279997 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, kBT. 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.
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.
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