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Biomedical Biomedical Applications of Applications of Nanotechnology Nanotechnology

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Page 1: Nanotechnology Bio

Biomedical Biomedical Applications of Applications of NanotechnologyNanotechnology

Page 2: Nanotechnology Bio

What is Nanotechnology?What is Nanotechnology?

The creation of useful, functional materials,The creation of useful, functional materials,devices, and systems through: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 AdvancesGoing Small for Big Advances””

Page 3: Nanotechnology Bio

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

Page 4: Nanotechnology Bio

Nanotechnology in MedicineNanotechnology 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.

NANOMEDICINENANOMEDICINENANOMEDICINE

Page 5: Nanotechnology Bio

J J LeukocLeukoc BiolBiol 2005;78:5852005;78:585

Targeted Drug DeliveryTargeted Drug Delivery

Page 6: Nanotechnology Bio

Risk Factors

CardiovascularCardiovascularDisease Disease

NeurologicalNeurologicalDisorder Disorder

Inflammation Inflammation

Obesity /Obesity /DiabetesDiabetes

TumorTumorAngiogenesis Angiogenesis / Metastasis/ Metastasis

NanotechnologyNanotechnology

Page 7: Nanotechnology Bio

Development of Development of In VivoIn Vivo and and In VitroIn Vitro

Inflammatory Disease Inflammatory Disease ModelsModels

Page 8: Nanotechnology Bio

•• Environmental ToxicantsEnvironmental Toxicants--Mediated Mediated Brain Inflammation.Brain Inflammation.

•• Bacterial InfectionBacterial Infection--Mediated Brain Mediated Brain Inflammation.Inflammation.

In Vivo Brain Inflammation ModelsIn Vivo Brain Inflammation Models

Page 9: Nanotechnology Bio

Environmental Toxicants & Brain InflammationEnvironmental Toxicants & Brain Inflammation

C57/BL6 MouseC57/BL6 Mouse

ChlorpyrifosChlorpyrifos[OP Pesticide][OP Pesticide]

Determination of proDetermination of pro--inflammatory mediatorsinflammatory mediators-- Cytokines: TNFCytokines: TNF--αα, IL, IL--11ββ, IL, IL--66-- ChemokineChemokine: MCP: MCP--11-- Adhesion molecules: EAdhesion molecules: E--selectin, ICAMselectin, ICAM--1, VCAM1, VCAM--11

Isolation of brain regionsIsolation of brain regions

Page 10: Nanotechnology Bio

0

5

10

15

20ControlCPF, 8 hCPF, 16 hCPF, 24 h

Rel

ativ

e Fo

ld In

duct

ion

(TN

F-α

mR

NA

/GA

PDH

mR

NA

)

*

**

*

*

*

*

**

*

HIP COR STR CER

Page 11: Nanotechnology Bio

0

5

10

15

20

25ControlCPF, 20 mg/kgCPF, 70 mg/kgCPF, 140 mg/kg

Rel

ativ

e Fo

ld In

duct

ion

(TN

F-α

mR

NA

/GA

PDH

mR

NA

)

** * *

*

* *

HIP COR STR CER

Page 12: Nanotechnology Bio

0

1

2

3

4

5

6ControlCPF, 4 hCPF, 8 hCPF, 24 h

R

elat

ive

Fold

Indu

ctio

n(I

L-6

mR

NA

/GA

PDH

mR

NA

)

*

*

**

* *

*

*

HIP COR STR CER

Page 13: Nanotechnology Bio

0

1

2

3

4

5

6

7ControlCPF, 20 mg/kgCPF, 70 mg/kgCPF, 140 mg/kg

R

elat

ive

Fold

Indu

ctio

n(I

L-6

mR

NA

/GA

PDH

mR

NA

)

*

*

*

*

*

HIP COR STR CER

Page 14: Nanotechnology Bio

0

1

2

3

4

5

6ControlCPF, 8 hCPF, 16 hCPF, 24 h

Rel

ativ

e Fo

ld In

duct

ion

(MC

P-1

mR

NA

/GA

PDH

mR

NA

)

*

*

*

*

*

*

HIP STR CER

Page 15: Nanotechnology Bio

0

1

2

3

4

5ControlCPF, 20 mg/kgCPF, 70 mg/kgCPF, 140 mg/kg

Rel

ativ

e Fo

ld In

duct

ion

(MC

P-1

mR

NA

/GA

PDH

mR

NA

)

*

*

* *

HIP STR CER

Page 16: Nanotechnology Bio

0.0

0.5

1.0

1.5

2.0

2.5

3.0 ControlCPF, 70 mg/kg

HIP COR STR CER

R

elat

ive

Fold

Indu

ctio

n(E

-sel

ectin

mR

NA

/GA

PDH

mR

NA

) * *

*

Page 17: Nanotechnology Bio

Bacterial Infection & Brain InflammationBacterial Infection & Brain Inflammation

C57/BL6 MouseC57/BL6 Mouse

LipopolysaccharideLipopolysaccharide[LPS][LPS]

Isolation of brain regionsIsolation of brain regions

Determination of proDetermination of pro--inflammatory mediatorsinflammatory mediators-- Cytokines: TNFCytokines: TNF--αα, IL, IL--11ββ, IL, IL--66-- ChemokineChemokine: MCP: MCP--11-- Adhesion molecules: EAdhesion molecules: E--selectin, ICAMselectin, ICAM--1, VCAM1, VCAM--11

Page 18: Nanotechnology Bio

0

5

10

15

20

25

30

35

40 ControlLPS

HIP COR STR CER

R

elat

ive

Fold

Indu

ctio

n(T

NF-α

mR

NA

/GA

PDH

mR

NA

) *

*

*

4 h Exposure4 h Exposure

0

5

10

15

20

25

30

HIP COR STR CER

**

*

24 h Exposure24 h Exposure

Page 19: Nanotechnology Bio

0

5

10

15

20

25

30

35

ControlLPS

HIP COR STR CER

Rel

ativ

e Fo

ld In

duct

ion

(IL

-6 m

RN

A/G

APD

H m

RN

A)

*

*

*

*

0

3

6

9

12

HIP COR STR CER

*

*

4 h Exposure4 h Exposure 24 h Exposure24 h Exposure

Page 20: Nanotechnology Bio

0

5

10

15

20

25ControlLPS

HIP COR STR CER

Rel

ativ

e Fo

ld In

duct

ion

(IL

-1β

mR

NA

/GA

PDH

mR

NA

) *

*

**

0

1

2

3

4

5

6

HIP COR STR CER *

*

*

4 h Exposure4 h Exposure 24 h Exposure24 h Exposure

Page 21: Nanotechnology Bio

0

20

40

60

80

100

120 ControlLPS

HIP COR STR CER

R

elat

ive

Fold

Indu

ctio

n(M

CP-

1 m

RN

A/G

APD

H m

RN

A)

*

*

*

*

0

5

10

15

20

HIP COR STR CER

*

*

*

4 h Exposure4 h Exposure 24 h Exposure24 h Exposure

Page 22: Nanotechnology Bio

0

3

6

9

12

15 ControlLPS

HIP COR STR CER

R

elat

ive

Fold

Indu

ctio

n(I

CA

M-1

mR

NA

/GA

PDH

mR

NA

)

**

*

*

0

2

4

6

8

10

HIP COR STR CER

*

*

*

*

4 h Exposure4 h Exposure 24 h Exposure24 h Exposure

Page 23: Nanotechnology Bio

0

5

10

15

20ControlLPS

HIP COR STR CER

R

elat

ive

Fold

Indu

ctio

n(E

-sel

ectin

mR

NA

/GA

PDH

mR

NA

)

*

*

* *

0

3

6

9

12

15

HIP COR STR CER

*

*

**

4 h Exposure4 h Exposure 24 h Exposure24 h Exposure

Page 24: Nanotechnology Bio

In Vitro Brain Inflammation ModelsIn Vitro Brain Inflammation Models

•• Brain Brain MicrovascularMicrovascularEndothelial CellsEndothelial Cells(BMEC)(BMEC)

•• AstrocytesAstrocytes

•• MicrogliaMicroglia

Page 25: Nanotechnology Bio

ProPro--inflammatory Stimuliinflammatory Stimuli[100 [100 ngng/ml of LPS]/ml of LPS]

Determination of proDetermination of pro--inflammatory mediatorsinflammatory mediators-- Cytokines: TNFCytokines: TNF--αα, IL, IL--11ββ, IL, IL--66-- ChemokineChemokine: MCP: MCP--11-- Adhesion molecules: EAdhesion molecules: E--selectin, ICAMselectin, ICAM--11

ProPro--inflammatory Stimuliinflammatory Stimuli--Mediated Mediated Inflammation in Brain CellsInflammation in Brain Cells

Brain CellsBrain Cells

Culture MediaCulture Media

Page 26: Nanotechnology Bio

Brain Microvascular Endothelial Cells (bEnd.3)Brain Microvascular Endothelial Cells (bEnd.3)

R

elat

ive

Fold

Indu

ctio

n(T

NF-α

mR

NA

/GA

PDH

mR

NA

)

0

20

40

60

80

100

120

Control 1 2 4 8

Exposure Time (hours)

*

*

**

Rel

ativ

e Fo

ld In

duct

ion

(IL

-1β

mR

NA

/GA

PDH

mR

NA

)0

2

4

6

8

10

Control 1 2 4 8

Exposure Time (hours)

*

*

Page 27: Nanotechnology Bio

Brain Microvascular Endothelial Cells (bEnd.3)Brain Microvascular Endothelial Cells (bEnd.3)

Rel

ativ

e Fo

ld In

duct

ion

(MC

P-1

mR

NA

/GA

PDH

mR

NA

)0

20

40

60

80

100

Control 1 2 4 8

Exposure Time (hours)

*

*

*

*

R

elat

ive

Fold

Indu

ctio

n(I

L-6

mR

NA

/GA

PDH

mR

NA

)

0

5

10

15

20

Control 1 2 4 8

Exposure Time (hours)

*

*

*

*

Page 28: Nanotechnology Bio

Brain Microvascular Endothelial Cells (bEnd.3)Brain Microvascular Endothelial Cells (bEnd.3)

Rel

ativ

e Fo

ld In

duct

ion

(E-s

elec

tin m

RN

A/G

APD

H m

RN

A)

0

5

10

15

20

25

30

Control 1 2 4 8

Exposure Time (hours)

*

*

*

*

Rel

ativ

e Fo

ld In

duct

ion

(IC

AM

-1 m

RN

A/G

APD

H m

RN

A)

0

3

6

9

12

Control 1 2 4 8

Exposure Time (hours)

*

*

Page 29: Nanotechnology Bio

Microglia (BVMicroglia (BV--2)2)

R

elat

ive

Fold

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ctio

n(T

NF-α

mR

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/GA

PDH

mR

NA

)

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Control LPS

*

Rel

ativ

e Fo

ld In

duct

ion

(IL

-1β

mR

NA

/GA

PDH

mR

NA

)0

1

2

800

900

1000

1100

Control LPS

*

Page 30: Nanotechnology Bio

Microglia (BVMicroglia (BV--2)2)

Rel

ativ

e Fo

ld In

duct

ion

(IL

-6 m

RN

A/G

APD

H m

RN

A)

0

1

2

6500

7000

7500

8000

Control LPS

*

Rel

ativ

e Fo

ld In

duct

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(MC

P-1

mR

NA

/GA

PDH

mR

NA

)0

10

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Control LPS

*

Page 31: Nanotechnology Bio

Astrocytes (C6)Astrocytes (C6)

R

elat

ive

Fold

Indu

ctio

n(T

NF-α

mR

NA

/GA

PDH

mR

NA

)

0

1

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360

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420

Control LPS

*

Rel

ativ

e Fo

ld In

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(IL

-6 m

RN

A/G

APD

H m

RN

A)

0

5

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45

Control LPS

*

Page 32: Nanotechnology Bio

Astrocytes (C6)Astrocytes (C6)

Rel

ativ

e Fo

ld In

duct

ion

(MC

P-1

mR

NA

/GA

PDH

mR

NA

)

0

10

20

30

40

50

60

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80

Control LPS

*

Page 33: Nanotechnology Bio

In Vitro Ischemia/Reperfusion ModelIn Vitro Ischemia/Reperfusion Model

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

Application Application

Page 34: Nanotechnology Bio

In Vitro Flow Chamber Systems In Vitro Flow Chamber Systems

Cone-Plate

Artificial Capillary

Orbital Shaker

Parallel-Plate

Page 35: Nanotechnology Bio

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

z

y

x

l

w

h

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

Page 36: Nanotechnology Bio

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

Page 37: Nanotechnology Bio

To eliminate these problems, we designed and developed a new double-layer PPFC

• The multilayer design onlyrequires 2D cutting, which iseasier and faster to manufactureand modify.

• Accepts up to four glass slides facing eachother so that the flow within the channel is exclusively formed by endothelial cells.

• Provides a total of 96 cm2 cellmonolayer per chamber.

• Placing glass slides in series shortens theduration of procedure.

Page 38: Nanotechnology Bio

The system becomes much simpler with the new chamber.

Page 39: Nanotechnology Bio

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.

Page 40: Nanotechnology Bio

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.

The new double-layer PPFC consists of separate layers of different materials and thicknesses

Page 41: Nanotechnology Bio

The new double-layer PPFC consists of separate layers of different materials and thicknesses

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 monolayersare placed between the gasket in the middle.

Placing endothelial monolayers on both sides of channel minimizes pressure loss while having a larger effective area.

Page 42: Nanotechnology Bio

The new double-layer PPFC consists of separate layers of different materials and thicknesses

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.

Page 43: Nanotechnology Bio

The new double-layer PPFC consists of separate layers of different materials and thicknesses

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.

Page 44: Nanotechnology Bio

PPFC

Flow Meter

Peristaltic Pump

Lower Reservoir

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

Upper Reservoir

Flow

Page 45: Nanotechnology Bio

• The streamlines near the lateral walls were not disturbedensuring that the lateral wall effects are negligible.

• The chamber clearly applies a uniform magnitude of shearstress throughout the entire surface where endothelial cellmonolayer will be placed.

Page 46: Nanotechnology Bio

RBE4

Static

Flow

HMEC-1

Page 47: Nanotechnology Bio

IL

-6 G

ene

Exp

ress

ion

(Rel

ativ

e Fo

ld In

duct

ion)

0.0

0.3

0.6

0.9

1.2

Static Flow

*

Gen

e E

xpre

ssio

n(R

elat

ive

Fold

Indu

ctio

n)0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4StaticFlow

*

ICAM-1 VCAM-1 E-selectin

*

*

Page 48: Nanotechnology Bio

In Vitro Ischemia/Reperfusion Model In Vitro Ischemia/Reperfusion Model

R

elat

ive

Fold

Indu

ctio

n(I

L-6

mR

NA

/ β-A

ctin

mR

NA

)

0

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7Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

Ischemia Reperfusion

Page 49: Nanotechnology Bio

In Vitro Ischemia/Reperfusion Model In Vitro Ischemia/Reperfusion Model

Rel

ativ

e Fo

ld In

duct

ion

(MC

P-1

mR

NA

/ β-A

ctin

mR

NA

)

0

1

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4

5Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

Ischemia Reperfusion

*

Page 50: Nanotechnology Bio

In Vitro Ischemia/Reperfusion Model In Vitro Ischemia/Reperfusion Model

Rel

ativ

e Fo

ld In

duct

ion

(IC

AM

-1 m

RN

A/ β

-Act

in m

RN

A)

0

1

2

3Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

Ischemia Reperfusion

Page 51: Nanotechnology Bio

In Vitro Ischemia/Reperfusion Model In Vitro Ischemia/Reperfusion Model

Rel

ativ

e Fo

ld In

duct

ion

(VC

AM

-1 m

RN

A/ β

-Act

in m

RN

A)

0

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6Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

*

Ischemia Reperfusion

Page 52: Nanotechnology Bio

In Vitro Ischemia/Reperfusion Model In Vitro Ischemia/Reperfusion Model

Rel

ativ

e Fo

ld In

duct

ion

(E-s

elec

tin m

RN

A/ β

-Act

in m

RN

A)

0

1

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4

5Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

**

Ischemia Reperfusion