Blood loss is the major cause of death in both civilian and battlefield traumas. Methods to staunch bleeding include pressure dressings and absorbent materials. For example, Quik-clot effectively halts bleeding by absorbing large quantities of fluid and concentrating platelets to augment clotting, but these treatments are limited to compressible and exposed wounds. An ideal treatment would halt bleeding only at the injury site, be stable at room temperature, be administered easily, and work effectively for internal injuries. We have developed synthetic platelets, based on ArgGly-Asp functionalized nanoparticles, that halve bleeding time after intravenous administration in a rat model of major trauma. The effects of these synthetic platelets surpass other treatments including recombinant factor VIIa, which is used clinically for uncontrolled bleeding. Synthetic platelets were cleared within 24 hours at a dose of 20 mg/ml, and no complications were seen out to 7 days after infusion, the longest time point studied. These synthetic platelets may be useful for early intervention in trauma and demonstrate the role that nanotechnology can have in addressing unmet medical needs.

A microvascular network is critical for the survival and function of most tissues. We have investigated the potential of neural progenitor cells to augment the formation and stabilization of microvascular networks in a previously uncharacterized three-dimensional macroporous hydrogel and the ability of this engineered system to develop a functional microcirculation in vivo. The hydrogel is synthesized by cross-linking polyethylene glycol with polylysine around a salt-leached polylactic-co-glycolic acid scaffold that is degraded in a sodium hydroxide solution. An open macroporous network is formed that supports the efficient formation of tubular structures by brain endothelial cells. After subcutaneous implantation of hydrogel cocultures in mice, blood flow in new microvessels was apparent at 2 weeks with perfused networks established on the surface of implants at 6 weeks. Compared to endothelial cells cultured alone, cocultures of endothelial cells and neural progenitor cells had a significantly greater density of tubular structures positive for platelet endothelial cell adhesion molecule-1 at the 6-week time point. In implant cross sections, the presence of red blood cells in vessel lumens confirmed a functional microcirculation. These findings indicate that neural progenitor cells promote the formation of endothelial cell tubes in coculture and the development of a functional microcirculation in vivo. We demonstrate a previously undescribed strategy for creating stable microvascular networks to support engineered tissues of desired parenchymal cell origin.

Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications.

Neural stem cells (NSCs) have the potential to replace the major cell types of the central nervous system (CNS) and may be important in therapies for injuries to and diseases of the CNS. However, for such treatments to be safe and successful, NSCs must survive and differentiate appropriately following transplantation. A number of polymer scaffolds have shown promise in improving the survival and promoting the differentiation of NSCs. To capitalize on the interaction between scaffolds and NSCs, we need to determine the fundamental material properties that influence NSC behavior. To investigate the role of material properties on NSCs, we synthesized a library of 52 hydrogels composed of poly(ethylene glycol) and poly(L-lysine) (PLL). This library of hydrogels allows independent variation of chemical and mechanical properties across a wide range of values. By culturing NSCs on this library, we have identified a subset of gels that promotes NSC migration and a further subset that promotes NSC differentiation. By combining the material properties of these subsets with the cell behavior, we determined that mechanical properties play a critical role in NSC behavior with elastic moduli promoting NSC migration and neuronal differentiation. Amine concentration is less critical, but PLL molecular weight also plays a role in NSC differentiation.

Angiogenesis precedes recovery following spinal cord injury (SCI), and its extent correlates with neural regeneration suggesting that angiogenesis may play a role in repair. An important precondition for studying the role of angiogenesis is the ability to induce it in a controlled manner. Previously, we showed that a coculture of endothelial cells (ECs) and neural progenitor cells (NPCs) promoted the formation of stable tubes in vitro and stable, functional vascular networks in vivo in a subcutaneous model. We sought to test whether a similar coculture would lead to formation of stable functional vessels in the spinal cord following injury. We created microvascular networks in a biodegradable two component implant system and tested the ability of the coculture or controls (lesion control, implant alone, implant plus ECs, or implant plus NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a four fold increase in functional vessels compared to the lesion control, implant alone, or implant plus NPCs groups and a 2 fold increase in functional vessels over the implant plus ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for formation of the blood spinal cord barrier (BSB). No other groups showed positive staining for the BSB in the injury epicenter. This work provides a novel method to induce angiogenesis following SCI and a foundation for studying its role in repair.