Search

It is estimated that 2.2 million people have glaucoma in the US and 67 million people worldwide. The majority of cases are associated with elevated intraocular pressure (IOP) and decreasing IOP eliminates or greatly reduces degeneration in most cases, including cases in which the IOP is in the normal range but optic neuropathy occurs. Timolol maleate has the longest record of safety and efficacy to lower IOP and is administered via eye drops one or more times per day. Unfortunately, compliance is poor across patient populations leading to degeneration. Patients typically see their ophthalmologist once every 3-4 months. If one could administer a long-acting treatment while in the doctor's office, one might overcome the compliance issue and effectively preserve sight. The critical step is to develop a formulation for timolol maleate that leads to sustained delivery for greater than 90 days and would permit a different treatment paradigm, namely subconjunctival administration once every 3-4 months. By using a 50 : 50 blend of PLGA 502H and PLA, this study was able to fabricate microspheres that delivered timolol maleate continually over 107 days, well within the time frame needed to make subconjunctival administration feasible and permit a new approach to treating glaucoma and diseases of the eye more broadly.

A critical shortcoming of current surface functionalization schemes is their inability to selectively coat patterned substrates at micrometer and nanometer scales. This limitation prevents localized deposition of macromolecules at high densities, thereby restricting the versatility of the surface. A new approach for functionalizing lithographically patterned substrates that eliminates the need for alignment and, thus, is scalable to any dimension is reported. We show, for the first time, that electropolymerization of derivatized phenols can functionalize patterned surfaces with amine, aldehyde, and carboxylic acid groups and demonstrate that these derivatized groups can covalently bind molecular targets, including proteins and DNA. With this approach, electrically conducting and semiconducting materials in any lithographically realizable geometry can be selectively functionalized, allowing for the sequential deposition of a myriad of chemical or biochemical species of interest at high density to a surface with minimal cross-contamination.

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.

Inhibition of the epidermal growth factor receptor (EGFR) has been shown to reduce tumor growth and metastases and promote axon regeneration in the central nervous system. Current strategies for inhibiting EGFR include the administration of reversible or irreversible small-molecule tyrosine kinase inhibitors (TKIs). However, to be effective in vivo constant and sustained delivery is required. This study explored the feasibility of encapsulating the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in poly(lactic-co-glycolic acid) (PLGA) microspheres to achieve sustained delivery of the TKI. We characterized microspheres prepared using three different emulsion methods: solid-in-oil-in-water, oil-in-water, and oil-in-water with co-solvent. Addition of a co-solvent increased the loading and release of AG1478, and significantly (P

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, QuikClot 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 Arg-Gly-Asp functionalized nanoparticles, which 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.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuroprotection and repair, but long-term delivery from polymer systems has been challenging. We investigated the role the chemistry of the polymer played in loading and delivery of BDNF via microspheres, which are suitable for minimally invasive administration.We synthesized polymers based on PLGA and PEG to determine what components augmented loading and delivery. We characterized microspheres fabricated from these polymers using a battery of tests, including sizing, in vitro release, and bioactivity of the BDNF using PC12 cells engineered to express the trkB receptor.We found that a triblock polymer of PLGA, PLL, and PEG led to the delivery of BDNF for periods of time greater than 60 days and that the BDNF delivered was bioactive. The microsphere size was amendable to injection via a 30 gauge needle, allowing minimally invasive delivery.PLGA-PLL-PEG leads to greater loading and longer-term delivery of BDNF than PLGA or a blend of the polymers. We hypothesize that the introduction of an amphiphilic PLGA-based polymer increases the interaction of the BDNF with the polymer and leads to release that more closely correlates with the degradation of the polymer.

Angiogenesis precedes recovery following spinal cord injury 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 the 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 + ECs or implant + NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a fourfold increase in functional vessels compared with the lesion control, implant alone or implant + NPCs groups and a twofold increase in functional vessels over the implant + ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for the formation of the blood-spinal cord barrier. No other groups have shown positive staining for the blood-spinal cord barrier in the injury epicenter. This work provides a novel method to induce angiogenesis following spinal cord injury and a foundation for studying its role in repair.

Poly(lactic-co-glycolic acid) (PLGA) is one of the more widely used polymers for biomedical applications. Nonetheless, PLGA lacks chemical moieties that facilitate cellular interactions and surface chemistries. Furthermore, incorporation of hydrophilic molecules is often problematic. The integration of polymer functionalities would afford the opportunity to alter device characteristics, thereby enabling control over drug interactions, conjugations, and cellular phenomena. In an effort to introduce amine functionalities and improve polymer versatility, we synthesized two block copolymers (PLGA-PLL 502H and PLGA-PLL 503H) comprised of PLGA and Poly(ε-carbobenzoxy-L-lysine) utilizing dicyclohexyl carbodiimide (DCC) coupling. PLGA-PLL micropsheres encapsulated approximately six-fold (502H) and three-fold (503H) more vascular endothelial growth factor (VEGF), and 41% (503H) more ciliary neurotrophic factor (CNTF) than their PLGA counterparts. While the amine functionalities were amenable to the delivery of large molecules and surface conjugations, they did not comprise polymer biocompatibility. With the versatile combination of properties, biocompatibility, and ease of synthesis, these block copolymers have the potential for diverse utility in the fields of drug delivery and tissue engineering.

Transplantation of Bcl-2-transduced human umbilical vein endothelial cells (ECs) in protein gels into the gastrocnemius muscle improves local reperfusion in immunodeficient mouse hosts with induced hind limb ischemia. We tested the hypothesis that incorporation of local, sustained growth factor delivery could enhance and accelerate this effect. Tissue engineering scaffolds often use synthetic polymers to enable controlled release of proteins, but most synthetic delivery systems have major limitations, most notably hydrophobicity and inefficient protein loading. Here, we report the development of a novel alginate-based delivery system for vascular endothelial growth factor-A165 (VEGF) that exhibits superior loading efficiency and physical properties to previous systems in vitro. In vivo, VEGF released from alginate microparticles within protein gels was biologically active and, when combined with EC transplantation, led to increased survival of transplanted cells at 28 days. The composite graft described also improved early (14 days) tissue perfusion and late (28 days) muscle myoglobin expression, a sign of recovery from ischemia, compared with EC transplantation and VEGF delivery separately. We conclude that our improved approach to sustained VEGF delivery in tissue engineering is useful in vivo and that the integration of high efficiency protein delivery enhances the therapeutic effect of protein gel-based EC transplantation.—Jay, S. M., Shepherd, B. R., Bertram, J. P., Pober, J. S., Saltzman, W. M. Engineering of multifunctional gels integrating highly efficient growth factor delivery with endothelial cell transplantation.

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.

Delivery of factors capable of promoting oligodendrocyte precursor cell (OPC) survival and differentiation in vivo is an important therapeutic strategy for a variety of pathologies in which demyelination is a component, including multiple sclerosis and spinal cord injury. Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that promotes both survival and maturation of a variety of neuronal and glial cell populations, including oligodendrocytes. Present results suggest that, although CNTF has a potent survival and differentiation promoting effect in vitro on OPCs isolated from the adult spinal cord, CNTF administration in vivo is not sufficient to promote oligodendrocyte remyelination in the glial-depleted environment of unilateral ethidium bromide (EB) lesions.

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.