Team B³Team B³Breaking through the Blood-Brain BarrierBreaking through the Blood-Brain Barrier

Mentor: Dr. Helim Aranda-EspinozaLibrarian: 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: T-cells called to brain as response to inflammation, easier to pass through BBB

METHODOLOGYBBB Model Filomicelles

OBJECTIVE ONE: CREATE A PHYSIOLOGICALLY REPRESENTATIVE BBB MODEL

Creating the BBB Model Testing Barrier Properties Disrupting the Barrier

CREATING THE BBB MODEL Consists of two parts

Creating a hydrogel with appropriate stiffness

Polyacrylamide (PA) 0.2 – 1.0 Kpa PA gels coated with ECM protein

Forming a HBMECs monolayer Cultured according to manufacturer’s protocol p2-5 plated on gels

Extracellular Matrix

Human Brain MicrovascularEndothelial Cells

TESTING BARRIER PROPERTIES TEER Testing

Using a Endohm Chamber and VoltohmeterStarting day 2 after plating

Adhesion proteinsVisualization of cell bordersPrimary and secondary antibody staining

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-β-catenin antibody (green).

DISRUPTING THE BARRIER TNF-α and IL-1α Concentration in increasing magnitude Representing different diseased states

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)

T-cells collected in tube (green)

Cells fed through separation column in magnetic field

Isolate T-cells

Create filomicelles

Filomicelle modifications

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 complexGlycoproteins gp41 and gp120 ORCD-3 antibody

Isolate T-cells

Create filomicelles

Filomicelle modifications

Filomicelle/T-cell complex

Unmodified filomicelle Filomicelle with dye Filomicelle with dye and targeting moeities

Isolate T-cells

Create filomicelles

Filomicelle modifications

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) Co-receptors

on T-cell

T-cell membrane

Isolate T-cells

Create filomicelles

Filomicelle modifications

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)

x5

Assessing Migration Insert in model will be removed Migrated complex and filomicelle will be in solution accumulated at bottom of well Measurements

Fluorescence microscopy ImageJ 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 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

TIMELINESpring 2011 – August 2011: Complete Objective 1Become familiar with techniques and protocols for

both BBB models and filomicelles productionCreate models with TNF-α and IL-1α

August 2011 – December 2011: Complete Objective 2Infuse dye into filomicelle and test fluorescenceCreate modified complex by adding glycoproteins or

antibody to filomicellesIsolate T-cells and create filomicelle/T-cell complex

TIMELINE (CONTINUED)December 2011-June 2012- Complete Objective 3Test the filomicelle/T-cell complex on models Collect data to see how much of the complex crossed

the barrier

June 2012-May 2013Analyze data and submit for publication in a peer-

reviewed 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

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QUESTIONS?