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Transcript of 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