nanotechnology bio

Biomedical Biomedical Applications of Applications of NanotechnologyNanotechnology

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””

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

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

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

Targeted Drug DeliveryTargeted Drug Delivery

Risk Factors

CardiovascularCardiovascularDisease Disease

NeurologicalNeurologicalDisorder Disorder

Inflammation Inflammation

Obesity /Obesity /DiabetesDiabetes

TumorTumorAngiogenesis Angiogenesis / Metastasis/ Metastasis

NanotechnologyNanotechnology

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

Inflammatory Disease Inflammatory Disease ModelsModels

•• 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

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

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

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

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

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

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

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

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

) * *

*

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

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

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

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

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

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

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

In Vitro Brain Inflammation ModelsIn Vitro Brain Inflammation Models

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

•• AstrocytesAstrocytes

•• MicrogliaMicroglia

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

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)

*

*

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)

*

*

*

*

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)

*

*

Microglia (BVMicroglia (BV--2)2)

R

elat

ive

Fold

Indu

ctio

n(T

NF-α

mR

NA

/GA

PDH

mR

NA

)

0

10

20

30

40

50

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

*

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

ion

(MC

P-1

mR

NA

/GA

PDH

mR

NA

)0

10

20

30

40

50

60

70

80

90

Control LPS

*

Astrocytes (C6)Astrocytes (C6)

R

elat

ive

Fold

Indu

ctio

n(T

NF-α

mR

NA

/GA

PDH

mR

NA

)

0

1

2

330

360

390

420

Control LPS

*

Rel

ativ

e Fo

ld In

duct

ion

(IL

-6 m

RN

A/G

APD

H m

RN

A)

0

5

10

15

20

25

30

35

40

45

Control LPS

*

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

70

80

Control LPS

*

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

In Vitro Flow Chamber Systems In Vitro Flow Chamber Systems

Cone-Plate

Artificial Capillary

Orbital Shaker

Parallel-Plate

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

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

• 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.

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.

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

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.

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.

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.

PPFC

Flow Meter

Peristaltic Pump

Lower Reservoir

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

Upper Reservoir

Flow

• 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.

RBE4

Static

Flow

HMEC-1

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

*

*

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

1

2

3

4

5

6

7Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

Ischemia Reperfusion

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

2

3

4

5Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

Ischemia Reperfusion

*

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

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

1

2

3

4

5

6Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

*

*

Ischemia Reperfusion

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

2

3

4

5Normal FlowIschemia/Reperfusion

0.5 1 6 12 24 (hours)

**

Ischemia Reperfusion