It is widely accepted that many phase transitions do not follow nucleation pathways as envisaged by the classical nucleation theory. Many substances can traverse intermediate states before arriving at the stable phase. The apparent ubiquity of multi-step nucleation has made the inverse question relevant: does multistep nucleation always dominate single-step pathways? Here we provide an explicit example of the classical nucleation mechanism for a system known to exhibit the characteristics of multi-step nucleation. Molecular resolution atomic force microscopy imaging of the two-dimensional nucleation of the protein glucose isomerase demonstrates that the interior of subcritical clusters is in the same state as the crystalline bulk phase. Our data show that despite having all the characteristics typically associated with rich phase behaviour, glucose isomerase 2D crystals are formed classically. These observations illustrate the resurfacing importance of the classical nucleation theory by re-validating some of the key assumptions that have been recently questioned.

It is shown that diffusion-limited classical nucleation theory (CNT) can be recovered as a simple limit of the recently proposed dynamical theory of nucleation based on fluctuating hydrodynamics (Lutsko, JCP 136, 034509 (2012)). The same framework is also used to construct a more realistic theory in which clusters have finite interfacial width. When applied to the dilute solution/dense solution transition in globular proteins, it is found that the extension gives corrections to the the nucleation rate even for the case of small supersaturations due to changes in the monomer distribution function and to the excess free energy. It is also found that the monomer attachement/detachment picture breaks down at high supersaturations corresponding to clusters smaller than about 100 molecules. The results also confirm the usual assumption that most important corrections to CNT can be acheived by means of improved estimates of the free energy barrier. The theory also illustrates two topics that have received considerable attention in the recent literature on nucleation: the importance sub-dominant corrections to the capillary model for the free energy and of the correct choice of the reaction coordinate.

In this work, the efficiency of bioinspired citrate-functionalized nanocrystalline apatites as nanocarriers for delivery of doxorubicin (DOXO) has been assessed. The nanoparticles were synthesized by thermal decomplexing of metastable calcium/citrate/phosphate solutions both in the absence (Ap) and in the presence (cAp) of carbonate ions. The presence of citrate and carbonate ions in the solution allowed us to tailor the size, shape, carbonate content, and surface chemistry of the nanoparticles. The drug-loading efficiency of the two types of apatite was evaluated by means of the adsorption isotherms, which were found to fit a Langmuir-Freundlich behavior. A model describing the interaction between apatite surface and DOXO is proposed from adsorption isotherms and ζ-potential measurements. DOXO is adsorbed as a dimer by means of a positively charged amino group that electrostatically interacts with negatively charged surface groups of nanoparticles. The drug-release profiles were explored at pHs 7.4 and 5.0, mimicking the physiological pH in the blood circulation and the more acidic pH in the endosome-lysosome intracellular compartment, respectively. After 7 days at pH 7.4, cAp-DOXO released around 42% less drug than Ap-DOXO. However, at acidic pH, both nanoassemblies released similar amounts of DOXO. In vitro assays analyzed by confocal microscopy showed that both drug-loaded apatites were internalized within GTL-16 human carcinoma cells and could release DOXO, which accumulated in the nucleus in short times and exerted cytotoxic activity with the same efficiency. cAp are thus expected to be a more promising nanocarrier for experiments in vivo, in situations where intravenous injection of nanoparticles are required to reach the targeted tumor, after circulating in the bloodstream.

Many mineralization processes occur in convection-free conditions. Understanding these processes requires knowledge of crystal nucleation and growth processes in gels or high viscous sol systems. In this work, the crystallization parameters of calcium carbonate in an agarose viscous sol using counterdiffusion crystallization were monitored as a function of time. Additionally, by comparing the precipitation parameters in the high viscous sol entrapping charged polypeptides, namely, poly-L-lysine (pLys), poly-Laspartate (pAsp), and poly-L-glutamate (pGlu), it was possible to establish the polypeptide capability to inhibit, or eventually promote, the calcium carbonate nucleation and/or crystal growth processes. The polymorphism and morphology of the precipitates indicate that pLys only influences the growth mechanism of calcium carbonate without affecting the nucleation process. On the contrary, pAsp and, to a minor extent, pGlu affect both nucleation and growth. The application of this analysis can be extended to other additives and macromolecules able to affect crystallization processes.

A two-variable stochastic model for diffusion-limited nucleation is developed using a formalism derived from fluctuating hydrodynamics. The model is a direct generalization of the standard Classical Nucleation Theory. The nucleation rate and pathway are calculated in the weak-noise approximation and are shown to be in good agreement with direct numerical simulations for the weak-solution/strong-solution transition in globular proteins. We find that Classical Nucleation Theory underestimates the time needed for the formation of a critical cluster by two orders of magnitude and that this discrepancy is due to the more complex dynamics of the two variable model and not, as often is assumed, a result of errors in the estimation of the free energy barrier.