Designing Multi-Layered Nanoparticles For Combination Gene And Drug Cancer Therapy.

Cancer continues to be a highly prevalent and lethal disease, despite significant advances in understanding tumor biology and developing new chemotherapies. Major obstacles in cancer chemotherapy are drug resistance and systemic toxicities. Potential strategies for addressing these problems include delivering combination therapies to overcome drug resistance, and utilizing synergistic agents to minimize dosing and subsequently drug toxicity. In turn, delivery can also be optimized to target the tumor site and consequently minimize systemic side effects. Polymer nanocarriers are gaining interest as vehicles for cancer therapeutics for their abilities to not only deliver multiple agents, but also target the tumor itself. Our goal is to design multi-layered polymer nanoparticles (MLNPs) for efficient delivery of small molecules and genetic material towards synergistically inhibiting tumor growth. The MLNPs were first optimized for transfection in vitro through delivery of plasmids encoding for luciferase (pLuc) and green fluorescent protein (pGFP). The particles were then evaluated for effective delivery of both a candidate small molecule, camptothecin (CPT), and a plasmid encoding for TNF related apoptosis inducing ligand (pTRAIL) (CT MLNPs). Co-delivery of CPT and pTRAIL via CT MLNPs were then evaluated for growth inhibition of brain, colorectal, and breast cancer cells in vitro. MLNPs were approximately 116 nm in diameter. They were able to delivery approximately 575 ng of plasmid per mg of particle, and between 0.1 mg to 0.01 µg of CPT per mg of particle. MLNPs were non-toxic, and human embryonic kidney cells (293T) transfected with pLuc loaded MLNPs expressed comparable amounts of luciferase as cells transfected with the gold standard lipid formulation, Lipofectamine 2000. Thirty-seven percent of transfected 293T cells expressed GFP 72 h after transfection. Studies on tumor death kinetics related to CPT exposure and pTRAIL transfection suggested that simultaneous transfection and drug exposure provided the greatest inhibition of cell growth. MLNPs were able to provide the optimal timing for delivery of both agents. Synergy analysis of co-delivering CPT and pTRAIL via CT MLNPs, using the Chou-Talalay method, provided a combination index at 50% inhibition ranging between 0.31 and 0.53 for all cell lines. These CI values indicate a synergistic interaction between the two agents. For obtaining a 50% effect level, co-delivery with MLNPs resulted in providing 3.14-7.38 fold reduction in CPT and 4.66 to 6.09 fold reduction in pTRAIL. These initial results support our hypothesis that MLNPs can deliver both small molecule and genetic agents towards synergistically inhibiting tumor growth

Novel polymer constructs for controlled release and presentation of topographic cues in support of neuronal cells

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (leaves 25-26).In order to improve nerve healing, a new treatment that uses conductive polymer scaffolds to bridge gaps between damaged nerve ends and deliver drugs controllably was explored. In order to optimize neuron growth across scaffolding a neuronal scaffold designed with an electrically conductive polymer, polypyrrole (PPy), will be used as a substrate to enhance nerve cell interaction in vitro. The polymer will be analyzed for the capacity to influence cellular phenotype, including via controlled biomolecular delivery and surface topography. This thesis showed that all these concepts are possible and begins to optimize these characteristics. Successfully optimization of these scaffold characteristics will provide a novel method for treating injury in the central nervous system.by Asiri Ediriwicikrema.S.B

von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel-Palade bodies

BACKGROUND: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. METHODS: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. RESULTS: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. CONCLUSIONS: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction

Using mixed objects in the training of object-based image classifications

Image classification for thematic mapping is a very common application in remote sensing, which is sometimes realized through object-based image analysis. In these analyses, it is common for some of the objects to be mixed in their class composition and thus violate the commonly made assumption of object purity that is implicit in a conventional object-based image analysis. Mixed objects can be a problem throughout a classification analysis, but are particularly challenging in the training stage as they can result in degraded training statistics and act to reduce mapping accuracy. In this paper the potential of using mixed objects in training object-based image classifications is evaluated. Remotely sensed data were submitted to a series of segmentation analyses from which a range of under- to over-segmented outputs were intentionally produced. Training objects were then selected from the segmentation outputs, resulting in training data sets that varied in terms of size (i.e. number of objects) and proportion of mixed objects. These training data sets were then used with an artificial neural network and a generalized linear model, which can accommodate objects of mixed composition, to produce a series of land cover maps. The use of training statistics estimated based on both pure and mixed objects often increased classification accuracy by around 25% when compared with accuracies obtained from the use of only pure objects in training. So rather than the mixed objects being a problem, they can be an asset in classification and facilitate land cover mapping from remote sensing. It is, therefore, desirable to recognize the nature of the objects and possibly accommodate mixed objects directly in training. The results obtained here may also have implications for the common practice of seeking an optimal segmentation output, and also act to challenge the widespread view that object-based classification is superior to pixel-based classification

Decellularization of Porcine and Primate Optic Nerve Lamina

The optic nerve lamina cribrosa is a mesh-like collagenous structure consisting of an extracellular matrix with varying porosity and glial cell distribution through which axons of retinal ganglion cells travel en route to the lateral geniculate nucleus. There has been significant interest in its structure with respect to its possible role in the pathophysiology of glaucomatous optic neuropathy. Our goal was to decellularize porcine and primate lamina to evaluate for a potential role as a three-dimensional scaffold for stem cell growth, regeneration, and delivery

Reconstruction Of An Outer Retina Using Electrospun Polycaprolactone (pcl) And Cells Derived From Human Embryonic Stem Cells.

As a step towards engineering an outer retina suitable for transplantation, we designed a biocompatible, biodegradable scaffold that allows retinal progenitor cells (RPCs) to form flat, laminar structures. The scaffold minimizes the exposure of retinal progenitors to extracellular matrix components that are not found in the retina, and enables co-culture with the retinal pigment epithelium. Scaffolds were formed from electrospun fibers of polycaprolactone (PCL). Scaffold thickness was varied by increasing the duration of collection during electrospinning; porosity and pore size distribution was adjusted by varying polymer concentration in solution. The porosity and thickness of the sheets were varied to optimize cell to cell contact formation and PCL degradation rate, respectively. RPCs derived from human embryonic stem cells (hESC-derived RPCs) were cultured to form neurospheres. Dissociated neurospheres were seeded onto +/- laminin coated PCL sheets in normal or low O2 incubators, and maintained in two types of serum free medium. Fluorescence labeling and confocal microscopy were used to assess tissue morphology. Individual PCL fibers were ~3 ìm thick, sheets varied from 20-200 ìm in thickness, and pores ranged from 5-100 ìm in diameter. The scaffolds degraded over 4-7 weeks. Scaffolds with pores 25-50 ìm in diameter were subsequently chosen for culture experiments. Biomechanical strength testing estimated a Young\u27s modulus of 0.071 megapascals (MPa). Confocal imaging confirmed that the RPCs penetrated the thickness of the scaffold irrespective of laminin coating and O2 level, and continued to express retinal markers such as Pax6, recoverin, and N-cadherin, with heightened expression in the differentiation medium. Cells adhered uniformly to laminin-coated PCL, but formed aggregates on uncoated PCL. Staining for the proliferative marker, Ki67, indicated active cell division in both medium types and O2 levels. Preliminary RPE: RPC co-culture experiments with laminin coated PCL in low oxygen conditions revealed polarization of N-cadherin, with concomitant stress fiber formation in the RPE monolayer. Overall, electrospun PCL polymers sustained the differentiated properties acquired by neurospheres and appear to be suitable scaffolds for reconstructing an outer retinal layer

Orbit Case #1 - That Looks Foreign to Me

This symposium has been created to highlight the surgical aspects of our field and to educate neuroophthalmologists and trainees through the presentation and discussion of complex surgical neuroophthalmic case scenarios