Team B Breaking through the BloodBrain Barrier Sakib

Team B³ Breaking through the Blood-Brain Barrier Sakib Adnan Regina Borsellino Alice He Somdutta Mukherjee Victor Peng Karthya Potti Kelly Shih Janina Vaitkus Victor Wang Rani Woo Robert Zhang Adam Zuber Mentor: Dr. Helim Aranda-Espinoza Librarian: Ms. Joscelyn Langholt

PROBLEM • The blood-brain barrier (BBB) does not allow drugs that treat neurological diseases, such as Alzheimer’s Disease, to cross from the bloodstream into the brain. • These diseases go untreated and become progressively worse.

PURPOSE • To use the body’s own immune system as a method of transporting drugs across the blood-brain barrier • Filomicelles as a vehicle for drug delivery • Attach filomicelles to T-cells to create filomicelle/T-cell complex. • Filomicelle/T-cell complex crosses blood-brain barrier as part of immune response

BACKGROUND • Blood-Brain Barrier (BBB): selectively permeable membrane that separates the brain from the bloodstream • Filomicelles: Di-block copolymers that can self assemble to form a vehicle for drug delivery

BACKGROUND • T-cells: immune cells with targeting receptors for filomicelle attachment • Immune response: Tcells called to brain as response to inflammation, easier to pass through BBB

METHODOLOGY BBB Model Filomicelles Make healthy BBB Model Infuse dye into filomicelles Make varying levels of compromised BBB using TNF-α and IL-1α Modify surface by adding glycoproteins gp 41 and gp 120, or CD-3 antibodies Control experiment with filomicelles Attach modified filomicelle to Tcell Test filomicelle/T-cell complex ability to permeate the BBB

OBJECTIVE ONE: CREATE A PHYSIOLOGICALLY REPRESENTATIVE BBB MODEL Creating the BBB Model Testing Barrier Properties Disrupting the Barrier Creating the BBB Model Testing Barrier Properties Disrupting the Barrier

CREATING THE BBB MODEL Consists of two parts Creating a hydrogel with Human Brain Microvascular Endothelial Cells appropriate stiffness Polyacrylamide (PA) 0. 2 – 1. 0 Kpa PA gels coated with ECM protein Forming a HBMECs monolayer Extracellular Matrix Cultured according to manufacturer’s protocol p 2 -5 plated on gels Creating the BBB Model Testing Barrier Properties Disrupting the Barrier

TESTING BARRIER PROPERTIES TEER Testing Using a Endohm Chamber and Voltohmeter Starting day 2 after plating Adhesion proteins Visualization of cell borders Primary and secondary antibody staining Creating the BBB Model Testing Barrier Properties HUVEC morphology at monolayer confluency on fibronectin -coated polyacrylamide gels. Scale bar indicate 50 µm. After monolayer formation, HUVECs were treated with Hoechst nuclear stain (blue) and cell borders are stained with anti-βcatenin antibody (green). Disrupting the Barrier

DISRUPTING THE BARRIER TNF-α and IL-1α Concentration in increasing magnitude Representing different diseased states Creating the BBB Model Testing Barrier Properties Disrupting the Barrier

OBJECTIVE TWO: CREATE A FILOMICELLE/T-CELL COMPLEX Isolate T-cells Create filomicelles Make two modifications to filomicelles Let filomicelles attach to T-cells Isolate T -cells Create filomicelles Filomicelle modifications Filomicelle/ T-cell complex

ISOLATING T-CELLS Isolate T-cells from human blood samples Currently writing IRB proposal Protocol involves magnetic labeling Anticipate no problems Magnetic labeling of non T-cells using microbeads (pink) Isolate T -cells Create filomicelles Filomicelle modifications Cells fed through separation column in magnetic field T-cells collected in tube (green) Filomicelle/ T-cell complex

CREATING THE FILOMICELLES Use two co-polymer in chloroform, rehydration techniques Takes ≈3 days We have contact with an expert in filomicelle development Isolate T -cells Create filomicelles Filomicelle modifications Filomicelle/ T-cell complex

FILOMICELLE MODIFICATIONS Modification 1: infusion of dye Purpose: to simulate a real drug inside the filomicelle carrier Modification 2: attachment of proteins to form Filomicelle/T-cell complex Glycoproteins gp 41 and gp 120 OR CD-3 antibody Isolate T -cells Create filomicelles Filomicelle modifications Filomicelle/ T-cell complex

Unmodified filomicelle Isolate T -cells Filomicelle with dye Create filomicelles Filomicelle modifications Filomicelle with dye and targeting moeities Filomicelle/ T-cell complex

FILOMICELLE/T-CELL COMPLEX Culture filomicelles Incubate filomicelles with T-cells & signaling molecules Two possible interactions: T-cell will engulf filomicelle (glycoproteins) Filomicelle will bind to outside of T-cell (antibody) Isolate T -cells Create filomicelles Co-receptors on T-cell Filomicelle modifications T-cell membrane Filomicelle/ T-cell complex

OBJECTIVE 3: TEST FILOMICELLE/T-CELL COMPLEX ON DIFFERENT BBB MODELS Test transmigration abilities of the complex in different BBB models, which represent different stages of disease Control: filomicelle + dye modification only Hypothesis: The filomicelle-T-cell complex will permeate through the BBB models more compared to the control

(BBB models with varying levels of permeability) x 5

ASSESSING MIGRATION Insert in model will be removed Migrated complex and filomicelle will be in solution accumulated at bottom of well Measurements Fluorescence microscopy Image. J Plate reader FACS

ANTICIPATED RESULTS - TESTING Filomicelle/T-cell complex will exhibit more permeability through each degree of disruption in BBB as compared to the control The control filomicelle does not have mechanism to pass through the BBB model T-cell conjugation assists transmigration through BBB permeability increases with increasing concentrations of TNF-α and IL-1α

POTENTIAL OBSTACLES Coagulation of filomicelles on membrane of BBB model Particles may get caught on the BBB model insert Filomicelle and T-cell attachment could dislodge while permeating through BBB Contamination

TIMELINE Spring 2011 – August 2011: Complete Objective 1 Become familiar with techniques and protocols for both BBB models and filomicelles production Create models with TNF-α and IL-1α August 2011 – December 2011: Complete Objective 2 Infuse dye into filomicelle and test fluorescence Create modified complex by adding glycoproteins or antibody to filomicelles Isolate T-cells and create filomicelle/T-cell complex

TIMELINE (CONTINUED) December 2011 -June 2012 - Complete Objective 3 Test the filomicelle/T-cell complex on models Collect data to see how much of the complex crossed the barrier June 2012 -May 2013 Analyze data and submit for publication in a peerreviewed journal (Fall 2012) Write Gemstone Thesis

BUDGET ≈ $25, 000 for supplies and materials ≈ $8, 000 for travel expenses to conferences Continuous grant application

ACKNOWLEDGEMENTS Dr. Aranda-Espinoza – Mentor Carlos Luna and Kim Stroka – Graduate Students Dr. Muro and Dr. Shah – Experts Gemstone staff

REFERENCES Banks W. Developing drugs that can cross the blood-brain barrier: applications to Alzheimer's disease. BMC Neurosci. 2008; 9 Suppl 3: S 2. Rubin L, Hall D, Porter S, et al. A cell culture model of the blood-brain barrier. J Cell Biol. Dec 1991; 115(6): 1725 -1735. Banks WA, Ercal N, Price TO. The Blood-Brain Barrier in Neuro. AIDS. Current HIV Research. 2006; 4(3): 259 -266. Butt AM, Jones HC, Abbott NJ. Electrical Resistance Across the Blood-Brain Barrier in Anaesthetized Rats: A Developmental Study. Journal of Physiology. 1990; 429: 47 -62. Stanness K, Westrum L, Fornaciari E, et al. Morphological and functional characterization of an in vitro blood-brain barrier model. Brain Res. Oct 1997; 771(2): 329 -342. Engler A, Bacakova L, Newman C, Hategan A, Griffin M, Discher D. Substrate compliance versus ligand density in cell on gel responses. Biophysical Journal. Jan 2004; 86(1): 617628. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science. Nov 18 2005; 310(5751): 1139 -1143. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. Aug 25 2006; 126(4): 677 -689 Stroka K. M. and Aranda-Espinoza H. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction. Blood. Submitted. Norman LL, Aranda-Espinoza H. Cortical Neuron Outgrowth is Insensitive to Substrate Stiffness. Cellular and Molecular Bioengineering. Dec 2010; 3(4): 398 -414. Geng Y, Dalhaimer P, Cai S, et al. Shape effects of filaments versus spherical particles in flow and drug delivery. Vol 2: Nature Nanotechnology; 2007: 249 -255. Dalhaimer P, Engler AJ, Parthasarathy R, Discher DE. Targeted Worm Micelles. Vol 5: American Chemical Society Biomacromolecules; 2004: 1714 -1719. Kim Y, Dalhaimer P, Christian DA, Discher DE. Polymeric worm micelles as nano-carriers for drug delivery. Vol 16: IOP Science Nanotechnology; 2005: S 484 -S 491. Qian J, Zhang M, Manners I, Winnik MA. Nanofiber micelles from the self-assembly of block copolymers. Trends Biotechnol. Feb 2010; 28(2): 84 -92. Simone EA, Dziubia TD, Discher DE, Muzykantov VR. Filamentous Polymer Nanocarriers of Tunable Stiffness that Encapsulate the Therapeutic Enzyme Catalase. Vol 10: Biomacromolecules; 2009: 1324 -1330. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell. 5 ed. New York: Garland Science; 2008. Briz V, Poveda E, Soriano V. [HIV entry into the cells--mechanisms and therapeutic possibilities]. Med Clin (Barc). 2006; 126(9): 341 -348. Engelhardt B. Molecular mechanisms involved in T cell migration across the blood-brain barrier. Journal Of Neural Transmission (Vienna, Austria: 1996). 2006; 113(4): 477 -485. Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, Berman JW. CCL 2/Monocyte Chemoattractant Protein-1 Mediates Enhanced Transmigration of Human Immunodeficiency Virus (HIV)-Infected Leukocytes across the Blood–Brain Barrier: A Potential Mechanism of HIV–CNS Invasion and Neuro. AIDS. The Journal of Neuroscience. 2006; 26(4): 9.

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