The effects of particle size, coating,

and reactivity on cell function

Tatsiana Mironava, Zhi Pan, Ying Liu, Nicole Elstein, Sowmya Sundaresh,
Miriam H. Rafailovich, Wilson Lee, Nadine Pernodet, Marcia Simon, Michael
Hadjiagarou

SUNY at Stony Brook, Stony Brook, NY 11794-2275

 Nanotechnology is a Strategic Field with Numerous
Applications in:

• Medicine (Tissue engineering, Drug

delivery, Diagnostics)
• Chemistry ( Cosmetics, Catalysis,
Filtration, Household, Foods)

• Electronics (Single Electron

Transistors, Spintronic, Hard Disks,
0,85 inch hard disk drive stores up to 4 GB of data.
MRAMs, Quantum dot lasers)

• Materials (Nanoparticle-strengthened
steels, Anti-reflection layers, Nanotube
field emission displays)

• Environment (Increased efficiency in

energy production, Reduction of energy
consumption, Recycling of batteries)
A “Lab-on-a-Chip” measures the reaction of up to 400
known genes to an applied substance.
 Hair from Mummies
Paints
Nanoparticles are used
ancient. Instruments to metallic
particles
observe them are new! for shine

Electron microscope image of hair dye

Carbon
Black in
Ancient
Egypt:
Makeup
 Carbon Black Today

Single walled nanotubes:

nanotubes
Extremely strong

Carbon Nanoparticle (atomic planes): Turbostatic

High resolution TEM image:

• Carbon Particles are crystalline and turbostatic.
•Lattice plains trap polymer chains.
• Confinement increases viscosity and strength
Maybe not so good!
Delamination of Rubber tires:
Exxon/Sid Richardson Collaboration
Effect of Carbon Black and Silica Fillers in Elastomer Blends Macromolecules,
2001. 34(20): p. 7056-65.

•Tires contain about 15 kinds of

rubber.
•Proper adhesion between
components is needed for safety
and performance.
Effect of Carbon Black on
rubber interfaces:
•Initially broad interface (100nm)
reduced to 20 nm with 1% carbon.
•Interfacial motion frozen at 2nm at
5% carbon.
Clay: another nanoparticle from
ancient times
Known to ancients to
have good materials
properties.

• Easy to process and

mold.
• Flame resistant (baked
in high temperature
kilns)
• Medicinal properties-
aids in wound healing
• Biblical-associated
with life.
Clay today: Natural montmorillonites Layers
AlSi Platelets
•Clays are layered aluminum silicate
compounds held together by ionic charges.
•In order to separate platelets they are
functionalized with Di-tallow molecules which
renders them hydrophobic and easy to exfoliate
d
in organic solvents.
•Exfoliation yields a very large surface to
d =24.2Å
volume ratio. www.nanoclay.com
HT: Hydrogenated
Di-tallow functional
group attached to
platelets.
 Can Plexiglass replace glass?

Glass shatters, weighs

more, difficult to mold.
BUT-has good thermal
properties.

PMMA SPN 5 wt% SPN 15 wt% SEN 5 wt% SEN 15 wt%

• Clays are smaller than the wavelength of light.

• Compound is optically clear, moldable, shatterproof.
• Is it still flammable?
RecyclingM. Si et al Macromolecules, 2006
Plastic recycling industry

Melt Mixed

PS/PMMA/
PETE/C20A
Clay

PS/PMMA/C20A PS/PMMA PS/PMMA/PET/C20A/C6A PS/PMMA/PET

Clay blend
7 GPa

Blend
5 GPa

Recycled
Blend
3 GPa
Polymers and Clay: Large variety of nanocomposites
• Enables synthetic substitutes of natural materials
i.e. glass, metals, wood, and fibers.
• Nanocomposites can be engineered to be flexible,
shatterproof, lightweight, and flame resistant.

Cotton? The step-assist on this 2002 GMC Which one is real wood?
Safari is made of an advanced
Glass or PET? TPO nanocomposite material.
 Disadvantages
• Polymers are much more flammable than the
materials they are replacing.

•They decompose easily into carbon,

hydrogen, and oxygen when burnt.

• They decompose easily when exposed to UV

light, from the sun…or corrosive chemical
from land fills.

…..leaving behind the less flammable and

more stable nanoparticles which enter the
environment.
Classifications of Nanoparticles:
1. Friable form: Loose powders

2. Encapsulated: Nanocomposites, circuits

3. Liquid suspensions: personal care, meds, ..

 National Science Foundation: 2003

Should we proceed?
 Cosmetics

• Bees BeautyAll Natural Products

For All Natural Beauty
• pureMinerals Ingredients
• 100% NATURAL mineral based makeup
that's actually good for your skin.
PROTECTS your skin from UVA and UVB
rays.
“Natural” Ingredients!
• bareBeauty BLUSH: Micronized
Titanium Dioxide, Micronized Zinc Oxide,
Iron Oxide, Bismuth Oxychloride, Boron
Nitride, Octyl Palmitate, Cyclomethicone,
Green Tea (camellia sinesis) Extract,
Mica, Superoxide Dismutase, Catalase,
Apple Extract (Pyrus Malus), Horseradish
(Cochlearia Armoracia), Phosolipids,
Retinyl Palmitate, Tocopheryl Acetate,
Lavender, Sandalwood, Geranium, Ylang
Ylang, Patchouli, Rosemary, Sage,
Cedarwood, Palmarose May contain:
Manganese Violet, Carmine, Chromium
Oxide Green, Ultramarine
• . Iron, Titanium, and Zinc in Base
Makeup
Flesh colored base makeup's usually
contain black iron oxide, red iron
oxide, and titanium dioxide and/or
zinc oxide which are white. The ratio
of these oxides determines the Reactive
shade.
Nanoparticles
Section 5 Health Hazard Information:
Routes of Entry: inhalation, ingestion and skin absorption.
Effects of Overexposure: Exposure to Gold Compounds
may be harmful. Can cause contact dermatitis. The
toxicological properties have not been thoroughly
investigated.
2171
20

By Dennis Simanaitis  •  Illustration by Richard Borge

May 2005

Technology Update: Nanotech — Friend or Foe?

They're tiny. They're everywhere. But are they user-
friendly or a threat to civilization?
Separate chemical Dermal Fibroblasts were cultured
effects from size with Au/Citrate nanoparticles:
effects: • Au is inert FDA approved for
internal use.
•Citrate is non-toxic
•Cultured with different types of
skin cells
•Skin contact is an important
point of entry

+ = ??
Primary tissue
culture cell
Damage to Organ: Study different cells

Model:
•The damage is different in each
type of cell…even cancer/normal/
stem cells

• HeLa or cell line models are not

appropriate

•Must use tissue culture cells:

Test cell function impact

•In vitro assay can help us model /

predict damage to organism
 O
O OH
Citrate Coated AuNPs AuCl4- + O-
O- O
H2 O
O-

Mean 40-50 min,

Diameter: 1000C
46 ± 5 nm
50nm

20

Mean
30 min,
N umber of particles

16
Diameter:

12
13 ± 1 nm 1000C

0
M ore
11

16
10

12
13
14
15

50nm
Diameter of particle, nm
Morphology of Dermal Fibroblasts: 13nm particles

control

0.13 mg/ml

Cell area is much

smaller in presence of
nanoparticles:

0.15mg/ml
 Effect on Cell Morphology

Au(46nm)

14000

12000
2 days

mm sq.
10000
6 days

Cell area,
8000

control 6000

4000

2000
0 10 20 30

Nanoparticle concentration, mg/ml media

Large decrease in cell size

being exposed to Au(46nm)
nanoparticles
10 g/ml
Nanoparticle Proliferation: Effective Doubling Time
Effective gold citrate 13 nm (4 days)
95
doubling time 90
gold citrate 13 nm (2 days)
gold citrate 13 nm (6 days)
increases 85 gold citrate 46 nm (4 days)
gold citrate 46 nm (2 days)
linearly with 80 gold citrate 46 nm (6 days)
particle

doubling time, hours

75

concentration. 70 46nm
13nm
 Slope for the 65

60
13 nm particles 55
is seven times 50

larger OR cells 45

can tolerate 40

seven times 35

more 13nm 0 20 40 60 80 100 120 140 160

nanoparticles concentration, g/ml

particles than
46 nm ones.
A. Where are the nanoparticles?

•Lysosome stained with

cathepsin D (red )
•Citrate coating removed.
•Particles do not enter
nucleus.
B. Where are the nanoparticles?
250
45 nm AuNPs

number of vacuoles per cell

13 nm AuNPs
13nm 200

142g/ml 150

100

50

0
3 days 6 days

46nm • “Equivalent “ toxicity occurs when the

NUMBER of vacuoles is the same.
20g/ml • Particles adsorb only to the vacuole
membrane.
• Vacuoles fill when membrane surface
is covered
• “Toxic” equivalence scales approx. with
the area subtended or ~ 1/d2 of
particles.
 The Vacuoles: time dependence

13 nm 45 nm

3 days
2 microns
100 nm 2 microns 100 nm

6 days

1.8 45 nm AuNPs
13 nm AuNPs number of particles/cluster per vacuole
200
vacuole size, micron

1.5 45 nm AuNPs
13 nm AuNPs
1.2
2 microns 100 nm 150 2 microns
100 nm

0.9 Mean vacuole size is 100

0.6 the same for 13nm Seven times more
0.3
and 46 nm particles 50 clusters per vacuole
after equivalent of the same size for
0.0
3 days 6 days incubation times. 0
3 days 6 days the 13nm particles.
How do the AuNPs
penetrate (1hr 46nm

incubation)?
The 46nm
particles enter by
endocytosis.
 13nm appear to
enter by diffusion
through the
membrane. 13nm
Diffusion is
more efficient.
Rate of
penetration is
seven times
higher for the
13nm particles.
 Endocytosis inhibition by PAO after 1hr incubation with AuNP

13 nm 45 nm
Not
inhibited

1 micron 200 nm
1 micron 200 nm

Inhibited

1 micron 200 nm

45 nm AuN Ps/ clusters ratio

1 micron
inhibited with PAO 200 nm
1 3 n m A u N P s/ c lu s te rs ra tio

inhibited with PAO not inhibited

not inhibited
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
inside outside inside outside
 Possible Mechanisms of NP Get into Cells
Lipid layer lowers surface tension.Amplitude of capillary
interfacial waves approx. 10-20nm .Ratchet effect – non
discriminate entry. [S. Sinha et al PRL 2007]

Grinnell* reported particles

could enter directly through
plasma membrane passages,
when particle size is small
*Lin et al., Molecular Biology of the Cell, 8 (1997) 59-71. enough: 10-15 nm.
Gold content per cell
1. Pre-soak AuNPs in 100% FBS at 37C for 24 hours.
2. Expose 13nm particles to 140 micro-grams and 46nm to 20 micro-
grams for 5 hours.
3. Use mass spectroscopy to measure Au content per cell.

• 13nm Particles
affected most.
100 • Penetration
45 nm non presoaked

mechanism reverted to

13 nm non presoaked
80 less efficient
45 nm presoaked

endocytosis.
Au, ng/cell

60 • Recognition of ECM
protein by membrane.
40
presoaked
13 nm

20

0 1 2 3 4
Proposed: Selective Adsorption of Proteins. Favors 45nm
• Adsorption of proteins from serum is selective and depends on
particle Rg and surface interactions.
• Small particles can not bind large proteins. Entropy of confinement
overcomes enthalpy.
• E=H-TS
• Hence large particles have more proteins/surface area than smaller
ones.
• Cell membrane recognizes the proteins and ingests particles by
endocytosis.
 Dermal fibroblasts (CF-31) on glass
red: actin filaments green: Vinculin (integrin receptors)

Receptors transmit tension

and exert traction forces

15 h
Vector diagram of each bead

• Surface distribution of forces exerted

by dermal fibroblast on HA surface.
• Extension of force below the
substrate surfae.

A 3-D view of the traction

forces
 Implications of Actin Deterioration:
Actin fibers (green)

• The Actin fibers distribute the tension across the cell

and exert traction forces on the substrate.

• The Actin Cytoskeleton interacts with the

Extracellular Matrix proteins to regulate cell functions
such as proliferation, adhesion, and differentiation

Broken Actin (dots) • As the Actin fibers in cells are harmed, cell growth
and various cellular functions will be drastically
affected; cell area, migration, cell modulus.
 Fibroblast migration from agarose
droplet on HA matrices

24h 20 min

@ 4oC @ 4oC

Hydrogel matrix Agarose-droplet Incubation in SF-DMEM

plated containing fibroblasts & 30 ng/ml PDGF

Photomicrographs analyzed using SPOT® software 18h

Out-migration = B – A @ 37oC

B: area of migration + agarose

droplet area
A: agarose droplet area
Impaired
Migration: May
have
consequences
in aging and
wound healing.

Pernodet, et al,
Small 2006, 2, No. 6, 766
•The actin fibers are irregular, thinner, and small
defects with nanoparticle inclusions are visible.
Defective fibers or reduced
actin expression?

3 days exposure
Au Au
Control (15) (45)

Beta-tubulin 55 kDa
nanoparticles

Actin 45 kDa
 Decreased ECM protein expression
Cells treated with 13 and 45 nm AuNPs for 3 days, recovered for 5 and 14 days.
• Overalldecrease 24% FN and 45% for Collagen
• Col/Fn ratio decreased 30%--correlated with hardening
of the ECM.
control control
exposed to 13 nm AuNPs exposed to 13 nm AuNPs
exposed to 45 nm AuNPs exposed to 45 nm AuNPs
100
100

80
80

% of fibronectin / cell
% collagen / cell

60 60

40 40

20 20

0 0
3 days exposure 5 days recovery 14 days recovery 3 days exposure 5 days recovery 14 days recovery

Collagen Fibronectin
 Principle of Shear Modulation Force Microscopy
The indentation h of an elastic substrate by a
hard spherical tip of radius R, is given in the
Hertz model by :
2/3
 L 
h   D 1/ 2 
 R 

since: 3 1  2
D E  2(1   )G
4 E
2
we have: 3 1  L 3
h( )
8 G R1 / 2

then: h  G 2 / 3
?
ΔX G
Johnson, K. L. Contact Mechanics. Cambridge: Cambridge University Press; 1985.
• CF-31 cells plated on 23% Sulfonated PS spun cast films produce
large ECM fibers .
• Allows for simultaneous measurement of cell/ECM/susbtrate
mechanics.

ECM
ECM

Focal Adhesion
Actin Stress Fiber
Nucleus
 ECM produced by fibroblasts

Control Nano:0.39 mg/ml Nano:1.20 mg/ml

3.0
ECM

2.5
• Cells exposed to higher
2.0
nanoparticle concentrations
nano:
produce thinner and fewer
Relative modulus

0.6 mg/ml
1.5
ECM fibers.
nano:
1.0 0.4 mg/ml • Thinner fibers have HIGHER
nano:
modulus (factor of 3)
0.5
0.2 mg/ml
control
• Fibers are distorted.
0.0
25 26
 Do Cells Recover?

Cells transmit nanoparticles to daughter cells upon dividing.

Particle concentration in the cell decreases if source is removed.
Since damage is a function of concentration—is it possible for
cells to recover??
 Cells recovery experiment
Different Remove media
concentrations on 3rd day after
of gold addition of
Cells are plated nanoparticles are nanoparticles Number of
added and replace with cells is
fresh media. counted.
Cells are
stained and
imaged

Day (-1) Day 0 Day 1 Day 3 Day 4,6 and 8

 Is there a critical concentration for recovery?
2000 cells were plated in each case at day zero
Recovery is much more difficult for 45nm particles, than 15 nm particles.
250
200
control
control 225
180 94.84 g/ml Au(15)/citrate 13.03 g/ml Au(40)/citrate
142.26 g/ml Au(15)/citrate 200 19.545 g/ml Au(40)/citrate
160
189.68 g/ml Au(15)/citrate 26.06 g/ml Au(40)/citrate

number of cells X 200

175
140
number of cells x 200

150
120

125
100
exposure recovery exposure recovery
80 100

60 75

40 50

20 25

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
day day

Particle diameter Approx. Critical concentration

(microgm/ml)
15nm 80
45nm 10
 Apoptosis
(3 and 6 days exposure)

100
100
80 3 days exposure
6 days exposure 80 3 days exposure
60 6 days exposure
60
40
40
20
apoptosis, %

apoptosis, %
20
1.5 1.5
1.0 1.0
0.5 0.5
0.0 0.0
0 25 50 75 100 125 150 175 200 225 0 5 10 15 20 25 30
13 nm AuNP concentration, g/ml 45 nm AuNP concentration, g/ml
Correct doubling time: subtract apoptotic
fraction from day 3.AuNPs Induce apoptosis
but do not affect doubling time.
100000
100000 control
control
13.03 g/ml Au(45)/citrate
94.84 g/ml Au(13)/citrate
19.55 g/ml Au(45)/citrate

cell number
142.26 g/ml Au(13)/citrate
26.06 g/ml Au(45)/citrate
cell number

189.68 g/ml Au(13)/citrate

10000

10000

1000

3 4 5 6 7 8 3 4 5 6 7 8

days days
 Cells recovery after gold citrate (13nm)
nanoparticles exposure (confocal microscope)
control 94.84 g/ml 142.26 g/ml

rd
ay

day
 Cells recovery after gold citrate (13nm)
nanoparticles exposure (mercury lamp)
control 94.84 g/ml 142.26 g/ml

rd
ay

8 day
 Decreased ECM protein expression
Cells treated with 13 and 45 nm AuNPs for 3 days, recovered for 5 and 14 days.
• Overalldecrease 24% FN and 45% for Collagen
• Col/Fn ratio decreased 30%--correlated with hardening
of the ECM.
control control
exposed to 13 nm AuNPs exposed to 13 nm AuNPs
exposed to 45 nm AuNPs exposed to 45 nm AuNPs
100
100

80
80

% of fibronectin / cell
% collagen / cell

60 60

40 40

20 20

0 0
3 days exposure 5 days recovery 14 days recovery 3 days exposure 5 days recovery 14 days recovery

Collagen Fibronectin
 Reactive nanoparticles: Titanium and Zinc Oxides
•TiO2 and ZnO
are main
component of
photovoltaic
cells which
collect the
electrons to
generate
electricity.

Cosmetics: "The particles are so small they let

visible light through and thus appear transparent,“
This means that sunscreen [containing these
nanoparticles] doesn't look like the war-paint of
other sun lotions”
Turney, Micronisers, CSIRO
 Titanium Dioxide Particles: TiO2
•Two common types used in commercial products; Rutile and
Anatase with refractive indexes of 2.77 and 2.58.
•Excellent reflectors for light ranging from the visible to the
infrared.
•Photocatalytic-emits an electron when exposed to UV light.
• Large production of free radicals that can react further and
degrade organics in matrix, especially DNA. (McBride, T.J., Preston,) B.D.,
Loeb, L.A. (1991) )
S u p e r o x id e
D iSs mu pu et ar os xe i d e
D is m u t a s e

O O2 O O 2 - .- . H ++
H H HO O2 . .
2 2 2

O2 → O2-. e -e -
H H2 O O2
2 2
O2-. + O2-. + 2H+ → H2O2 + O2 C a ta la s e
B oC v ai nt ae l aS s ee r u m A l b u m i n
B o v in e S e r u m A l b u m in
h v = 3 .2 e V
O2-. + H2O2 → O2 + OH- + .OH
h v = 3 .2 e V

h++
h

O OH H- - . O. H
O H
D M S O
M Da nM n Si t Oo l
M a n n it o l
 TiO2 with different dispersions

a
Anatase

Rutile b 400

350
(110)
(211)
TiO2 Rutile
Sample 2
(preferred)
In te n s ity (c o u n ts )

300

250
(101)
200
(200) (301)

c
(111) (220)
150
(112)
(002)
100 (210) (310)

50

-50
20 30 40 50 60 70 80
2 Theta Angle (deg)
 PHYSICAL SUNSCREENS
OTC Panel
U.S. Federal Register, 43FR38206, August 25, 1978

“As a physical sunscreen agent, titanium

dioxide (TiO2) is a safe, opaque and effective
product that provides a barrier to sun-sensitive
individuals against sunburns, because it
reflects and scatters UVA and UVB radiation
290-400 nm) rather than absorbing the rays...”

54
 What about the Photostability
of chemical Sunscreens ??
.

After all, a molecule that absorbs UV radiation

must convert that energy into something.

• radiative decay (emission)

• nonradiative decay (heat)
• photochemical changes

55

Sunscam
 Think sunscreen protects against
News:
cancer? Think again.
By Michael Castleman
May/June 1998 Issue
…Skin Cancer Foundation (SCF) makes its
annual appeal to the public to use
sunscreen. As people heed their warning
this year, few will remember the report that
made headlines in February. According to
a survey of new research by epidemiologist
Marianne Berwick of the Memorial Sloan-
Kettering Cancer Center in New York,
there is no evidence that sunscreen offers
any real protection against malignant
melanoma, ... "It's not safe to rely on
sunscreen," Berwick told the press.
The SCF …. telling consumers that
"sunscreen should continue to be an
integral part of a comprehensive
program" to prevent melanoma “
Sunscreen:
•Introduced in 1940 as “tanning lotion”
• In 1960 changed name to
“Sunscreen”.
•$18M in 1970, $500M in 1996
• Melanoma: 1/250 in ’80 and 1/84 in ’96
• Rises at the rate of 6% per year or one
of the fastest rising cancers in the US.
Protection:
The SCF adds that sunscreen alone “ is
not enough. You need to wear
protective clothing..”
The only proven way is to prevent
melanoma is to cover up..and..think
twice before you slap on
sunscreen. .
 SKIN CANCER STUDY
Nambour town, Queensland, Australia.
Green et al., The Lancet, 354 (1999) 723-729

1621
I n i t ia l p a r t i c i p a n t s

404 408 416 393

a s s ig n e d a s s ig n e d a s s ig n e d a s s ig n e d
d a ily s u n s c r e e n p lu s d a ily s u n s c r e e n p lu s n o s u n s c re e n n o s u n s c re e n
b e ta c a r o te n e p la c e b o b e ta c a ro te n e p la c e b o

354 338 357 334

e x a m in e d f o r e x a m in e d f o r e x a m in e d f o r e x a m in e d f o r
s k in c a n c e r s k in c a n c e r s k in c a n c e r s k in c a n c e r

SUNSCREEN NO SUNSCREEN
65 Basal-cell carcinoma 63 Basal-cell carcinoma
22 Squamous-cell carcinoma 25 Squamous-cell carcinoma
57
Bauer, J et al. Effect of sunscreen and
clothing on the number of melanocytic
nevi in 1,812 German children attending
day care. American Journal of
Epidemiology, Vol. 161, April 2005, pp.
620-627
•Germany: 1812 children studied
• no correlation bet incidence of
nevi and sunscreen
• Italy: 681 children bet ages of 7-
12
• 41% lower rate of nevi among
children who used protective
clothing
• 68% higher rate of nevi among
those using sun screens.
UVA (99.5%): 320-400nm: UVB (0.50%): 280-320nm:
•Penetrates deeply to • Causes sunburn and UVC ( ~0 ):
malanocytes. premature aging. •absorbed by
•Implicated in melanoma. • Basal carcinoma. atmosphere:
• Rating: 10% of UVB rating • SPF 50: filters 97% 200-280nm
• Very
dangerous, but
absorbed in the
atmosphere and
skin.
•Sunscreen
focuses on
the UVB
region.
• UVA is most
likely to
interact with
cells.
 • Main active
ingredients in
INCI Name: Benzophenone-8
UVA & UVB range INCI Name: Ethylhexyl sunblock are of
Methoxycinnamate
Mainly in UVB
molecules with
benzine rings.
• High UV absorption.
• High toxicity.
• Minimize their
concentration, while
INCI Name: Cinoxate INCI Name: Octocrylene
UVB range UVB range providing maximum
INCI Name: Butyl
SPF.
Methoxydibenzoyl • Introduce
ethane
UVA range
reflectors, i.e. TiO2
Initial step of proposed photolysis of avobenzone (i.e.
Parsol 1789). Note that DBM is a radical photoinitiator of
polymerization reactions
61
Sayre et al., personal communication, March 2004; and Photochem Photobiol., 81 (2005) 452-456
 What happens to DNA or Chromosomes?

• DNA:
Lambda DNA 48,502 bp
1Kb DNA ladder (0.5-10kb) (Biolabs, Inc.)
• Human chromosomes (Cold Spring Harbor)
• UV source:
UVA(350nm);UVB(300nm);UVC(250nm)
(2mW/cm2)
• Gel electrophoresis:
1% Agarose gel; 1x Tris-borate-EDTA buffer;
6V/cm.
• Surface electrophoresis.
 Irradiation Procedure
• TiO2 solution (2 mg/ml in 1x TBE);
• DNA solution (50 μg/ml in 1x TBE);
• 5 μl of each TiO2 and DNA solution mixed in microtube;
• UV irradiation for different time duration at room temp.

2 cm
 Gel electrophoresis
1kb λ TiO2 #2 λ TiO2 #2 λ TiO2 #2
ladder λ UVA UVA UVA UVB UVB UVB UVC UVC UVC
4h 4h 4h 4h 4h 4h 1h 1h 1h
Human chromosome DNA, Sample T1660By (Collaboration
with Eli Hatchwell, MD, Cold Spring Harbor)

Si substrate
buffer
electrode Loops
DNA drop or gel
Tails

Trains

No electric filed with electric filed

E
Surface electrophoresis
2000000
Lambda
250000
Lambda DNA
DNA 200000
50μg/ml
1500000
50μg/ml UVC 1h
Intensity, a.u.

Intensity, a.u.
150000

1000000

100000

500000
50000

0 0

0 1000 2000 3000 4000 0 1000 2000 3000 4000

Time, sec Time, sec

Lambda DNA 1000000

Lambda DNA
250000

50μg/ml 50μg/ml
200000
TiO2 800000
modified TiO2
UVC 1h
Intensity, a.u.

UVC 1h
Intensity, a.u.

150000 600000

100000 400000

50000 200000

0 0

0 1000 2000 3000 4000 0 1000 2000 3000 4000

Time, sec Time, sec

 Breakage of Chromosome DNA in gel

300 μm

Chromosome DNA with TiO2

20 Chromosome DNA 25
exposed to UV 1hr.
20
15

Roundness: 0.62±0.12 Roundness: 0.5±0.19

Counts
Counts

15
10

10

5
5

0 0
0 20 40 60 80 100 120 0 10 20 30 40 50 60 70 80 90
Feret Diameter, m Feret Diameter, m
 Rutile: Clustered TiO2
b 6000

5000
a
4000
cell area (sq.pixels)

3000

2000

1000
control TiO2: TiO2:
0.4
0.8
0
Day 6
control 0.4 mg/ml

c 4000

3500

3000

2500
cell number

2000

1500

1000
control TiO2:
500 0.4
TiO2:
0 0.8
6 days
• TiO2(with talc) 0.1 mg/ml

• TiO2(with talc) 0.3 mg/ml Anatase:

Damage
to cells in
the
absence
of light?
• TiO2(with talc) 0.5mg/ml

Anatase: Actin and cells are

destroyed with increasing
concentration.
 Apoptosis control positive control
(day 7):
Destruction is
faster than
TiO2 / talc TiO2
apoptosis
process!
talc #2
 Migration
Migration

Agarose-droplet Incubation in SF-DMEM

containing fibroblasts & 30 ng/ml PDGF

Average Distance of Migration vs. Condition 31control

31tio2
400000

350000
Migration is altered!!!
300000
Average Distance Migrated (sq. pixels)

250000
CF-31 Control CF-31 TiO2
200000

150000

100000

50000

0
conditions
 Clustered TiO2
d e

control 0.4 mg/ml

Average Distance of Migration vs. Condition
Average Distance Migrated (sq. pixels)

400000

350000 50

300000

250000 Collagen gel contraction ratio (%) 40

200000

150000 30

100000
control 0.4 mg/ml
20
50000 TiO2
0
conditions 10

0
control TiO2 #1 TiO2 #2
 Western blot – actin assay
Same cell number Same protein amount

actin

c
l

/tal
t ro

talc

#2
2
T iO
con

2
T iO
western-blot results (same cell number) western-blot results (same protein amount)

6000 6000
5000 5000
4000 4000
OD
OD

3000 3000
2000 2000
1000 1000
0 0
control TiO2 talc 50/50 #2 control TiO2 talc 50/50 #2
different particles different particles
Where are the particles? • TiO2
particles enter
in a similar
manner
• Much larger
clusters are
present in
vacuoles.
• Vacuole
membrane is
eventually
ruptured.
• Particles are
seen to cross
nuclear
membrane
after 6 days
incubation.
12 hours: Dermal Fibroblasts
 Are there good nanoparticles?
• Large aspect ratio-do not penetrate cells.
• Coated and non reactive polymer which
absorb the emitted electrons or free
radicals.
• Cancer cell are more sensitive. Potential for
use as targeted cancer therarpy
Surface Modified Nano TiO2 Concept Model
Oligomeric Proanthocyanidins (opc)

&
Poly[Methyl Vinyl Ether/malei Acid]
• Extremely high density
brush: effective at electron
capture.
Nano TiO2 • Entropically hindered
from interacting with the
cell membrane.
• Super hydrophobic.
• ROS production
Triethoxysilylethyl surpressed.
Polydimethylsiloxyethyl Hexyl • Cell penetration
Dimethicone
prevented.

77
control Particles do not enter or adhere
to cell membranes.
Some are seen floating.
Low concentration dependence.
Control
20000 Coated TiO2 (0.4)
18000 Coated TiO2 (0.8)
16000 Nano TiO2 (0.4)

c e ll c o u n t
Nano TiO2 (0.8)
14000
0.4 mg/ml 12000
10000
8000
6000
4000
2000
0
0 2 4 6 8 10 12
Time (Days)
20000

18000

16000
0.8 mg/ml 14000

12000
cell number 10000

8000
control Coated TiO2: Coated TiO2:
6000 0.4 0.8
4000

2000

0
11 days
 Montmorilonite Clays
NO CO2 WITH CO2
30000

PMMA
Cells incubated on PMMA 25000 W/ CLAY
with Clays PMMA
20000 W/ CLAY

cell density (cells/cm2)

Results: 15000

control
PMMA

10000
• On a 3 day growth curve,
osteoblast growth rate more than 5000 PMMA

tripled on pure PMMA surfaces. 0

PMMA

• PMMA is used as a filler for

implants.
•Metal surface have poor adhesion
for cells and MMA is polymerized to
“fit” the joint.
 General principal: Other polymers
Cell adsorption on the clay surfaces
Fibroblast cell (CF31) 48h
sodium clay
PDMS
organoclay
PDMS

PDMS

PDMS

PDMS 5% clay
composite

- Cell growth curve on different surfaces.

- Western Blot tests to determine which
PDMS proteins are expressed by the presence of clays
 Effects of Phosphates on Clay
50000

45000

40000

35000
0%
30000 0.5%
Cell Count

1%
25000
2%
20000 2% K
Control
15000

10000

5000

0
Day 0 Day 1 Day 3
 Conclusion
• Our cells were not designed to protect
themselves against nanoparticles less
than a few tens of nm in diameter.
• There is a legitimate cause for concern in
using these materials, especially in
solutions or airborne.
• Nanoparticles can be very useful if once
we fully understand their interactions with
cells
 Aging?

Do the effects become more severe in cells that are already weakened?
Aging effect?

CF-31 compared to Neonatal and CF-75 dermal fibroblasts

or or =?
+
 Migration

Average Distance of Migration vs. Cell Age

neocont
400000 neotio2
31cont
350000 31tio2
75cont
Average Distance Migrated (sq. pixels)

300000 75tio2

250000 Neo Control Neo TiO2

200000

150000

100000

50000

0
conditions

CF-75 Control CF-75 TiO2

 Keratinocyte Cell Growth Curve (TiO2 )
Control

160000
Control
TiO2 (0.05mg/ml)
140000
TiO2-FBS TiO2- FBS
120000

100000
Cell Count

80000

60000
TiO2
40000

20000

0
0 2 4 6 8 10
Growth Days
 Concentration Dependence of
Keratinocyte cell count (TiO2): Higher tolerance

50000
C e ll C o u n t ( D a y 6 )

40000

30000

20000

10000

0
Different Concentration
TiO2 TiO2 TiO2-FBS TiO2-FBS
TiO2 TiO2-FBS
1.0 mg/ml 0.1 mg/ml 0.01 mg/ml 1.0 mg/ml 0.1 mg/ml 0.01 mg/ml
TEM Image of Keratinocyte cells (TiO2)
 Recovery: Keratinocytes with 3T3
600000
Ctrl (P5) Concentration: 0.1 mg/ml for 2 days
TiO2
500000 TiO2-FBS
Ctrl (P6)
TiO2 (P5)
400000
TiO2-FBS
Cell Count

300000 (P6)

200000

100000

0
1 2 3 4 5 6 7 8 9 10
Growth Days
 Passage 5 -- Control

Day7

Day5
Passage 5 -- Day 7
TiO2 with FBS TiO2
 TiO2

3T3 cells
after 2 days

Control TiO2-FBS
Day 7: Re plated (w/o 3T3 cells) after
exposure 2 days to TiO2 (0.1mg/ml).
Control TiO2 TiO2-FBS
 Skin Penetration ?
Non-UV UV

Apply to fresh surgical cast off

There was no detectable difference between the two
samples using an optical microscope.
 Skin Penetration
Non-UV UV
TiO2 particles
are fluorescent.

Non-UV UV

Titanium
Dioxide
 Adsorption in tissue is
specific to duct lining.

Milk duct
 Are there good nanoparticles?
• Large aspect ratio-do not penetrate cells.
• Coated and non reactive polymer which absorb
the emitted electrons or free radicals.
• Could be recognized by enzymes and
metabolized properly.
• Biodegradable.
• Cancer cell are more sensitive. Potential for use
as cancer therarpy
Surface Modified Nano TiO2 Concept Model
Oligomeric Proanthocyanidins (opc)

&
Poly[Methyl Vinyl Ether/malei Acid]

Nano TiO2

Triethoxysilylethyl
Polydimethylsiloxyethyl Hexyl
Dimethicone

97
98
 Polymer Brush Calculation – Surface Modified TiO2
α = β (VTiO2/V#2) = β rTiO23 / r#23
α = ρ#2/ρTiO = 0.23 β=M#2/MTiO2 = 0.14
2
ρ1= Density of TiO2 M1 = Mass of TiO2
ρ2= Density of Functionalized TiO2 M2 = Mass of functionalized TiO2

r#2≈ 11nm

Mass of the shell ≈ 8.37 E-18 g,

Mass/ Chain ≈ 6.64E- 21g/chain Table 1. Molecular weight, pH, and
Total # of chains ≈ 1,259 density of each individual component

grafting density ≈ 0.5 chains per nm2

Radius of gyration (Rg)≈ 3nm

99
Possible Solution: Coat individual TiO2 particles to
prevent emission of the electron
• Coat TiO2 particles with polymers consistent
30nm
with the cosmetic industry. Use known coating
chemistries using UV light, sonolysis etc.
• Adsorb radical scavengers such as Lycopene.

Pink to
White: Free
radicals

TiO2, coated TiO2 UV

In situ UV-polymerization of MMA

 Effects of Phosphates on Clay
50000

45000

40000

35000
0%
30000 0.5%
Cell Count

1%
25000
2%
20000 2% K
Control
15000

10000

5000

0
Day 0 Day 1 Day 3
 Conformational changes in Fibrinogen
E domain
D D domain

αC domain

Sodium clay surface Organoclay surface

FB 2μg/ml 10min FB 2μg/ml 10min

globular trinodular

L = ~33 nm
L = ~46 nm
h= 1.5 nm h = 1.1 nm
scale bar = 200 nm
 Fibrinogen fiber formation in the absence of thrombin.
Surface unfolds molecule exposing A knob which initiates fiber formation.

I-2 I-8 ΦII

Organoclay

2μm 2μm 2μm

Mica clay

2μm 2μm 2μm

Cell Morphology on Fibrinogen Fibers: recognition
• Clays are beneficial in wound healing.
I-2 (0.05 mg/ml)

I-2 (4 mg/ml)
Cell migration 24 h on fibrin

Cell migration 24 h on fibrinogen

 Thrombin: Fibrin Fibrinogen

fibrinopeptides A
Fibrinogen
fibrinopeptides B
fibrinopeptides A
+Thrombin
cleavage
D-D contact

fibrinopeptides B +Thrombin + Thrombin

cleavage
 Conclusion
• Our cells were not designed to protect
themselves against nanoparticles less
than a few tens of nm in diameter.
• There is a legitimate cause for concern in
using these materials, especially in
solutions or airborne.
• Nanoparticles can be very useful if once
we fully understand their interactions with
cells
 Fibroblast migration from agarose
droplet on HA matrices

24h 20 min

@ 4oC @ 4oC

Hydrogel matrix Agarose-droplet Incubation in SF-DMEM

plated containing fibroblasts & 30 ng/ml PDGF

18h
@ 37oC
Photomicrographs analyzed using SPOT® software

Out-migration = B – A
B: area of migration + agarose
droplet area
A: agarose droplet area
SEM image of TiO2 particles
• Large particles
are Talc.
• Smaller ones
(~10nm) are
TiO2 particles.
• Titanium
dioxide is used
in most
sunscreens to
help prevent UV
radiation burns.
• Zinc is also
used in
sunscreens as
a UV blocking
agent.
 Biomedical: Variable size and magnetic (A. Ulman)
30 nm 15 nm 10 nm

25 nm
25 nm
Fe 25 nm Fe2O3 Fe2O3
•The magnetic stent, demonstrates that
magnetic microspheres carrying
therapeutic substances can be delivered
to magnetized cardiovascular implants
by simple intra-arterial injection
(Benjamin Yellen et al 2004).
• MRI contrast enhancement
• Targeted drug delivery
• Reported Autoimmune problems after
extended chemotherapy (Jack Kovach,
MD-Stony Brook Breast Cancer Center)
 A direct consequence on Cell Function:
Phagocytosis
Bacteria
Macrophages: their function is
to digest foreign particles:
Professional Phagocytes
• Too many vacuoles with Normal Macrophage
nanoparticles inhibit
phagocytosis.
• Macrophages lose their
ability to phagocyte
bacteria. (38% impairment)

Phagocyte / Au

Bacteria: Yersinia pseudotuberculosis

 Keratinocyte Cell Growth Curve (TiO2 )
Control

160000
Control
TiO2 (0.05mg/ml)
140000
TiO2-FBS TiO2- FBS
120000

100000
Cell Count

80000

60000
TiO2
40000

20000

0
0 2 4 6 8 10
Growth Days
 Concentration Dependent Keratinocyte cell Growth (TiO2):
FBS coating

50000
C e ll C o u n t ( D a y 6 )

40000

30000

20000

10000

0
Different Concentration
TiO2 TiO2 TiO2-FBS TiO2-FBS
TiO2 TiO2-FBS
1.0 mg/ml 0.1 mg/ml 0.01 mg/ml 1.0 mg/ml 0.1 mg/ml 0.01 mg/ml
TEM Image of SCC13 cells (TiO2)
TEM Image of Keratinocyte cells (TiO2-FBS)

Fibrin Clot &
Re-epithelialization
(Within min / hours)
(C): Adapted from: http://www.residentnet.com/private/shrink2.htm
Normal adult wound healing
(B) Granulation Tissue
(day 4-6)
Fibroblast activation and
(C) Wound remodeling
migration is the
(day 7-10)
rate-limiting step in granulation
tissue formation
Adapted from: Singer AJ and Clark RAF. The NEJM,
1999, 341 (10): 738-746
McClain SA et al. (1996) Am J Pathol. 149(4):1257-70.