(286e) Controlled Magnetite Nanoparticle Morphology for Better Cellular Uptake: Experiments and Models

            Aqueous
dispersions containing water-insoluble magnetite (Fe₃O₄)
nanoparticles are widely used in various biomedical applications, like
hyperthermia, magnetic labeling of cells, enhanced contrast in MRI of tissues etc.
In these applications, the final diameter and morphology of the nanoparticles
(either isolated primary particles or aggregates of primary particles) are the
most important parameters determining the efficacy of cellular uptake of these
particles. Several experimental reports suggest that Fe₃O₄ nanoparticles
synthesized in the aqueous medium can be either of isolated or an aggregated
morphology. It is of interest to explain the particle formation mechanism, so
that one can optimize the experimental conditions to achieve nanoparticles of a
specific diameter and morphology, which would determine colloidal stability and
shelf-life of these particles or their dispersions, for most effective cellular
uptake of these formulations. To this end, we develop the mechanism of Fe₃O₄
nanoparticle formation (a water-insoluble material) in an aqueous medium, the
resultant structure of the particulate-dispersion and its uptake by human
cells.  

  Methodology
and results: Time-scales, mechanism, morphologyy and uptake

We have conducted both chemical coprecipitation and thermal
decomposition methods for nanoparticle synthesis. Different biocompatible
coating agents such as carboxymethyl cellulose (CMC), dextran, citric acid or
poly (acrylic) acid (PAA) were used to coat the particles, in order to disperse
them and achieve a stable aqueous dispersion. The crystal structure and
diameter of Fe₃O₄ nanoparticles was confirmed by X-Ray
Diffractometer (XRD). Fourier transform infra red (FTIR) spectroscopy of
particles showed that the coating agents were chemically adsorbed on the
particle surface. Transmission electron microscopy (TEM), atomic force
microscopy (AFM) and dynamic light scattering (DLS) of coated particles
together gave both core particle diameter and the hydrodynamic diameter in the
dispersion. Now, in absence of a coating agent, particles synthesized by the
coprecipitation route always resulted in an aggregated morphology, having core,
primary particle diameter in the range of 6 - 9 nm. However, with citric acid
coating; nearly completely dispersed, coated particles were achieved by first
treating the uncoated particles with tetra methyl ammonium hydroxide (TMAOH),
followed by citric acid coating of the treated particles. In contrast, PAA
coated particle were still aggregated. On the other hand, in thermal
decomposition route, completely isolated and monodisperse PAA coated particles
having average core particle diameter of 6 nm with a standard deviation of 1.1
nm were obtained.

Therefore,
to understand the aforesaid contrasting morphology of PAA coated particles
obtained via the two routes, a time-scale based mechanisms of particle
formation for each route was developed. The relative rates (or in other words
the characteristic time scales) of each of the individual steps leading to
particle formation, namely, adsorption of coating agent on a nanoparticle,
diffusion of particle on a polymer chain, Brownian collision and coagulation of
particles etc., were estimated a-priori. We find that the coagulation time
scale in thermal decomposition route is much smaller than the experimental
aging time, resulting in complete coagulation and formation of isolated PAA
coated particles. In contrast, during coprecipitation, absence of coagulation
during the experimental aging time resulted in formation of aggregates of
particles. Thus from our mechanistic study, we conclude that the high
temperature synthesis of Fe₃O₄ in thermal decomposition
leads to faster coagulation rate and results in isolated nanoparticle
formation.

Coated
particles in a dispersion require a long shelf-life for in-vivo use.
Stability against sedimentation depends on the number of primary particles in
an aggregate (single primary particle, two particles forming a dimer, and
higher aggregates). We calculated the aggregate number density distribution of
our synthesized particles
in the aqueous
dispersion using Monte Carlo simulation. The
simulation was performed by calculating the total interaction potential between
two nanoparticles as a function of their interparticle distance, and applying a criterion for the two particles to aggregate; the criterion being
that the minimum depth of the secondary minimum in the total interaction
potential must be at least equal to thermal energy,k_BT. Total
interaction potential curve for citric acid coated particles showed a secondary
minimum (formation of reversible aggregates) and dextran coated particles
showed a primary minimum (formation of permanent aggregates). The former is due
to low shell thickness possessed by citric acid coated particles and latter is
due to bridging attraction of dextran coated particles. PAA coated particles
did not show either primary or secondary minimum, because of its larger shell
thickness, thereby justifying the completely isolated particles obtained in
experiments. Number density distribution predicted by simulation showed that
citric acid coated particles will be in the form of moderate aggregates,
dextran in complete aggregate and PAA in isolated form. Simulation predictions
were compared with experimental results and they showed good agreement with
each other. This new approach of determining the states of particles in
dispersions provides a-priori information in choosing an appropriate
coating agent for magnetic nanoparticles, in order to achieve isolated
particles in dispersions.

Finally,
dispersions containing different morphology of coated particles were incubated
(in-vitro) with human hepatoma cell lines (HepG2), in order to
understand the effect of morphology and surface charge of the coated
nanoparticles on cellular uptake. Better cell viability was observed with
citric acid and dextran coated nanoparticles, compared to PAA coated particles.
We find that the neutral dextran coated particles in the form of aggregates
were not taken up by the cells, whereas negatively charged and nearly isolated
citric acid coated particles showed very good uptake. Rate of uptake of citric
acid coated particles in cells was fitted to a mathematical model based on a
two stage mechanism of particle adsorption and internalization. Based on this
model, the average mass of Fe (in the form of nanoparticles) internalized by a
single HepG2 cell was predicted. From this analysis, we conclude that the
coated particles having isolated morphology and a surface charge can lead to
high cellular uptake.

Conclusions

Individual (primary) nanoparticle diameter, charge and
morphology of an ensemble of these nanoparticles - either in an isolated or an
aggregated form - are key parameters dictating colloidal stability and
shelf-life of aqueous particulate dispersions aimed towards long-term
usage of these dispersions for in-vivo cellular uptake. Our
time-scale based nanoparticle formation mechanism models primary particle
diameter, and interparticle potential-driven Monte Carlo simulation predicts
their equilibrium state of aggregation and dispersion stability. This in
conjunction with a model of cell-particle interaction explains experimental
data on coated magnetite nanoparticle uptake by HepG2 cells, correctly
predicting contrasting behavior of different coating agents on extent of
uptake. We find that citric acid coated, negatively
charged particles, having a nearly isolated morphology, leads to a high
cellular uptake; this compared to dextran coated particles, which are large
aggregates of uncharged particles. The experimentally validated
model is therefore useful in designing synthesis conditions to achieve
controlled size, shape and aggregation of primary nanoparticles, aimed towards
maximum uptake of solid particles, or extending further, in elucidating
diffusion and accessibility issues of guest molecules in porous nanoparticles. The
simulation results can also help in screening of a proper coating agent to
achieve any desirable state of aggregation of particles in the dispersion,
aimed towards optimum features in cellular uptake of nanoparticles.