Indian Journal of Experimental Biology
Vol. 48, October 2010, pp. 1037-1042
Interaction of soot derived multi-carbon nanoparticles with lung surfactants and
their possible internalization inside alveolar cavity
Pradip Kumar & H B Bohidar*
Nanomaterials and Nanocomposites Laboratory, School of Physical Sciences
Jawaharlal Nehru University, New Delhi 110 067, India
A systematic investigation of interaction of multi-carbon nanoparticles, obtained from soot, with dipalmitoyl
phosphatidylcholine (DPPC), a clinical pulmonary phospholipid surfactant, sold under trade name “Survanta”, was
undertaken to establish a model for internalization of these nanoparticles inside alveolar cavity. In vitro experiments
were carried out to establish the phospholipid assisted dispersion mechanism of carbon nanoclusters (size ≈150 nm, zeta
potential ≈ -15 mV) in water. Results obtained from an array of experimental methods, like dynamic laser light
scattering, electrophoresis, UV-absorption spectroscopy, surface tension studies and transmission electron microscopy,
revealed that the carbon nanoparticles interacted with DPPC predominantly via hydrophobic interactions. Selective
surface adsorption of DPPC molecules on nanoparticle surface was found to be strongly dependent on the concentration
of the phospholipid. DPPC, a gemini surfactant, formed a rigid monolayer around the carbon nanocluster even at
nanomolar concentration and provided excellent stability to the dispersion. Based on the experimental data it is proposed
that the free-energy gain involved in the hydrophobic interactions will facilitate the internalization of these nanoparticles
on the inner wall of the alveolar cavity.
Keywords: Alveolar cavity, Dipalmitoyl phosphatidylcholine, Dynamic light scattering, Internalization model,
Multi-carbon nanoparticles
With the rapid and uncontrolled industrialization and
urbanization the concentration of respirable
suspended particulate matter (RSPM, typical size
10 nm to 1000 nm) in ambient air has increased many
folds in the recent past all over the world. Fumes
emitted by industrial chimneys and internal
combustion engines contain a variety of carbonaceous
nanostructures which constitute a major part of RSPM
available in air1. It has been well established that
accessibility of these suspended particles to various
parts of the respiratory pathway is strongly size
dependent. As per International Commission for
Radiation Protection2 specifications particles of size
between 1-10 µm get deposited inside trachea and
bronchus, while smaller particles of size 10-100 nm
reach up to the bronchioles and alveoli. An integrated
picture reveals that the micron and nanosized particles
get deposited in the respiratory pathway with similar
propensity with larger ones mostly present in the
upper and smaller ones in the lower portion of the
respiratory tract. The dynamics of respiration is
governed by the synchronized swelling (inhalation)
and deswelling (exhalation) of millions of alveolar
sacs present inside the lungs. The internal surface of
the lungs is lined with lung surfactant dipalmitoyl
phosphatidylcholine (DPPC) which forms a monolayer
that separates the alveolar surface from the inner
cavity3 (Fig. 1). The presence of this phospholipid
_________________
*Correspondent author
Telephone: +91 11 2670 4637 & +91 11 2671 7562
Fax: +91 11 2674 1837
E-mail: [email protected]
Fig. 1—Structure of alveoli. The O2-CO2 occurs across a 1µm
thick phospholipid membrane. DPPC is the main surfactant
present inside alveoli. The strong electrostatic repulsion between
charged head groups prevents alveolar collapse in the exhaled
(de-swollen) state.
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INDIAN J EXP BIOL, OCTOBER 2010
surfactant ensures that the interfacial tension at the
inner alveolar surface is reduced, there by, enabling
expansion of alveoli with minimum expense of
energy. In the exhaled state (minimum alveolar
volume), the interfacial tension is very small whereas
in the fully expanded state (inhaled state) it is close to
25 dyn/cm. At the liquid-air interface the surface
tension of water is about 70 dyn/cm. In addition, the
presence of DPPC monolayer prevents full collapse of
the alveolar sacs in their exhaled state4. Presence of
inhaled nanosized particles can severely hinder the
overall function of the alveolar sac. This makes it
imperative to develop a molecular level mechanism in
order to understand the lung surfactant and
nanoparticle interaction.
Pulmonary surfactants, on the other hand, are a
complex mixture of lipids, phospholipids and
surfactant specific proteins5. These surface active
lipoprotein complexes are formed by type II alveolar
cells. While the surfactant specific proteins play the
role of optimal functioning of the surfactants, the
most abundant (70-80%) phospholipid, dipalmitoyl
phosphatidylcholine (DPPC), is the main surfactant
responsible for the surface tension reduction at the
liquid-air interface along the bronchoalveolar
surface6-8. The biological activity of DPPC is
manifested only when it is used in conjunction with
surfactant proteins SP-B and SP-C. Pulmonary
surfactant treatment has shown significant
improvement in surface tension behaviour of
bronchoalveolar lavage fluid, static lung compliance
and oxygenation of blood9-11. The commercial
pulmonary surfactant, Survanta is an animal derived,
FDA approved surfactant which is extracted from
minced cow lung with additional DPPC, palmitic acid
and tripalmitin; it also contains SP-B and SP-C in
unspecified amount. The molecular structure of DPPC
surfactant is depicted in Fig. 2.
Carbon nanoparticles and clusters associated with
size 50-100 nm present in air and when inhaled, these
can reach the alveolar cavity and interact with the
lung surfactant molecules. Such interaction can lead
to internalization of these particles inside the cavity
affecting the CO2-O2 exchange process that occurs
across the 1µm thick alveolar membrane walls. In this
communication,
the
concentration
dependent
interaction of lung surfactant, DPPC, with carbon
nanoparticles and clusters generated from carbon soot
is reported. Experimental data pertaining to zeta
potential, interfacial tension, UV-spectroscopy and
size of DPPC coated nanoparticles were obtained
which was used to model the internalization of carbon
nanoparticles inside alveolar cavity.
Materials and Methods
The methodology adopted for preparation of
carbon nanoparticles is described elsewhere12,13.
Survanta was purchased from Abbott Laboratories
Ltd., USA. For DPPC no confirmed CMC value could
be found in the literature, but it is expected to be quite
low14 (≈ 0.5 nM). The commercial preparation of
pulmonary surfactant, Survanta is too turbid, so we
used four different dilution ratios and prepared
solutions with dilutions: 50, 100, 200 and 300, and
these were used for dispersion of carbon
nanoparticles. Typically, 0.01% (w/v) powder of
carbon nano-material, obtained from lamp soot
(deposited on copper plate12) was added to the
surfactant solutions. The resultant dispersions were
mixed by continuous stirring with magnetic stirrer for
approximately 6 h and each sample was sonicated for
10 min. Then all samples were centrifuged for 20 min
at 7500 rpm to remove the big clusters and the
supernatant was used for further studies. The final
concentrations of CNPs in supernatant were
determined from UV-visible absorption spectra.
The particle size measurements were performed by
using dynamic light scattering (DLS) technique
(mostly at scattering angle = 90°, laser wavelength =
632.8 nm) on a digital correlator (PhotoCor
Instruments, USA). The samples were loaded into 5
ml cylindrical borosilicate glass cells and sealed. In
all measurements the difference between measured
and calculated baseline was not allowed to go beyond
± 0.1%. Scattered light obtained from sample was
converted into an intensity autocorrelation function
by a digital correlator. The measured intensity
Fig. 2—Chemical structure of dipalmitoylphosphatidylcholine (DPPC).
KUMAR & BOHIDAR: MULTI-CARBON NANOPARTICLES & LUNG SURFACTANTS
autocorrelation functions were analyzed by the
CONTIN regression software after ensuring that the
relaxation time distribution function did not contain
more than one distribution. Further details about DLS
and data analysis can be found elsewhere15. Average
particle size, morphology and size distribution of
MCNP were examined by a Fei-philips, Morgagni
268D transmission electron microscope (TEM)
(Digital TEM with image analysis system and
maximum magnification = × 280,000) operating at a
voltage 100 kV. The aqueous dispersion was drop –
cast onto a carbon-coated copper grid, and the grid
was air dried at room temperature (20°C) before
loading into microscope.
Electrophoresis measurements were performed on
a zeta potential instrument (ZC-2000, Microtec,
Japan). In order to minimize the influence of
electrolysis on the measurements, molybdenum (+)
and platinum (-) plates were used as electrodes. Also,
during the measurements, the cell chamber tap on
molybdenum electrode was kept open to release the
air bubbles, for the purpose of reducing their effects
on particle movement. During the measurements, the
molybdenum anode was cleaned each time as it
turned from a metallic to blue–black color. The
instrument was calibrated against 10-4 M AgI solution
to meet the pre-measurement conditions set by
manufacturer. The nominal distribution of zeta
potential is expected to be in the range -40 to -50 mV.
If we use the zeta potential (ζ) as an approximation of
the surface potential φ of a uniformly charged sphere,
the theory gives
ζ ≈ φ = 4π (σ/εκ)
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poorly soluble nanoparticle clusters dispersed in water
(Fig. 3). The typical cluster size was 150 nm and zeta
potential was close to -15 mV. These nanoparticles
have various sizes and conformations which allows us
to designate these as multi-carbon nanoparticles
(MCNP).
Figure 4 depicts the TEM picture of these particles
which shows asymmetric clusters of 30 nm size
monomers held together via hydrophobic interactions.
These clusters are highly polydisperse which supports
the DLS observations.
Survanta is a turbid preparation and it contains
surfactant proteins (SP-B and SP-C) in unspecified
amounts. The average cluster size of surfactant coated
nano-particles in these samples was measured
(Fig. 5). The increase in cluster size (≈145-200 nm)
Fig. 3—Particle size distribution of multi-carbon nanoparticles.
Notice the large polydispersity.
… (1)
where σ is the surface charge density of the particle
and ε and κ are the dielectric constant and DebyeHückel parameter of the solution, respectively. The
relationship between mobility (µ) and zeta potential
(ζ) is ζ = 4π (µη/ε). Then µ can be written as µ = σ/ηκ
where η is the viscosity of the solution. This is known
as Smoluchowski formula which is used to calculate
the zeta potential from electrophoretic mobility data16.
UV-spectroscopy data were collected by using a Cecil
model
CE-7200
(Cecil
Instrument,
UK)
spectrophotometer. Rectangular 5ml quartz cuvettes
were used to hold the samples.
Results and Discussion
The carbon nanoparticles were characterized by
DLS to ascertain their particle size distribution for the
Fig. 4—Typical TEM snapshot of MCNP clusters gives a
representation of polydispersity in size of dispersed particles in
solution. (scale bar = 100 nm).
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INDIAN J EXP BIOL, OCTOBER 2010
was typically 40%. The corresponding particle size
distribution (Fig. 6) indicates presence of
polydispersity in the dispersion. A small fraction of
scattering moieties had size ≈ 25-50 nm may be due
to the DPPC micelles present. Thus, there was coexistence of clusters adsorbed with DPPC surfactant
molecules and the free DPPC micelles.
The size of DPPC coated MCNPs deduced through
DLS studies was cross-checked with the same obtained
from TEM (Fig. 7). The picture shows existence of even
smaller micelles (size ≈ 10 nm) of DPPC uniformly
distributed in a dispersion of large clusters. The size of the
largest cluster seen in this picture has a size ≈ 100 nm and
the smallest has size ≈ 10 nm indicating wide
polydispersity. The UV spectra taken from the dispersions
indicated the specificity of interactions between MCNP
and Survanta. A typical spectrum is shown in Fig. 8
(dilution = 100). A sharp peak at 197 nm was recorded for
DPPC, and in presence carbon nanoclusters a broader
peak was observed at the same wavelength. This implied
that the surface of nanoclusters was heavily coated with
DPPC. Identical spectra were observed for other samples
that had various degrees of dilution. The 224 nm peak of
MCNP was completely missing from the spectra
indicating that all the nanoparticles were coated with
DPPC molecules. The corresponding zeta potential data
are shown in Fig. 9.
The combinations of UV absorption and zeta
potential data imply that the DPPC surfactants were
adsorbed on the carbon nanoclusters that resulted in the
increase in the size and surface charge of these clusters.
Since, DPPC is a surfactant with long hydrophobic tails,
preferential hydrophobic interaction between DPPC
molecules and carbon clusters permits the draining of
the hydrophobic tails onto the cluster surface. Such a
mechanism is consistent with the results presented.
Fig. 5—Variation of average diameter of carbon nanoclusters
dispersed in different Survanta solutions with different dilutions.
Fig. 6—Particle size distribution of clusters of MCNP dispersed in
Survanta solutions at different dilutions deduced from DLS data.
Internalization model
The interaction between gemini surfactant, DPPC,
and carbon nanoclusters was largely associative
innature. The surface charge on the surfactant coated
KUMAR & BOHIDAR: MULTI-CARBON NANOPARTICLES & LUNG SURFACTANTS
nanoclusters and the cluster size increased with
surfactant concentration indicating preferential
adsorption of DPPC on cluster surface that was
primarily driven by hydrophobic forces. Data imply
favourable hydrophobic interaction between DPPC
with any hydrophobic surface. Preferential draining of
the long aliphatic hydrocarbon chains on the carbon
nanocluster surface resulted in providing excellent
dispersion stability to these preparations. Thus, it is
possible to argue that when MCNPs enter the air
cavity of the alveoli (Fig. 10), these will immediately
get internalized in the phospholipid membrane which
provides ideal hydrophobic conditions for such
entrapment. The free-energy gain in such process
(DPPC adsorbing onto MCNP surface) is typically
1041
given by, ∆G = RT ln CMC, which comes to 10 kcal/mol. The effect of interfacial tension on
surfactant concentration was studied and the results
are presented in Fig. 11. It is clearly seen that the
Fig. 9—Variation of zeta potential shown as function of dilution
(water/Survanta) for carbon nanoclusters coated with the
pulmonary surfactant.
Fig. 10—The carbon nanoparticles reaching the cavity of the alveoli
(left) get internalized (right) in the phospholipid membrane due to
strong hydrophobic interactions. Such a process is energetically favored.
Fig. 7—Typical TEM snapshot of a nanocluster coated with
Survanta. (scale bar = 100 nm).
Fig. 8—UV-visible absorption spectra of MCNP dispersion
Survanta solution that had a dilution=100.
Fig. 11—Interfacial tension measured as function of DPPC
concentration. Note that the surface tension increases by about
20 dyn./cm due to the presence of CNPs.
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INDIAN J EXP BIOL, OCTOBER 2010
surface tension rises by 20 dyn/cm due to the presence
on carbon nanoparticles. Such an increase would lead
to more energy expensive inhalation.
Conclusions
The interaction of carbon nanoparticles and clusters
with lung surfactants was studied and it was found
that these surfactant molecules selectively adsorb onto
the hydrophobic carbon nanoparticle surface driven
by strong hydrophobic interactions. Such a process is
energetically favored. This conclusion permits
modeling of carbon nanoparticle internalization inside
alveolar cavity and it is proposed that the entrapment
of the nanoparticles occur along the inner wall of the
alveolar sac which is strongly hydrophobic. Such
localization of carbon nanoparticles can severely
hinder oxygen-carbon dioxide exchange.
Acknowledgement
This work was supported by a DRS research grant
of University Grants Commission, Government of
India. PKG acknowledges receipt of research
fellowship from the same organization.
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