Andrew J. Sawyer

Dilation and degradation of the brain extracellular matrix enhances penetration of infused polymer nanoparticles.

This study investigates methods of manipulating the brain extracellular matrix (ECM) to enhance the penetration of nanoparticle drug carriers in convection-enhanced delivery (CED). A probe was fabricated with two independent microfluidic channels to infuse, either simultaneously or sequentially, nanoparticles and ECM-modifying agents. Infusions were performed in the striatum of the normal rat brain. Monodisperse polystyrene particles with a diameter of 54 nm were used as a model nanoparticle system. Because the size of these particles is comparable to the effective pore size of the ECM, their transport may be significantly hindered compared with the transport of low molecular weight molecules. To enhance the transport of the infused nanoparticles, we attempted to increase the effective pore size of the ECM by two methods: dilating the extracellular space and degrading selected constituents of the ECM. Two methods of dilating the extracellular space were investigated: co-infusion of nanoparticles and a hyperosmolar solution of mannitol, and pre-infusion of an isotonic buffer solution followed by infusion of nanoparticles. These treatments resulted in an increase in the nanoparticle distribution volume of 51% and 123%, respectively. To degrade hyaluronan, a primary structural component of the brain ECM, a pre-infusion of hyaluronidase (20,000 U/mL) was followed after 30 min by infusion of nanoparticles. This treatment resulted in an increase in the nanoparticle distribution of 64%. Our results suggest that both dilation and enzymatic digestion can be incorporated into CED protocols to enhance nanoparticle penetration.

Engineering Cellular Response Using Nanopatterned Bulk Metallic Glass

Nanopatterning of biomaterials is rapidly emerging as a tool to engineer cell function. Bulk metallic glasses (BMGs), a class of biocompatible materials, are uniquely suited to study nanopattern–cell interactions as they allow for versatile fabrication of nanopatterns through thermoplastic forming. Work presented here employs nanopatterned BMG substrates to explore detection of nanopattern feature sizes by various cell types, including cells that are associated with foreign body response, pathology, and tissue repair. Fibroblasts decreased in cell area as the nanopattern feature size increased, and fibroblasts could detect nanopatterns as small as 55 nm in size. Macrophages failed to detect nanopatterns of 150 nm or smaller in size, but responded to a feature size of 200 nm, resulting in larger and more elongated cell morphology. Endothelial cells responded to nanopatterns of 100 nm or larger in size by a significant decrease in cell size and elongation. On the basis of these observations, nondimensional analysis was employed to correlate cellular morphology and substrate nanotopography. Analysis of the molecular pathways that induce cytoskeletal remodeling, in conjunction with quantifying cell traction forces with nanoscale precision using a unique FIB-SEM technique, enabled the characterization of underlying biomechanical cues. Nanopatterns altered serum protein adsorption and effective substrate stiffness, leading to changes in focal adhesion density and compromised activation of Rho-A GTPase in fibroblasts. As a consequence, cells displayed restricted cell spreading and decreased collagen production. These observations suggest that topography on the nanoscale can be designed to engineer cellular responses to biomaterials.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Distribution of polymer nanoparticles by convection-enhanced delivery to brain tumors.

Glioblastoma multiforme (GBM) is a fatal brain tumor characterized by infiltration beyond the margins of the main tumor mass and local recurrence after surgery. The blood-brain barrier (BBB) poses the most significant hurdle to brain tumor treatment. Convection-enhanced delivery (CED) allows for local administration of agents, overcoming the restrictions of the BBB. Recently, polymer nanoparticles have been demonstrated to penetrate readily through the healthy brain when delivered by CED, and size has been shown to be a critical factor for nanoparticle penetration. Because these brain-penetrating nanoparticles (BPNPs) have high potential for treatment of intracranial tumors since they offer the potential for cell targeting and controlled drug release after administration, here we investigated the intratumoral CED infusions of PLGA BPNPs in animals bearing either U87 or RG2 intracranial tumors. We demonstrate that the overall volume of distribution of these BPNPs was similar to that observed in healthy brains; however, the presence of tumors resulted in asymmetric and heterogeneous distribution patterns, with substantial leakage into the peritumoral tissue. Together, our results suggest that CED of BPNPs should be optimized by accounting for tumor geometry, in terms of location, size and presence of necrotic regions, to determine the ideal infusion site and parameters for individual tumors.

A peptide-morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model

Management of skin wound infections presents a serious problem in the clinic, in the community, and in both civilian and military clinical treatment centers. Staphylococcus aureus is one of the most common microbial pathogens in cutaneous wounds. Peptide-morpholino oligomer (PMO) conjugates targeted to S. aureus gyrase A mRNA have shown the ability to reduce bacterial viability by direct site-specific mRNA cleavage via RNase P. As a treatment, these conjugates have the added advantages of not being susceptible to resistance due to genetic mutations and are effective against drug resistant strains. While this strategy has proven effective in liquid culture, it has yet to be evaluated in an animal model of infected surface wounds. In the present study, we combined PMO conjugates with a thermoresponsive gel delivery system to treat full-thickness mouse cutaneous wounds infected with S. aureus. Wounds treated with a single dose of PMO conjugate displayed improved healing that was associated with increased epithelialization, reduced bacterial load, and increased matrix deposition. Taken together, our findings demonstrate the efficacy and flexibility of the PMO conjugate drug delivery system and make it an attractive and novel topical antimicrobial agent.

Astrocyte-Derived Thrombospondin-2 Is Critical for the Repair of the Blood-Brain Barrier

Thrombospondin (TSP)-2-null mice have an altered brain foreign body response (FBR) characterized by increases in inflammation, extracellular matrix deposition, and leakage of the blood-brain barrier (BBB). In the present study, we investigated the role of TSP-2 in BBB repair during the brain FBR to mixed cellulose ester filters implanted in the cortex of wild-type (WT) and TSP-2-null mice for 2 days to 8 weeks. Histological and immunohistochemical analysis revealed enhanced and prolonged neuroinflammation in TSP-2-null mice up to 8 weeks after implantation. In addition, recovery of the BBB was compromised and was associated with increased gelatinolytic activity and low levels of collagen type IV in the basement membranes of TSP-2-null blood vessels. An analysis of protein extracts from implantation sites revealed elevated levels of matrix metalloproteinase (MMP)-2 and MMP-9 in TSP-2-null brains. TSP-2-null astrocytes secreted higher levels of both MMPs in vitro compared with their WT counterparts. Furthermore, TSP-2-null astrocytes were deficient in supporting the recovery of barrier function in WT endothelial cells. Finally, Western blot analysis of astrocytes and brain endothelial cells revealed TSP-2 expression only in the former. Taken together, our observations suggest that astrocyte-derived TSP-2 is critical for the maintenance of physiological MMP-2 and MMP-9 levels during the FBR and contributes to the repair of the BBB.

The effect of inflammatory cell-derived MCP-1 loss on neuronal survival during chronic neuroinflammation

Intracranial implants elicit neurodegeneration via the foreign body response (FBR) that includes BBB leakage, macrophage/microglia accumulation, and reactive astrogliosis, in addition to neuronal degradation that limit their useful lifespan. Previously, monocyte chemoattractant protein 1 (MCP-1, also CCL2), which plays an important role in monocyte recruitment and propagation of inflammation, was shown to be critical for various aspects of the FBR in a tissue-specific manner. However, participation of MCP-1 in the brain FBR has not been evaluated. Here we examined the FBR to intracortical silicon implants in MCP-1 KO mice at 1, 2, and 8 weeks after implantation. MCP-1 KO mice had a diminished FBR compared to WT mice, characterized by reductions in BBB leakage, macrophage/microglia accumulation, and astrogliosis, and an increased neuronal density. Moreover, pharmacological inhibition of MCP-1 in implant-bearing WT mice maintained the increased neuronal density. To elucidate the relative contribution of microglia and macrophages, bone marrow chimeras were generated between MCP-1 KO and WT mice. Increased neuronal density was observed only in MCP-1 knockout mice transplanted with MCP-1 knockout marrow, which indicates that resident cells in the brain are major contributors. We hypothesized that these improvements are the result of a phenotypic switch of the macrophages/microglia polarization state, which we confirmed using PCR for common activation markers. Our observations suggest that MCP-1 influences neuronal loss, which is integral to the progression of neurological disorders like Alzheimers and Parkinson disease, via BBB leakage and macrophage polarization.

Nanoparticle-based evaluation of blood–brain barrier leakage during the foreign body response

OBJECTIVE The brain foreign body response (FBR) is an important process that limits the functionality of electrodes that comprise the brain-machine interface. Associated events in this process include leakage of the blood-brain barrier (BBB), reactive astrogliosis, recruitment and activation of microglia, and neuronal degeneration. Proper BBB function is also integral to maintaining neuronal health and function. Previous attempts to characterize BBB integrity have shown homogeneous leakage of macromolecules up to 10 nm in size. In this study, we describe a new method of measuring BBB permeability during the foreign body response in a mouse model. APPROACH Fluorescent nanoparticles were delivered via the tail vein into implant-bearing mice. Tissue sections were then analyzed using fluorescence microscopy to observe nanoparticles in the tissue. Gold nanoparticles were also used in conjunction with TEM to confirm the presence of nanoparticles in the brain parenchyma. MAIN RESULTS By using polymer nanoparticle tracers, which are significantly larger than conventional macromolecular tracers, we show near-implant BBB gaps of up to 500 nm in size that persist for at least 4 weeks after implantation. Further characterization of the BBB illustrates that leakage during the brain FBR is heterogeneous with gaps between at least 10 and 500 nm. Moreover, electron microscopy was used to confirm that the nanoparticle tracers enter into the brain parenchyma near chronic brain implants. SIGNIFICANCE Taken together, our findings demonstrate that the FBR-induced BBB leakage is characterized by larger gaps and is of longer duration than previously thought. This technique can be applied to examine the BBB in other disease states as well as during induced, transient, BBB opening.

Matricellular proteins in drug delivery: Therapeutic targets, active agents, and therapeutic localization

Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell-matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases.

Regulation of cell-cell fusion by nanotopography

Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.