Ryan A. Ortega

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

BackgroundDrug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA.ResultsMonodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature.ConclusionsThis work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

A mathematical model of superparamagnetic iron oxide nanoparticle magnetic behavior to guide the design of novel nanomaterials

Superparamagnetic iron oxide nanoparticles (SPIONs) exhibit unique magnetic properties that make them highly efficacious as MR imaging contrast agents and laboratory diagnostic tools. The complexity of SPION magnetic behavior and the multiple parameters affecting this behavior complicate attempts at fabricating particles suited for a particular purpose. A mathematical model of SPION magnetic properties derived from experimental relationships and first principles can be an effective design tool for predicting particle behavior before materials are fabricated. Here, a novel model of SPION magnetic properties is described, using particle size and applied magnetic field as the primary variable inputs. The model is capable of predicting particle susceptibility and non-linear particle magnetization as well as describing the vector magnetic field produced by a single particle in an applied field. Magnetization values produced by the model agree with recent experimental measurements of particle magnetizations. The model is used to predict the complex magnetic behavior of clustered magnetic particles in simulated in vivo environment; specifically, interactions between the clusters and water molecules. The model shows that larger particles exhibit more linear magnetic behavior and stronger magnetization and that clusters of smaller particles allow for more numerous SPION–water molecule interactions and more uniform cluster magnetizations.

Nylon 6,6 Nonwoven Fabric Separates Oil Contaminates from Oil-in-Water Emulsions

Industrial oil spills into aquatic environments can have catastrophic environmental effects. First responders to oil spills along the coast of the Gulf of Mexico in the southern United States have used spunbond nylon fabric bags and fences to separate spilled oil and oil waste from contaminated water. Low area mass density spunbond nylon is capable of sorbing more than 16 times its mass in low viscosity crude oil and more than 26 times its mass in higher viscosity gear lube oil. Nylon bags separated more than 95% of gear lube oil contaminate from a 4.5% oil-in-water emulsion. Field testing of spunbond nylon fences by oil spill first responders has demonstrated the ability of this material to contain the oily contaminate while allowing water to flow through. We hypothesize that the effectiveness of nylon as an oil filter is due to the fact that it is both more oleophilic and more hydrophilic than other commonly used oil separation materials. The nylon traps oil droplets within the fabric or on the surface, while water droplets are free to flow through the fabric to the water on the opposite side of the fabric.

The Synthesis Map Is a Multidimensional Educational Tool That Provides Insight into Students’ Mental Models and Promotes Students’ Synthetic Knowledge Generation

Synthesis mapping uses a novel, multidimensional presentation tool to allow students to create a detailed map of their hierarchal knowledge structures. It can be used as formative assessment to reveal students’ strengths, misconceptions, and organizational schema and as a summative assessment to test students’ understanding of course material.

Abstract 3630: Immunoengineering of tumor associated macrophages using targeted, siRNA delivering nanoparticles

Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Dysregulation of NF-κB signaling is implicated in creating a pro-tumor phenotype in TAMs. NF-κB signaling consists of a classical pathway and a less understood alternative pathway. Suppression of NF-κB activation in TAMs is predicted to decrease local smoldering inflammation and inhibit the pro-tumor TAM phenotype. However, recent studies have reported anti-tumor macrophage behavior induced by activation of the classical NF-κB pathway. We have successfully used mannosylated polymer nanoparticles (Mn-NP) to deliver siRNA for RNAi of NF-κB proteins in bone marrow derived macrophages (BMDMs) and mouse TAMs in vivo. Mannose serves as a macrophage targeting ligand via the mannose receptor (CD206). This work aims to characterize these nanomaterials for targeted delivery of functional nucleic acid sequences into macrophages in vitro and in vivo. These studies will also use the Mn-NP to investigate the effects of specific manipulation of NF-κB activation on macrophage phenotype. Mn-NPs are micelles formed by self-assembly of triblock polymers with a poly(BMA-co-PAA-co-DMAEMA) core, a DMAEMA siRNA condensing block, and an azide-containing outer block for further functionalization. Alkyne functionalized mannose is then “clicked” on the end of the polymer and presented on the micellar surface. In vitro cell studies carried out with BMDMs from mice that express luciferase as a reporter of total NF-κB activity. Mn-NP delivery of siRNA (50 nM for 24 hrs) against each of several NF-κB pathway proteins confirms efficacious knockdown of total NF-κB activity, comparable to transfection with the commercial agent Lipofectamine. Mn-NPs are also highly biocompatible in vitro and exhibit no significant liver or kidney toxicity in mice at doses of 5 mg/kg. The mannosylated carrier effectively delivers cy3 fluorescently labeled nucleotides to TAMs in multiple model systems, including in primary mammary tumors arising spontaneously in polyoma mice and an implanted ovarian tumor model, . Currently, studies are focused on a potential therapeutic strategy that involves RNAi of an inhibitor of the classical NF-κB pathway, IκBα, in order to strategically activate the classical pathway in TAMs. Quantitative RT-PCR analysis of macrophages treated with Mn-NP formulated with siRNA against IκBα show a decrease in M2 markers, a decrease in smoldering inflammation associated cytokines, and an increase in immune cell recruiting chemokines. Citation Format: Ryan Ortega, Whitney Barham, Oleg Tikhomirov, Kavya Sharman, Fiona Yull, Todd Giorgio. Immunoengineering of tumor associated macrophages using targeted, siRNA delivering nanoparticles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3630. doi:10.1158/1538-7445.AM2014-3630

Abstract 3010: The role of NF-kappaB in mammary tumor initiation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Nuclear factor kappa B signaling is accepted as a major component of pro-survival signaling within breast cancer cells. However, less is known about how NF-kappaB could contribute to breast tumor initiation. Inflammatory signaling can be activated within the breast by such common factors as obesity, involution following lactation, aging, and psychological stress. Thus, understanding how activated NF-kappaB could lead to the initiation of breast tumors would provide opportunities to develop prevention strategies. We generated a doxycycline-inducible mouse model to study the effects of constitutively active IKK2 on ductal morphogenesis. In this model, Tet-o-cIKK2 mice are crossed with MMTV-rtTA transgenics to target inducible expression specifically to the mammary epithelium. Expression of cIKK2 in transplanted mammary tissue results in enlarged terminal end-buds, increased lateral branching, and intraductal hyperplasia. Transgene induction for only 3 days in a 6 week old female induces filling of ductal lumens, hypercellularity and loss of strict basal/luminal structure. Quantitative PCR of mammary tissue reveals up-regulated markers of macrophage infiltration such as CCL2 and F4/80 and markers of inflammation including Cox-2, TNF-alpha, and CXCL1. Interestingly, constitutive IKK2 activation also results in a decrease in hormone receptor levels (ERα and PR). Immunofluorescent staining shows that activation of classical NF-kappaB increases nuclear p65, as expected. However, nuclear p52 is also increased in transgene positive cells, suggesting that a subset of the effects resulting from activation of classical NF-kappaB are mediated by cross talk with the alternative pathway. Taken together, we find that activation of NF-kappaB in mammary epithelium is sufficient to induce hyperplastic growth and loss of ductal structure. Ongoing studies seek to define what additional factors, such as the right hormonal context, will cause the lesions to progress to malignancy. Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Halina Onishko, Linda Connelly, Fiona E. Yull. The role of NF-kappaB in mammary tumor initiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3010. doi:10.1158/1538-7445.AM2014-3010

Abstract 3981: Reprogramming tumor associated macrophages toward an anti-tumor phenotype by targeting the NF-κB pathway using novel targeted nanotherapeutics.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create an inflammatory, pro-tumor niche. Activation of the NF-κB pathway has been implicated in creating a pro-tumor phenotype in TAMs. Manipulation of TAM phenotype is a new approach to engage anticancer immunity, but has been limited by a lack of methods capable of therapeutic delivery to TAMs in vivo. We have successfully utilized mannosylated polymer nanoparticles capable of disrupting the endosomal compartment to deliver siRNA for RNAi of NF-κB proteins into bone marrow derived macrophages (BMDMs) derived from transgenic mice with a GFP/Luciferase reporter of NF-κB activity (NGL). In in vitro studies, the nanoparticles are comparable to commercial transfection agents using both gene and protein level readouts for knockdown. The transfection protocol utilizing these novel vehicles has been optimized with respect to transfection time, siRNA dose, and siRNA:polymer ratio with the intent to inform in vivo experiments. The presence of serum does not significantly affect transfection efficiency in vitro, presumably due to an almost neutral particle surface charge. Preliminary in vivo studies have revealed no significant particle toxicity. Delivering siRNA specific to the p52/p100 protein in the alternative pathway achieves knockdown of total NF-κB activity by approximately 80% in NGL BMDMs stimulated by TNF-α. By targeting proteins in the classical pathway, we have decreased total NF-κB activity by approximately 50% in the same model. While inhibition of NF-κB activity may be desirable in some contexts, we recently reported that induced activation of NF-κB in macrophages can result in anti-tumor activity. We have delivered a liposomal formulation of muramyl tripeptide (Mifamurtide), a synthetic derivative of a bacterial cell wall peptide and an activator of macrophages, to NGL BMDMs. Mifamurtide delivery increases both NF-κB activity, and the production of reactive oxygen species, indicating a preliminary mechanistic explanation for the therapeutic potential of NF-κB activation. Mifamurtide is used clinically in the European Union to treat osteosarcoma, potentially providing an avenue for rapid clinical translation of NF-κB modulating therapy for other tumor types. However, Mifamurtide has the potential to activate multiple pathways simultaneously. A more elegant approach would be to target knockdown of an NF-κB inhibitor to macrophages to mediate pathway specific activation. In preliminary studies we have demonstrated the ability to increase total NF-κB activity by treating NGL macrophages with nanoparticles carrying siRNA against the IκBα inhibitor of NF-κB. Our data provides evidence that delivering siRNA specifically to macrophages to modulate their functions using nanoparticles has potential as a therapeutic approach to cancer treatment. Citation Format: Ryan A. Ortega, Whitney Barham, Bharat Kumar, Shann S. Yu, Fiona Yull, Todd D. Giorgio. Reprogramming tumor associated macrophages toward an anti-tumor phenotype by targeting the NF-κB pathway using novel targeted nanotherapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3981. doi:10.1158/1538-7445.AM2013-3981

Abstract 1545: Education of macrophages through modulation of NF-kappaB: an opportunity for targeted therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Macrophages are a plastic cell type, capable of adapting to numerous signals within their environment. As part of the innate immune system, macrophages were traditionally considered anti-tumor (M1), but it has been well established that macrophages can also help to create a pro-tumor, pro-metastatic tumor niche (M2). NF-κB transcription factors can regulate both pro- (MMPs, VEGF) and anti-tumor (iNOS) downstream targets within macrophages, suggesting that modulation of NF-κB may play a role in the two different macrophage phenotypes. However, our understanding of NF-κB signaling specifically within macrophages during tumor progression is limited. To this end, we have developed murine transgenic models that enable us to induce expression of an activator or dominant inhibitor of NF-κB in macrophages by adding doxycycline to the drinking water of mice. We have combined these novel transgenics with the polyoma model of mammary cancer for our studies. We have recently shown that activation of NF-κB in macrophages significantly limits metastasis in a tail vein model of tumor progression. In this model, constitutive IKK2 activity within macrophages leads to an anti-tumor immune response including altered immune cell populations within the lung microenvironment, changes in chemokine and cytokine expression and rapid killing of tumor cells during the seeding phase mediated by reactive oxygen species. Our current work has extended these findings to an orthotopic mammary tumor model. Again, we find that activation of NF-κB in macrophages results in decreased primary tumor growth and decreased tumor seeding into the blood. To model this activation in vitro, we have utilized immortalized bone marrow derived macrophages from IκBα knock-out mice compared to a wild type line. IκBα KO macrophages display changes in morphology and adhesion relative to wild type macrophages. This correlates with increased cytotoxic behavior in co-culture with polyoma tumor cells, mirroring the in vivo phenotype of the cIKK2 expressing macrophages. Given these findings, we believe that targeted activation of NF-κB signaling in macrophages could be harnessed to overcome the education of macrophages by tumor cells, and could be exploited as a novel targeted therapy. Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Linda Gleaves, Halina Onishko, Taylor Sherrill, Linda Connelly, Timothy S. Blackwell, Fiona E. Yull. Education of macrophages through modulation of NF-kappaB: an opportunity for targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1545. doi:10.1158/1538-7445.AM2013-1545