Transcript Breaking through the Blood

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 gp41 and gp120, 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
appropriate stiffness
Human Brain Microvascular
Endothelial Cells
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
 p2-5 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-β-catenin 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 gp41 and gp120 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)
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

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

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QUESTIONS?