Nanotechnology Bio

Biomedical
Applications of
Nanotechnology

What is Nanotechnology?
The creation of useful, functional materials,
devices, and systems through:

1. Controlling and manipulating matter on the
nanometer-length scale (1-100 nm), and
2. Exploiting novel phenomena and properties
(physical, chemical, biological, mechanical,
electrical) at the nanoscale.

“Going Small for Big Advances”
“Going Small for Big Advances”

Potential market for nanotechnology ?
$1 trillion business within the next 10 to 15 years.

Nanotechnology in Medicine

• Biological imaging for medical diagnostics.
• Advanced drug delivery systems.
• Biosensors for airborne chemicals or other
toxins.
• Regenerative medicine:
More durable, rejection-resistant
artificial tissues and organs.

NANOMEDICINE
NANOMEDICINE

Targeted Drug Delivery

J Leukoc Biol 2005;78:585

Cardiovascular Obesity /
Disease Diabetes

Risk Factors Inflammation Nanotechnology

Tumor
Neurological
Angiogenesis
Disorder
/ Metastasis

Development of
In Vivo and In Vitro
Inflammatory Disease
Models

In Vivo Brain Inflammation Models

• Environmental Toxicants-Mediated
Brain Inflammation.

• Bacterial Infection-Mediated Brain
Inflammation.

Environmental Toxicants & Brain Inflammation

Isolation of brain regions

C57/BL6 Mouse

Determination of pro-inflammatory mediators
- Cytokines: TNF-α, IL-1β, IL-6
- Chemokine: MCP-1
Chlorpyrifos
- Adhesion molecules: E-selectin, ICAM-1, VCAM-1
[OP Pesticide]

20 *
Control
(TNF-α mRNA/GAPDH mRNA)
CPF, 8 h
CPF, 16 h
Relative Fold Induction

15 CPF, 24 h
*

10
*

5 * *
* *
* *
*
0
HIP COR STR CER

25
(TNF-α mRNA/GAPDH mRNA) Control
CPF, 20 mg/kg *
CPF, 70 mg/kg
20

CPF, 140 mg/kg

15

10
* * *
5 * *
*

0
HIP COR STR CER

6
Control *
CPF, 4 h
(IL-6 mRNA/GAPDH mRNA)
5 CPF, 8 h

CPF, 24 h

4 *
*

3 * *
*
2 *
*

1

0
HIP COR STR CER

7
Control
CPF, 20 mg/kg
*
6 CPF, 70 mg/kg

CPF, 140 mg/kg
5

4 * *

3 *
*
2

1

0
HIP COR STR CER

6 *
Control
(MCP-1 mRNA/GAPDH mRNA)
CPF, 8 h
5 CPF, 16 h

CPF, 24 h

4 *
*

3 * *
*
2

1

0
HIP STR CER

5
(MCP-1 mRNA/GAPDH mRNA) Control *
CPF, 20 mg/kg *
CPF, 70 mg/kg
4

CPF, 140 mg/kg

3
*
*
2

1

0
HIP STR CER

3.0 *
(E-selectin mRNA/GAPDH mRNA)
Control
* CPF, 70 mg/kg
2.5

*
2.0

1.5

1.0

0.5

0.0
HIP COR STR CER

Bacterial Infection & Brain Inflammation

Isolation of brain regions

C57/BL6 Mouse
- Chemokine: MCP-1
Lipopolysaccharide
- Adhesion molecules: E-selectin, ICAM-1, VCAM-1
[LPS]

40 30
* Control

35
LPS *
25

*
30 * *
20
25
*
20 15

15
10
10
5
5
0 0
HIP COR STR CER HIP COR STR CER

4 h Exposure 24 h Exposure

35 12

30
Control * *
LPS

25 9

20
* 6
15 *
10
* 3 *
5

0 0


25 6
* Control *
(IL-1β mRNA/GAPDH mRNA)

LPS
5
20

4
*
15
3 * *
10 * 2
*
5
1

0 0


120 20
Control

LPS
* *
100

* 15
80 *
*
60 10

40 * *
5
20

0 0


15 Control
10
(ICAM-1 mRNA/GAPDH mRNA)

* LPS
*
*

12 * 8

9 6 * *

6 * 4
*
3 2

0 0


20 15

Control
LPS
* *

12
15 * *
9 *
10
* * 6

5
*
3

0 0


In Vitro Brain Inflammation Models

• Brain
Microvascular
Endothelial Cells
(BMEC)

• Astrocytes

• Microglia

Pro-inflammatory Stimuli-Mediated
Inflammation in Brain Cells

Pro-inflammatory Stimuli
[100 ng/ml of LPS]

Culture Media

Brain Cells

- Chemokine: MCP-1
- Adhesion molecules: E-selectin, ICAM-1

Brain Microvascular Endothelial Cells (bEnd.3)

120 10
*

(IL-1β mRNA/GAPDH mRNA)
100 *
8

80
6
60 *
4
40

* 2
20
*
*
0 0
Control 1 2 4 8 Control 1 2 4 8

Exposure Time (hours) Exposure Time (hours)


20 100
*

*

80


15 *

60
*
10
* 40
*

5
20
*
*
0 0



30 * 12

(ICAM-1 mRNA/GAPDH mRNA)
*
25


* 9
20 *
15 6
*
10
3
5 *
0 0



0
10
20
30
40
50

Control
*

LPS
0 (IL-1β mRNA/GAPDH mRNA)
1
2
800
900
1000
1100
Microglia (BV-2)

Control
*

LPS


0
1
2
6500
7000
7500
8000

Control
*

LPS
0 (MCP-1 mRNA/GAPDH mRNA)
10
20
30
40
50
60
70
80
90
Microglia (BV-2)

Control
*

LPS


0
1
2
330
360
390
420

Control
*

LPS
0
5
10
15
20
25
30
35
40
45
Astrocytes (C6)

Control
*

LPS


0
10
20
30
40
50
60
70
80

Control
*
Astrocytes (C6)

LPS

In Vitro Ischemia/Reperfusion Model

A Novel Design of Double-
Layer Parallel-Plate Flow
Chamber & Its Biomedical
Application

In Vitro Flow Chamber Systems

Cone-Plate Orbital Shaker

Artificial Capillary Parallel-Plate

Parallel-plate flow chambers (PPFC) have been
most commonly used for its simplicity of concept
x

y
l
h

w z

Flow enters the parallel plates at the origin and exits
where x equals the length of the chamber, l

Conventional PPFCs have shown weaknesses and
problems in several aspects of its design

To eliminate these problems, we designed and
developed a new double-layer PPFC
• Accepts up to four glass slides facing each
other so that the flow within the channel is
exclusively formed by endothelial cells.
• Provides a total of 96 cm2 cell
monolayer per chamber.

• Placing glass slides in
series shortens the
duration of procedure.
• The multilayer design only
requires 2D cutting, which is
easier and faster to manufacture
and modify.

The system becomes much simpler with the new chamber.

The new double-layer PPFC consists of separate
layers of different materials and thicknesses
Acrylic sheets of 0.08 inch thickness

Acrylic sheets of 0.5 inch thickness

Each acrylic layer was cut
by Laser Computer Aided
Modeling and Manufacture
(LaserCAMM) machine.

The system is a
computerized laser cutter
that uses a laser beam to
cut sheet materials into
intricate patterns with a
high degree of accuracy.

Silicone gaskets of 0.03 inch thickness

Silicone gasket of 0.01 inch thickness

Silicone gaskets of 0.03
inch thickness serve as a
firm grip for glass slides.

The silicone gasket in the
middle constitutes the
channel height, h, and the
width, w.

Glass slides to fill up space

Glass slides which will have
cells seeded

Up to four glass slides can
be entered in a chamber.
Glass slides or
endothelial monolayers
are placed between the
gasket in the middle.
Placing endothelial
monolayers on both sides
of channel minimizes
pressure loss while having
a larger effective area.


Media enters through
the inlets.
Fills up a small reservoir
formed in the gasket.
Spreads evenly across
width through a thin slit.
Flows across the
endothelial monolayer.
Escapes the chamber
through the thin slit, the
small reservoir, and the
outlets.

To set up the chamber bubble-free,
the layers are installed in the order
from the bottom to the top layers
where the flow channel, reservoirs
are filled up with media by means of
syringe as each layer is piled up.

A flow loop system provides a constant
hydrostatic pressure to the PPFC

Upper
Reservoir
Flow

Flow
Meter

Peristaltic
Pump

Lower
PPFC Reservoir

• The streamlines near the lateral walls were not disturbed
ensuring that the lateral wall effects are negligible.
• The chamber clearly applies a uniform magnitude of shear
stress throughout the entire surface where endothelial cell
monolayer will be placed.

RBE4 HMEC-1

Static

Flow

1.4
1.2 Static
1.2 Flow

(Relative Fold Induction)
(Relative Fold Induction)
IL-6 Gene Expression

Gene Expression
0.9 1.0

0.8
0.6
* *
0.6
*
0.4
0.3
*
0.2

0.0 0.0
Static Flow ICAM-1 VCAM-1 E-selectin

7
Normal Flow

(IL-6 mRNA/β-Actin mRNA)
6 Ischemia/Reperfusion *
5

4

3

2

1

0
0.5 1 6 12 24 (hours)

Ischemia Reperfusion

5
Normal Flow *
(MCP-1 mRNA/β-Actin mRNA)
Ischemia/Reperfusion
4 *

3

2

1

0
0.5 1 6 12 24 (hours)


3

(ICAM-1 mRNA/β-Actin mRNA)
Normal Flow
*

2

1

0
0.5 1 6 12 24 (hours)


6

(VCAM-1 mRNA/β-Actin mRNA)
Normal Flow
5
Ischemia/Reperfusion *

4

3
*
2

1

0
0.5 1 6 12 24 (hours)


5

(E-selectin mRNA/β-Actin mRNA)
Normal Flow
*
4 *

3

2

1

0
0.5 1 6 12 24 (hours)


Nanotechnology Bio

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