Jeremy L. Steinbacher

Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells

BackgroundExposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression.ResultsMicroarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 106μm2/cm2) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 106μm2/cm2) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (FOS, ATF3, IL6 and IL8) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells.ConclusionsCristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses in vitro are suggested as predictive in determining the pathogenicity of potentially harmful particulates.

Use of Bifunctional Ureas to Increase the Rate of Proline-Catalyzed α-Aminoxylations

The rate of the proline-catalyzed alpha-aminoxylation of aldehydes is significantly increased in the presence of a bifunctional urea. Structure-activity relationship data indicate that both an amine and a urea are crucial for rate enhancement. The evidence presented herein suggests that this rate enhancement originates from the hydrogen bonding interaction between the bifunctional urea and an oxazolidinone intermediate to increase the rate of enamine formation. Proline derivatives that are incapable of forming oxazolidinones exhibit no rate enhancement in the presence of the bifunctional urea. The rate enhancement is general for a variety of aldehydes, and the faster reactions do not reduce yields or selectivities.

Enhanced Uptake of Porous Silica Microparticles by Bifunctional Surface Modification with a Targeting Antibody and a Biocompatible Polymer

Strategies were developed by which mesoporous microparticles were modified on their external surfaces with tetraethylene glycol (TEG), a protein, or both, leaving the pore surfaces available for modification with a separate moiety, such as a dye. Only particles bifunctionally modified with both TEG and a cell-specific antibody were taken up specifically by a targeted cancer cell line. In contrast to similarly functionalized nanoparticles, endocytosed microparticles were not contained within a lysosome.

Thiourea/Proline Derivative-Catalyzed Synthesis of Tetrahydrofuran Derivatives: A Mechanistic View

A thiourea/proline derivative-catalyzed synthesis of linear α-substituted tetrahydrofuran/pyran derivatives starting with lactol substrates is presented. This study demonstrates the utility and potential complications of using (thio)urea/proline cocatalysis as each of these catalysts is necessary to provide the observed reactivity, but a time-dependent decrease in enantioselectivity is observed. New mechanistic insights into (thio)urea/proline cocatalysis are presented.

Gd-labeled microparticles in MRI: in vivo imaging of microparticles after intraperitoneal injection.

Particulate materials have recently been studied for in vivo applications such as imaging, drug delivery, and even combined therapy/imaging agents for the simultaneous detection and treatment of diseases.[1–4] Nanoparticles have received much attention in the drug delivery field;[5, 6] however, because they can enter organelles and distribute widely throughout the body,[7–10] there is some uncertainty about the acute and/or chronic toxicities of nanoparticles. Microparticles, with their larger size, may be a less toxic alternative that still can be modified with the same array of functionalities as nanoparticles. We recently showed that acid-prepared mesoporous spheres (APMS),[11, 12] composed of silica and with an average diameter of 1.5 µm, are readily taken up by cells when modified with tetraethylene glycol (TEG) on their external surfaces. These microparticles also readily deliver chemotherapeutic agents in vitro and in vivo,[13, 14] and can be bifunctionally modified with both TEG and antibodies for targeted uptake by cancer cells.[15] A unique feature of our microparticles is that they are not present within endosomes after uptake but instead are directly surrounded by cytosol, in contrast to nanoparticles. Thus, molecular cargo including DNA and RNA is released directly to the cytosol and is not subject to the enzymatic and pH-based degradation mechanisms within endosomes. Our current interests include studies on the molecular mechanisms and treatment of malignant mesothelioma (MM)[16] and tumors of the intraperitoneal (IP) cavity, such as pancreatic and ovarian cancers.[17] To visualize our particles in vivo, we used a known ligand to immobilize gadolinium(III) to the pores of APMS and imaged animals via magnetic resonance imaging (MRI).[18] Nanoparticle-based MRI contrast agents have been reported recently;[19, 20] however, we report here for the first time that porous silica microparticles containing a Gd chelate can be injected IP and tracked in real-time by MRI. Surprisingly, a fraction of the particles were excreted via the urine, a finding we confirmed by analyzing the urine and tissues of the treated animals.

Adsorption and Release of siRNA from Porous Silica

Porous silica particles are potential transfection agents for nucleic acid-based therapies because of their large specific surface areas and pore volumes and the ease with which they can be chemically modified to maximize the loading of cargo and to effect targeting in vivo. Here, we present a systematic study of the effects of pore size and pore modification on the adsorption and release of short, interfering RNA (siRNA) from a mesoporous silica particle developed in our laboratory. Using adsorption isotherms and release experiments, we found that the short polyamine diethylenetriamine was the best chemical modification for achieving both the adsorption and release of large amounts of siRNA. The degree of functionalization with diethylenetriamine caused drastic changes in the loading capacity and binding strength of siRNA to silica with relatively large pores (8 nm and larger), but the degree of functionalization had a weaker effect in narrow pores (4 nm). Multilayer adsorption could occur in materials with large pores (15 nm). Release experiments showed that intermediate pore sizes and intermediate degrees of functionalization resulted in the best compromise between maximizing loading (from strong adsorption) and maximizing release. Capillary electrophoresis and quantitative, real-time PCR demonstrated that siRNA was released intact and that these particles functioned as a transfection agent of mammalian cells in vitro.

Increased efficacy of doxorubicin delivered in multifunctional microparticles for mesothelioma therapy

New and effective treatment strategies are desperately needed for malignant mesothelioma (MM), an aggressive cancer with a poor prognosis. We have shown previously that acid‐prepared mesoporous microspheres (APMS) are nontoxic after intrapleural or intraperitoneal (IP) administration to rodents. The purpose here was to evaluate the utility of APMS in delivering chemotherapeutic drugs to human MM cells in vitro and in two mouse xenograft models of MM. Uptake and release of doxorubicin (DOX) alone or loaded in APMS (APMS‐DOX) were evaluated in MM cells. MM cell death and gene expression linked to DNA damage/repair were also measured in vitro. In two severe combined immunodeficient mouse xenograft models, mice received saline, APMS, DOX or APMS‐DOX injected directly into subcutaneous (SC) MM tumors or injected IP after development of human MMs peritoneally. Other mice received DOX intravenously (IV) via tail vein injections. In comparison to DOX alone, APMS‐DOX enhanced intracellular uptake of DOX, MM death and expression of GADD34 and TP73. In the SC MM model, 3× weekly SC injections of APMS‐DOX or DOX alone significantly inhibited tumor volumes, and systemic DOX administration was lethal. In mice developing IP MMs, significant (p < 0.05) inhibition of mesenteric tumor numbers, weight and volume was achieved using IP administration of APMS‐DOX at one‐third the DOX concentration required after IP injections of DOX alone. These results suggest APMS are efficacious for the localized delivery of lower effective DOX concentrations in MM and represent a novel means of treating intracavitary tumors.

Simplified Mesofluidic Systems for the Formation of Micron to Millimeter Droplets and the Synthesis of Materials

We present and validate simple mesofluidic devices for producing monodisperse droplets and materials. The significance of this work is a demonstration that simple and complex droplet formulations can be prepared uniformly using off-the-shelf small-diameter tubing, barbed tubing adapters, and needles. With these simple tools, multiple droplet-forming devices and a new particle concentrator were produced and validated. We demonstrate that the droplet-forming devices could produce low-dispersity particles from 25 to 1200 μm and that these results are similar to results from more complicated devices. Through a study of the fluid dynamics and a dimensional analysis of the data, we have correlated droplet size with two dimensionless groups, capillary number and viscosity ratio. The flow-focusing device is more sensitive to both parameters than the T-junction geometry. The modular character of our mesofluidic devices allowed us to rapidly assemble compound devices that use flow-focusing and T-junction devices in series to create complex droplet-in-microcapsule materials. This work demonstrates that flow chemistry does not require complicated tools, and an inexpensive tool-kit can allow anyone with interest to enter the field.

Controlled Synthesis of Silica Capsules: Taming the Reactivity of SiCl4 Using Flow and Chemistry

Monodisperse silica microcapsules are typically fabricated using hard templating methods. Though soft templating methods are known, none yet provides a fast and easy method to produce monodisperse capsules. Herein, we describe a mesofluidic strategy whereby monodisperse droplets of reactive silica precursors are formed using a snap-off mechanism via a T junction. Both the mesofluidic system and the composition of the reactive silica formulation are critical features. Using solid- and solution-state 29Si nuclear magnetic resonance, scanning electron microscopy, and optical microscopy, we have developed models for why some formulations form exploding capsules, why some capsules contain crystalline materials, and why some capsules have thin or thick walls.

A multifunctional mesothelin antibody-tagged microparticle targets human mesotheliomas.

Pleural and peritoneal mesotheliomas (MMs) are chemoresistant tumors with no effective therapeutic strategies. The authors first injected multifunctional, acid-prepared mesoporous spheres (APMS), microparticles functionalized with tetraethylene glycol oligomers, intraperitoneally into rodents. Biodistribution of APMS was observed in major organs, peritoneal lavage fluid (PLF), and urine of normal mice and rats. After verification of increased mesothelin in human mesotheliomas injected into severe combined immunodeficient (SCID) mice, APMS were then functionalized with an antibody to mesothelin (APMS-MB) or bovine serum albumin (BSA), a nonspecific protein control, and tumor targeting was evaluated by inductively coupled plasma mass spectrometry and multifluorescence confocal microscopy. Some APMS were initially cleared via the urine over a 24 hr period, and small amounts were observed in liver, spleen, and kidneys at 24 hr and 6 days. Targeting with APMS-MB increased APMS uptake in mesenteric tumors at 6 days. Approximately 10% to 12% of the initially injected amount was observed in both spheroid and mesenteric MM at this time point. The data suggest that localized delivery of APMS-MB into the peritoneal cavity after encapsulation of drugs, DNA, or macromolecules is a novel therapeutic approach for MM and other tumors (ovarian and pancreatic) that overexpress mesothelin.