INCREASING ENDOTHELIAL LAYER PERMEABILITY BY COUPLING SONOPORATION WITH MICROBUBBLES

DARWIN KWOK12, BRANDON HELFIELD1, XUCAI CHEN1, FLORDELIZA S. VILLANUEVA1

1.   Center for Ultrasound Molecular Imaging and Therapeutics, University of Pittsburgh Medical Center 2. Carnegie Mellon University, Carnegie Institute of Technology, Biomedical Engineering Department

INTRODUCTION/BACKGROUND IN TODAY’S STANDARD of clinical care, the most common treatments for cancer include surgery, chemotherapy, and radiation therapy – all of which results in immediate post-operational trauma.1 •  Researchers have pioneered a non-invasive method of cancer treatment called

“targeted therapy”. •  One difficulty in this method is the inaccessibility of certain locations in which

cancer bodies can manifest. •  For example, a tumor evolving in the brain will be protected from drug delivery

due to the impermeability of the blood-brain barrier.

MICROBUBBLES are created as a phospholipid micelle coated with a cell-specific antibody and filled with a therapeutic gene or drug. •  Microbubbles are clinically employed as intravascular contrast agents for

ultrasound imaging •  Microbubbles are typically encapsulated by phospholipids, between 1 to 10

microns in diameter, oscillate in an ultrasound field (Figure 1). •  Recently, ultrasound-stimulated microbubbles have been employed as a

potential approach to targeted therapeutic delivery.2,3 •  The mechanisms of ultrasound-coupled delivery remains poorly understood.

Figure 1: An individual 2.88 μm phospholipid encapsulated gas microbubble situated in water, insonicated with a 1 MHz, 8-cycle tapered pulse at 0.25 MPa. Video is recorded with 120x magnification at 10.83 Mfps. (courtesy of Brandon Helfield)

SHEAR STRESS caused by the coupling of sonoporation causes the reorganization of the intercellular junctions of endothelial cells. •  Sonoporation potentially increases vascular and cellular permeability by

oscillating microbubbles adjacent to the endothelial layer. (Figure 2) •  The primary function of tight junctions is to prevent the passage of molecules

and ions between the gaps of adjacent cells, forcing materials to actually enter cells by diffusion or active transport.

Figure 2: Ultrasound application on a microbubble delivering a therapeutic drug to a target tissue

HYPOTHESIS The application of sonoporation (ultrasound and microbubble coupling) on an in vitro representation of an endothelium layer leads to intercellular junction reorganization resulting in an increased permeability of the arterial endothelium. OBJECTIVE: To determine the ultrasound parameters and microbubble concentration that permits the highest drug diffusion rate through a monolayer of human umbilical vein endothelial cells (HUVECs).

METHODS/PROTOCOL INVERTED CELL PLATING is required to plate HUVECs on the bottom side of a transwell (since microbubbles on the bottom solution will float upwards to the cells). •  1 x 106 HUVECs in 600 μL of EBM-2 media is plated on multiple transwells •  Each transwell is 24 mm in diameter with pore sizes of 3.0 μm •  An apparatus comprised of an elevated petri dish lid is used to prevent media from

perforating through the membrane by applying a counteractive upwards tensile force •  The apparatus (Figure 3) is incubated for 12 hours before re-inverting into a 2.5 mL of

EBM-2 in a 6-well transwell (Figure 4) with 1.5 mL of EBM-2 added above the transwell DELIVERY

DIFFUSION PROFILING is performed after the plated transwells have been incubated for 4 days to allow cell growth and monolayer development •  Four transwell conditions will be evaluated: 1) Non-plated, 2) HUVEC-plated,

3) HUVEC-plated w/ 30 sec US (0.25 MPa), 4) HUVEC-plated w/ 30 sec US (0.50 MPa) •  2.5 uL of a 0.010 g/mL FITC-Dextran is dissolved in 2.5 mL of EBM-2 media below the

transwell, and 1.5 mL EBM-2 with no fluorescein is added above. •  50 μL is taken from the solution above the transwell at t = 0 hours and t = 1 hour and

placed into a 384-well plate for overhead fluorescent imaging to determine the concentration of FITC-Dextran diffused above (translated with a calibration curve).

•  Hoechst stain is applied after experimentation, and 5 images at 20x magnification are taken with a DAPI filter to confirm cell density.

RESULTS

Figure 5: Graphical representation of final FITC-Dextran concentrations on top transwell between transwell conditions

•  With ultrasound data (n = 2), without ultrasound data (n = 3)

RESULTS DAPI FLUORESCENCE IMAGING is conducted on each of the aforementioned seeded-conditions. The following images are taken at 20x mag with 0.5 s exposure (Figure 6)

CONCLUSION •  Inverted transwell cell culture is made more efficient by using a novel approach

and apparatus (Figure 3) in retaining media above the membrane. •  Diffusion rate of FITC-Dextran is correlated to the increase in ultrasound

pressure. •  At high pressures of ultrasound (~0.50 Mpa), sonoporation begins to damage

cell monolayer integrity. •  Analogous cell densities between transwells treated with mild ultrasound and

transwells not treated with ultrasound suggests cell migration and dissociation of tight junctions.

FUTURE WORK •  Determine optimal ultrasound pressure and duration that minimizes cell damage

while influencing tight junction dissociation. •  Introduce a secondary layer of cells (on the topside of the transwell membrane)

to create a cellular bilayer to better mimic in vivo conditions. •  Find a suitable Cell-Tracker dye that will evaluate cell coverage rather than a cell

density basis using Hoechst staining (Cell-Tracker Orange proved ineffective). •  Use modified approach to create a diffusion profile curve of transwell conditions

REFERENCES 1.   National Institutes of Health, Cancer Treatment. (n.d.), from http://www.cancer.gov/about-cancer/treatment. 2.   Carson AR, McTiernan CF, Lavery L, Grata M, Leng X, Wang J, Chen X, Villanueva FS. Ultrasound-targeted microbubble

destruction to deliver siRNA cancer therapy. Cancer Res. 2012;72:6191–9. 3.   Kopechek JA, Carson AR, Mctiernan CF, Chen X, Hasjim B, Sen M, Grandis JR, Villanueva FS. Ultrasound Targeted

Microbubble Destruction- Mediated Delivery of a Transcription Factor Decoy In- hibits STAT3 Signaling and Tumor Growth. Theranostics 2015;5:1–10.

ACKNOWLEDGEMENTS

A special thanks to Dr. Flordeliza Villanueva for her continued support throughout this project. A big thanks to Brandon Helfield and

Xucai Chen for their dedicated help and advice in every step of this project.

Figure 3: Inverted cell seeding (right) will allow cells to be exposed to the microbbubles present on the bottom solution (left)

Figure 4: The seeded HUVECs on the inverted transwell (left) is flipped into a well of 2.5 mL EBM-2 (right) after incubating for 12 hr

Figure 6: DAPI fluorescence imaging of HUVEC-plated transwells treated with no ultrasound (left), 0.25 MPa ultrasound (center), and 0.50 MPa ultrasound.

DRUG DELIVERY APPLICATIONS

100 μm

100 μm

100 μm

2.12E-03

3.06E-04 5.03E-04

1.79E-03

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

3.00E-03

No Cell w/ Cell w/ US (0.25 MPa) w/ US (0.50 MPa)

Conc

entra

tion

(g/m

L)

Transwell Condition

Comparison of Final Concentrations between Treatments