Bioengineering

Receptor-targeted nanoparticles for enzyme replacement therapy of genetic Pompe disease


Authors: Laura Northrup, Janet Hsu, Tridib Bhowmick, and Silvia Muro
Department or Program: BIOE
Presented by: Janet Hsu
Abstract: Polymer nanoparticles (NPs) bearing targeting moieties are promising platforms for drug delivery. Yet, delivery of enzymes by targeted NPs is a relatively unexplored strategy with a wide range of applications, including treatment of enzyme deficiencies. As an example, we explored NPs for enzyme replacement therapy (ERT) of Pompe disease. This is a lysosomal storage disorder (LSD) characterized by multiorgan dysfunctions affecting mainly the liver, heart, and skeletal muscle, caused by a genetic deficiency of acid α-glucosidase (α-Glu) and lysosomal accumulation of glycogen. ERT by intravenous injection of recombinant α-Glu is the current treatment for Pompe disease, yet, high doses are employed leading to production of antibodies to the injected enzyme. To minimize doses and improve ERT, we designed a prototype platform consisting of 100 nm FITC-polystyrene NPs coated with antibodies to a cell surface receptor overexpressed in many pathologies, intercellular adhesion molecule (ICAM)-1, and α-Glu (anti-ICAM/α-Glu NPs). Fluorescent microscopy verified successful targeting and internalization (>70%) of anti-ICAM/α-Glu NPs in turanose-treated endothelial cells (a Pompe disease model) within 30 minutes. NP uptake occurred by CAM-endocytosis mediated by ICAM-1. Over 50% of anti-ICAM/α-Glu NPs were transported to lysosomes by 5 hours, resulting in a significant reduction of the glycogen storage. Coupling 125I-α-Glu to anti-ICAM NPs increased the amount of enzyme delivered to the liver and heart by 18- and 6-fold, respectively, 30 minutes after intravenous injection in mice. Delivery to other target organs was also improved. These results suggest that delivery of α-Glu by ICAM-1-targeted NPs may enhance ERT for Pompe disease.

Signaling Molecule Expression of Chondrocytes Isloated from Distinct Cartilage Zones


Authors: Emily E Coates and John P. Fisher
Department or Program: BIOE
Presented by: Emily Coates
Abstract: While initial research strategies attempted to model articular cartilage as homogenous, more recent work recognizes cartilage as a complex tissue comprised of three major zones with distinct extracellular matrix organization and cellular phenotype. Regeneration of healthy cartilage can only be complete with the formation of all cartilage zones. For this to be possible, phenotypical differences between chondrocyte cell populations must be understood. Our research motivation is to understand differences in molecular signaling in each zone of articular cartilage. To this end, we isolated bovine chondrocytes from each zone and used an alginate bead model to culture cells in three dimensional scaffolds. We used this model to examine cell viability based on radial location within the alginate bead. In addition, we investigated differences in extracellular matrix production and signaling molecule expression between cartilage zones. To examine the cellular effects of encapsulation we have studied gene expression of signaling molecule insulin-like growth factor (IGF-1)and IGF-1 binding protein. To understand the effects of signaling molecules on chondrocyte phenotype we measured gene expression of type II and type I collagen. Results show differences in both matrix production and signaling molecule expression based on cartilage zone.

Effect of Media Flow Rate on the Osteogenic Differentiation of Human Mesenchymal Stem Cells in a Novel Bioreactor System


Authors: Andrew B. Yeatts, Elyse M. Geibel and John P. Fisher
Department or Program: BIOE
Presented by: Andrew Yeatts
Abstract: In vitro culture techniques must be improved in order to increase the feasibility of cell based tissue engineering strategies. It is well documented in the literature that in vitro culture of cells in three dimensional scaffolds is limited by nutrient transport. Therefore we propose to develop a novel bioreactor system for the culture of human mesenchymal stem cells (hMSCs) in three dimensional scaffolds and to investigate the effect the system parameters have on the osteogenic differentiation of these cells. This system utilizes a novel yet elegant design to create a more effective environment for cell culture without the typical drawbacks associated with more complicated perfusion systems such as leaking, high pressure, and bacterial contamination. In our design, hMSCs in the bioreactor system will be encapsulated in alginate beads which will be tightly packed in a tubular growth chamber. Media is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds, eliminating air pockets and enhancing nutrient transfer while exposing the cells to shear stress. The entire system can be sterilized via autoclave and is housed within a standard cell culture incubator. Preliminary results demonstrate an increased amount of cell proliferation as compared to static controls throughout a twelve day culture period and that the flow rate of media through the growth chamber enhances the osteogenic differentiation of hMSCs as shown by an increase in BMP-2 expression on day eight in the bioreactor as compared to a static control. These results represent a significant advance in the use of bioreactors for tissue engineering applications.

Osteogenic signal expressions of rat bone marrow stromal cells can be controlled by fabrication parameters of photo-crosslinked diethyl fumarate/poly(propylene fumarate) composite scaffolds


Authors: Kyobum Kim1, Anqi Lu1, Rob Breithaupt2, David Dean3, Antonios G. Mikos4, and John. P. Fisher2
Department or Program: ENCH
Presented by: Kyobum Kim
Abstract: Diethyl fumarate (DEF) can be incorporated within poly(propylene fumarate) (PPF) to increase crosslinking density and stiffness of composite scaffolds due to the creation of additional bridges between PPF polymer chains. Using these composites, we hypothesize that increasing DEF contents and increasing pore size would promote osteogenic signal gene expression by augmenting substrate rigidity and facilitating nutrient transport, therefore enhancing the osteogenic differentiation of progenitor cells. Therefore, we investigated the effect of DEF content and pore sizes on osteogenic signal expressions of rat bone marrow stromal cells (BMSCs) on PPF/DEF macroporous composite scaffolds. BMSCs were transplanted onto scaffolds fabricated with 100:0, 90:10, 75:25, and 67:33 ratio of PPF:DEF as well as 180-300 and >500 µm pore sizes. Results demonstrated that incorporation of DEF decreased sol fraction in composite scaffolds, and therefore increased level of crosslinking density. In addition, 75:25 of PPF:DEF ratio showed highest compressive modulus and offset yield strength. Initial metabolic activity of cells in DEF incorporated scaffolds was similar to those in the PPF control. However, DEF dissolved in culture media showed a negative effect on monolayer cell for 4 hrs. Reverse transcription-polymeric chain reaction data on day 8 demonstrated that osteogenic signal gene expression including fibroblast growth factor-2, transforming growth facor beta-1, vascular endothelial growth factor, and matrix metalloproteinases-13 could be controlled by both DEF contents and pore size. These signals altered early osteogenic differentiation, demonstrated by alkaline phophatase, but did not significantly alter late osteogenic differentiation, demonstrated by osteocalcin expression. Therefore, we concluded that incorporate DEF amount and pore size of macroporous composite scaffolds are critical parameters to control osteogenic signal gene expression during early osteogenic differentiation of progenitor cell population.

Using Native AI-2 Processing Enzymes to Disrupt Bacterial Communication in a bioMEMS Device


Authors: Varnika Roy, Xiaolong Luo , Gregory F. Payne, Gary W. Rubloff , William E. Bentley
Department or Program: MOCB
Presented by: Varnika Roy
Abstract: Bacteria communicate via a phenomenon known as quorum sensing (QS) which is facilitated by small signaling molecules called autoinducers. The autoinducer AI-2 is seen in over 70 bacterial species. In this study native AI-2 degrading enzymes are used to disrupt bacterial communication in a bioMEMS device. Degradation of AI-2 to inhibit a QS based bacterial response is medically relevant as QS facilitates undesirable pathogenic responses in bacteria. The use of biological microelectromechanical systems (bioMEMS) provides the added advantage of having a distinct platform for studying the bacteria under the controlled conditions of micro devices. The bioMEMS device is fabricated with microchannels on a glass slide with patterned gold electrodes. The bio-polymer chitosan is electrodeposited on the negatively biased electrodes. The device is bio-functionalized with enzymes (LsrK-Tyr, LsrG-Tyr), which are known to phosphorylate and cleave AI-2 in the Escherichia coli QS circuitry in a sequential manner. The enzymes are covalently conjugated onto the chitosan by activation of the C-terminal tyrosine tag using tyrosinase. AI-2 introduced into the channel is consecutively phosphorylated and degraded by the enzymatic assembly line, after which it is exposed to AI-2 sensitive GFP reporter cells on the device. This work provides a reusable platform to study QS modulation. It holds promise for the development of a new generation of antimicrobials based on targeting communication in a population of cells and puts forth the idea of study of a microbiome in a controlled MEMS environment.

Isoelectric Focusing-Multiplexed Reversed Phase Liquid Chromatography Polymer Microchip with Integrated High-Pressure Valves


Authors: Jikun Liu, Chien-Fu Chen, Chien-Cheng Chang, Don L. DeVoe
Department or Program: ENME
Presented by: Jikun Liu
Abstract: A cyclicolefin polymer (COP) microchip supporting parallel 2-D peptide separations is described. By combining isoelectric focusing (IEF) as a first dimension and multiplexed reversed phase liquid chromatography (mRPLC) as a second dimension, the system enables efficient high-throughput fractionation prior to mass spectrometry in support of peptide mass fingerprinting for global proteomic analysis from highly limited specimens. The IEF-mRPLC chip incorporates high pressure micro shut-off valves, allowing uniform sample transfer and gradient elution from each micro LC column, and ensuring hydrodynamic isolation between the separation dimensions. The utility of the initial microchip is demonstrated by separation of a bovine serum albumin (BSA) tryptic digest in a chip containing a five channel RPLC array. Coupling between the on-chip separations and MALDI-MS is also described, with a 2.7-fold increase in number of identified peptides achieved over the direct analysis of BSA digest without front-end separation.

Planar Phospholipids Membrane Formation in Open Well Thermoplastic Chips


Authors: C. Shao, M. Colombini, D. L. DeVoe
Department or Program: ENME
Presented by: Chenren Shao
Abstract: Membrane-bound proteins have a large hydrophobic trans-membrane region, rendering difficulty of study at traditional immune essay systems. Planar Phospholipids Membrane (PPM) mimics the native environment for ion channels in vitro and has been widely used as an electrophysiological study platform since 1960s. Typically by painting or monolayers folding method, PPMs form across an aperture on a hydrophobic supporting layer, which separates aqueous buffer in two chambers. The miniaturization of PPM system through micro fabrication techniques has provided additional advantages over traditional bulk system, including small sample volume, easy formation process through spontaneous thinning, high bandwidth of electrophysiological measurements, and high throughput by arraying a large number of PPM units in a single microchip. In particular, the small sample volume involved, typically 2µL~20µL in each side, is essential for quick execution of temperature change and fluidic perfusion. Here we present a series of efforts to achieve a smaller platform of planar phospholipids membrane. A layer of polyvinylidene chloride (12.5μm thick) is sandwiched by thermal bonding with two polycarbonate wafer and has an aperture (50~120μm in diameter) hosting a PPM. By manually painting with lipid solution (1,2-diphytanoyl-sn-glycero-3-phosphocholine, DPhyPC in hexanol/hexadecane mixture), stable PPM can form reproducibly and provide enough lifetime to host an ion channel test more than two hours. Gramicidin channels in 3DMSO/2DMF are added to verify that PPM is thin enough to host proteins with their characteristic behavior that cannot be confused with a non-specific conductance. Ceramide is one kind of lipids that can form stable channels in outer mitochondrial membranes and regulate various cell activities such as apoptosis. To fully investigate the regulation mechanism, we are interested to see how ceramide channels interact with different ligands, such as trehalose. A perfusion channel is incorporated to the microsystem stated above and provides addition and removal pathway of 50mM trehalose. Flowrate of 0.4μL/min guarantees a quick execution of fluidic condition change without the rupture of membrane.