potential for local generation of APC at the site of islet infusion, during the initial period of elevated thrombin production.

Interface between hemostasis and adaptive immunity.

Stress induced activation or denudation of the endothelium elicits arrest and activation of platelets with attendant triggering of coagulation, culminating in a physical barrier to limit blood loss. Recently, coagulation-activated osteopontin, chemerin, and protease-activated receptor signaling, as well as platelet-derived molecules including platelet factor 4, serotonin, P-selectin, and CD154 (CD40L) have been revealed as new links between hemostasis and adaptive immunity. The initiation of hemostasis establishes a local state of inflammation that serves as an adjuvant system for antigen presentation, consequently influencing the onset and functional characteristics of an evolving adaptive immune response. In this context, the hemostatic system and its associated signaling pathways warrant further study as novel therapeutic targets that may enhance, abrogate, or otherwise selectively direct the adaptive immune response.

Biomolecular surface engineering of pancreatic islets with thrombomodulin.

Islet transplantation has emerged as a promising treatment for Type 1 diabetes, but its clinical impact remains limited by early islet destruction mediated by prothrombotic and innate inflammatory responses elicited upon transplantation. Thrombomodulin (TM) acts as an important regulator of thrombosis and inflammation through its capacity to channel the catalytic activity of thrombin towards generation of activated protein C (APC), a potent anticoagulant and anti-inflammatory agent. We herein describe a novel biomolecular strategy for re-engineering the surface of pancreatic islets with TM. A biosynthetic approach was employed to generate recombinant human TM (rTM) bearing a C-terminal azide group, which facilitated site-specific biotinylation of rTM through Staudinger ligation. Murine pancreatic islets were covalently biotinylated through targeting of cell surface amines and aldehydes and both islet viability and the surface density of streptavidin were maximized through optimization of biotinylation conditions. rTM was immobilized on islet surfaces through streptavidin-biotin interactions, resulting in a nearly threefold increase in the catalytic capacity of islets to generate APC.

Immobilization of Actively Thromboresistant Assemblies on Sterile Blood‐Contacting Surfaces

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.