Douglas J. Goetz

Hyaluronan microspheres for sustained gene delivery and site-specific targeting

Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting.

Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo

We exploited leukocyte–endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium in vitro and in vivo. Leukocyte–endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under in vitro flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium in vivo, and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium in vivo due to the addition of a targeting ligand. The leukocyte–inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.

Particle Diameter Influences Adhesion under Flow

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.

Wnt5a is expressed in murine and human atherosclerotic lesions

Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO(2) (SC-CO(2)), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO(2) pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO(2) were higher than the untreated samples 12% glucose yield (12 g/100g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO(2) pretreatment at 3500 psi and 150°C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO(2) treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.

Phenyl methimazole inhibits TNF-α-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells

Proinflammatory cytokine (e.g., TNF-α)-induced expression of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of the vascular endothelium and a consequent increase in leukocyte adhesion are key aspects of pathological inflammation. A promising therapeutic approach to diminish aberrant leukocyte adhesion is, therefore, to inhibit cytokine-induced ECAM expression at the transcription level. Several studies suggest that methimazole, a compound used clinically to treat autoimmune diseases, such as Graves’ disease, may also diminish pathological inflammation by suppressing ECAM expression. In this study we probed the hypothesis that a derivative of methimazole, phenyl methimazole (compound 10), can reduce cytokine-induced ECAM expression and consequent leukocyte adhesion. We found that compound 10 1) dramatically inhibits TNF-α-induced VCAM-1 mRNA and protein expression in human aortic endothelial cells (HAEC), has a relatively modest inhibitory effect on TNF-α induced E-selectin expression and has no effect on ICAM-1 expression; 2) significantly reduces TNF-α-induced monocytic (U937) cell adhesion to HAEC under in vitro flow conditions similar to that present in vivo; 3) inhibits TNF-α-induced IFN regulatory factor-1 binding to VCAM-1 promoter; and 4) reduces TNF-α-induced IRF-1 expression in HAEC. Combined, the results indicate that phenyl methimazole can reduce TNF-α-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and that this contributes significantly to reduced monocytic cell adhesion to TNF-α-activated HAEC.

Ligand coated nanosphere adhesion to E- and P-selectin under static and flow conditions.

AbstractThe heterogeneous distribution of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of vascular endothelium provides an opportunity to deliver drugs to select tissues. The targeting could be achieved by using carriers whose outer surface has a ligand for a selectively expressed ECAM. The carriers would interact with the endothelium in a fluid dynamic environment and in many of these schemes nanoparticles would be used. It is unclear what role various parameters (e.g., ligand–ECAM chemistry, fluid shear) will have on the adhesion of the nanoparticles to the endothelium. To facilitate studies in this area, we have developed a prototypical in vitro model that allows investigation of nanoparticle adhesion. We coated polystyrene nanospheres with a humanized mAb (HuEP5C7.g2) that recognizes the ECAMs E- and P-selectin. Adhesion assays revealed that HuEP5C7.g2 nanospheres exhibit augmented, specific adhesion to selectin presenting cellular monolayers and that the adhesion can be affected by the fluid shear. These results; (i) strongly suggest that HuEP5C7.g2 could be used to target nanoparticles to selectin presenting endothelium; (ii) demonstrate that fluid shear can affect nanoparticle adhesion; and (iii) define a system which can be used to study the effects of various system parameters on nanoparticle adhesion.

Expression and Functional Significance of Adhesion Molecules on Cultured Endothelial Cells in Response to Ionizing Radiation

Objective: Upregulation of adhesion molecules on endothelial cells following irradiation has been shown, but the functional significance of this upregulation in various endothelial cell lines is not clear. We have developed an in vitro flow model to study the functional consequences of the radiation‐induced upregulation of E‐selectin and intercellular adhesion molecule (ICAM‐1).

Phenylmethimazole Decreases Toll-Like Receptor 3 and Noncanonical Wnt5a Expression in Pancreatic Cancer and Melanoma Together with Tumor Cell Growth and Migration

Purpose: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo. Experimental Design: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated. Results: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation. Conclusions: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.