Prof. Dr. Nelson Dur

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AULA 5-2 NANOTUBOS DE CARBONO NANOMEDICINA
Prof. Dr. Nelson Durán IQ-UNICAMP CURSO
QF-435-SEGUNDO SEMENTRE 2008 NANOMATERIAIS
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Bianco, Applications of carbon nanotubes in drug
delivery. Current Opinion in Chemical Biology
2005, 9674679
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human epidermal keratinocytes (HEKs)
Monteiro-Riviere et al. Surfactant effects on
carbon nanotube interactions with human
keratinocytes. Nanomedicine Nanotechnology,
Biology, and Medicine 1 (2005) 293 299
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These MWCNTs induced the release of the
proinflammatory cytokine interleukin 8 (IL-8)
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In conclusion, these studies demonstrate that
Pluronic F127 is relatively nontoxic to HEKs in
culture but does not increase the cytotoxicity of
MWCNTs. In contrast, MWCNTs dosed alone in the
aqueous cell culture medium caused significantly
more IL-8 release than when MWCNTs were dosed in
Pluronic F127. MWCNTs were equally cytotoxic to
HEKs with or without the presence of a
surfactant. Studies that model nanotube behavior
in nonbiological systems suggest that increased
dispersion would result in an enhanced ability to
interact with cells. Our studies suggest that,
although dispersion occurred with surfactant,
biological interactions did not correlate with
this property.
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Tian e al. Cytotoxicity of single-wall carbon
nanotubes on human Wbroblasts. Toxicology in
Vitro 20 (2006) 12021212
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Fig. 2. Binding energy intensity of samples with
and without purification process (A) the peaks C
1s and Fe 2p 3/2 in the binding energy intensity
are present in SWCNTs before purification (B)
only carbon, peak C1s, is present in purified
SWCNTs.
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Fig. 3. The effect of refned carbon nanomaterials
upon the survival of human fibroblast cells (A)
cells treated with 25 g/ml of CG, MWCNT, CB, AC
and SWCNTs, for 15 days. Three replicate plates
were used for each data point and the experiments
were performed at least three times (B) cells
treated with SWCNTs in concentrations of 0.8,
1.61, 3.125, 6.25, 12.5, 25, 50 and 100 g/ml, for
15 days.
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Fig. 5. Effect of SWCNTs human fibroblast cells
(A) scanning electron microscopy image of
dispersed SWCNTs over the substrate, they have
the sharpest shape, amid the five nanomaterials,
due to a rather large aspect ratio (B) change
in cell spreading seen on samples treated with
SWCNTs.
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Fig. 6. Morphology of human fibroblast cells
observed under transmission electron microscopy
images (A) typical normal cell (B) cell
treated with SWCNTs.
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Fig. 7. Phase contrast microscopy images showing
the distribution of fibronectin and P-cadherin in
normal and SWCNT-treated cells (A) normal cell
(B) fibronectin (red), P-cadherin (green) and
cell nucleus (blue) of a normal cell (C)
SWCNT-treated cell (D) fibronectin (red),
P-cadherin (green) and cell nucleus (blue) of a
SWCNT-treated cell.
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Fig. 8. Phase contrast microscopy images showing
the distribution of FAK protein in normal and
SWCNT-treated cells (A) normal cell (B) FAK
(green) and cell nucleus (blue) in a normal cell
(C) SWCNT-treated cell (D) FAK (green) and
cell nucleus (blue) in a SWCNT-treated cell.
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Fig. 9. Phase contrast microscopy images showing
the distribution of Factin in normal and
SWCNT-treated cells (A) F-actin (red) and cell
nucleus (blue) in a normal cell (B) F-actin
(red) and cell nucleus (blue) in a SWCNT-treated
cell.
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Their results are twofold. Firstly, it was found
that surface area is the variable that best
predicts the potential toxicity of these refined
carbon nanomaterials, in which SWCNTs induced the
strongest cellular apoptosis/necrosis. Secondly,
it was found that refined SWCNTs are more toxic
than its unrefined counterpart. For comparable
small surface areas, dispersed carbon
nanomaterials due to a change in surface
chemistry, are seen to pose morphological changes
and cell detachment, and thereupon
apoptosis/necrosis. Finally, it was proposed a
mechanism of action that elucidates the higher
toxicity of dispersed, hydrophobic nanomaterials
of small surface area.
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osteosarcoma ROS 17/2.8 cells
Zanello et al. Bone Cell Proliferation on Carbon
Nanotubes. Nano Lett. 2006, Vol. 6, No. 3 562-567
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Figure 3. Morphology of ROS 17/2.8 cells cultured
on AP-SWNTs (A-C), AP-MWNTs (D-F), and control
cultures on glass cover slips (G-I), as seen with
SEM. (A) Osteoblast colony on AP-SWNTs. (B) A
flat cell body of a single cell extends over
almost the entire field of observation the cell
nucleus protrudes in the center. (C) Tape-like
cytoplasmic prolongations (arrow) extend from the
flat body of a ROS 17/2.8 cell (a portion of it
shown at the left upper corner of the picture) on
an evenly distributed AP-SWNT substrate.
(D) Osteoblast colony on AP-MWNTs. The nanotubes
aggregate unevenly in areas of the glass surface
(notice bundles on CNTs on the right). (E) Image
of a single ROS 17/2.8 cell on AP-MWNTs. A round
single-cell body extends thin neurite-like
cytoplasmic prolongations (arrow) that reach the
nanotube bundles. (F) SEM micrographs at higher
magnification show a detail of long threadlike
cytoplasmic prolongations (arrow) that extend
from the round body of a single ROS 17/2.8 cell
(partially seen on the left, upper corner),
interweave with, and reach individual AP-MWNTs.
(G) ROS 17/2.8 cell colony cultured on glass. (H)
Image of a single cell obtained at higher
magnification. (I) Detail of a portion of the
cell cytoplasm (covering the left upper half of
the picture) in contact with the glass surface
no cytoplasmic prolongations are observed.
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As shown in Figure 3A-C, ROS 17/2.8 cell bodies
grew flat on AP-SWNTs, similar to their growth on
glass (Figure 3G-I). Typical cell diameters for
flat ROS 17/2.8 osteoblasts was of the order of
40 ím, which resembled osteoblasts found on the
surface of natural bone. However, cell bodies
were spherical when cultured on AP-MWNTs. They
developed long threadlike cytoplasmic
prolongations, as seen in Figure 3D-F. Round cell
bodies on MWNTs measured approximately 15 ím in
diameter. This cell morphology resembles that of
osteocytes, the fully differentiated osteoblasts
embedded in the bone matrix. Osteocytes connect
and communicate with neighbor cells by means of
thin cytoplasmic prolongations that run across
the mineralized matrix. Interestingly, a similar
neurite-like growth pattern was described for
neuronal cultures on AP- and functionalized
MWNTs.17 SEM observations performed at high
magnification revealed the morphology of physical
contacts between cell and CNT materials, as shown
in Figure 3C, F, and I. Spherical osteoblasts on
AP-MWNTs grew long threadlike cytoplasmic
prolongations that reached the nanotube bundles
as a way to anchor to a discontinuous,
three-dimensional substrate (Figure 3F). These
thin pseudopods had diameters in the range of
10-20 nm, close in size to MWNT diameters.
Alternatively, flat osteoblasts on AP-SWNTs grew
shorter, tape-like cytoplasmic prolongations that
adhered to the more evenly distributed layer of
nanotubes (Figure 3C). Nanometer-scale
cytoplasmic prolongations were not observed in
ROS 17/2.8 cells grown on glass (Figure 3I).
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It was found that ROS 17/2.8 cells cultured on
glass produced cubic crystals after the first
week in culture (Figure 4A). These crystals
(100-500 nm in length) dispersed at random in
intercellular spaces, suggesting that they were
not a case of nonspecific ectopic mineralization.
On the contrary, ROS 17/2.8 cells cultured on
SWNTs produced plate-shaped crystals (100-1000 nm
in length, and approximately 20 nm thick) similar
in shape to HA crystals found in woven bone,
which aggregated in clusters outside the cells
(Figure 4B). Although plate-shaped crystals
aggregated on the nanomaterials in a disordered
fashion, our results indicate that CNTs
constitute a suitable substrate for deposition of
a mineralized matrix.
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Figure 5 shows current-to-voltage relations and
raw current traces of a voltage-gated, outward
rectifying chloride current that activates at
depolarizing potentials in ROS 17/2.8 cells
culture. Chloride current amplitude was reduced
by 200 ?M 4,4-diisothiocyanatostilbene-2,2-disul
fonic acid (DIDS, Sigma), a specific Cl- channel
blocker applied to the bath (data not shown). It
was not found any statistically significant
differences in Cl- current amplitudes and voltage
sensitivity in ROS 17/2.8 cells grown on AP-SWNTs
and AP-MWNTs. However, current amplitudes
measured from cells on AP-MWNTs at membrane
potentials over 40 mV were slightly larger than
those recorded from AP-SWNT cultures, as shown in
the same figure
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High-threshold, voltage-activated (HVA) calcium
currents were recorded from ROS 17/2.8 cells
cultured on AP-SWNTs and AP-MWNTs, as shown in
Figure 6. We found that HVA Ca2 current
amplitudes obtained at 20 mV, the membrane
voltage value for maximal activation, were ca.
twofold larger in osteoblasts cultured on
AP-MWNTs than AP-SWNTs. This Ca2 current was
enhanced significantly by 0.5 íM S(-) BayK 8644
(Sigma, Figure 6, left panel), a dihydropyridine
agonist specific for L-type Ca2
channels. Calcium currents were completely
blocked by 500 íMCd2 added to the bath at the
end of the experiment (not shown). Inward Ca2
currents recorded in AP-SWNT and AP-MWNT cultures
resembled an L-type Ca2 channel involved in
exocytosis of bone materials described in ROS
17/2.8 cells cultured on plastic dishes. Large
Ca2 current amplitudes obtained in AP-MWNTs
correlate with the changes in cell morphology
found in this nanomaterial substrate
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Normal plasma membrane electrical functions are
necessary for osteoblasts to maintain exocytotic
activities, and therefore bone formation. Here it
was proved that electrical activities of the
osteoblast membrane are maintained, and Ca2
channel functions enhanced, in cells grown on
neutral CNTs, confirming a degree of
biocompatibility of AP-SWNTs and AP-MWNTs. This
is the first description of electrical activities
of ion channels in a cell type cultured on CNTs.
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Their results provide insight into the
understanding of the degree of biocompatibility
between live cells and CNTs, and the real
possibilities for CNTs to be used as an
alternative material for the treatment of bone
pathologies that lead to bone loss, with the
potential for the regrowth of normal bone. It was
found that osteoblasts grow and produce
mineralized bone when cultured on electrically
neutral CNTs. This growth is diminished by
chemical modifications that introduce a net
electric charge to the CNTs. In addition, we
verified that ROS 17/2.8 cells retain electrical
properties necessary for adequate secretion of
bone materials when cultured on CNTs. These
results suggest that electrically neutral CNTs
can be considered as potential filling materials
for the treatment of injured bone. It was
concluded that CNTs sustain osteoblast growth and
bone formation, and thus represent a potential
technological advance in the field of bone
bioengineering. CNTs show promising
biocompatibility with osteoblast cells, and they
appear to modulate the cell phenotype.
Application of CNTs to bone therapy may lead to
the development of new bonegraft materials and
techniques.