(600d) Surface-Induced Nucleation Strategies: Seeking Symmetries between Self-Assembly of Heteronucleants and Crystals

Nucleation represents the core of a variety of processes occurring in nature, such as ice or cloud formation, but it is also responsible of amyloid aggregation leading to Alzhemeir’s disease or even creation of virus capside. The formation of new phase in defined environment as a consequence of self-assembly and ordering processes represents a fil-rouge for all these phenomena. More specifically, nucleation can occur homogeneously or heterogeneously, depending on whether aggregation mechanism involves the presence of single or multiple phases. In the latter case, stable nuclei are formed taking advantage of favorable interactions with foreign surfaces which lower the energetic cost for new interface area formation. However, heterogeneous nucleation often occurs in uncontrolled manner because of impurities in the system, leading to significantly different crystallization outcomes. This aspect is pivotal to pharmaceutical and biopharmaceutical industries as nucleation not only affects process kinetics, but also crystalline form, habit and size selection, as well as downstream processes.

Recently, the role of external surface features on crystallization of active pharmaceutical ingredients (APIs) is being investigated by relating surface chemistry and morphology to nucleation kinetics and polymorph selection. Here, we present an organic approach to the study of heteronucleants quality attributes in order to relate them to crystallization of small molecule drugs. The complete and diversified characterization of surface properties represents the core of this contribution, as the interplay of complex and different aspects of a surface is a key-aspect when promoting or inhibiting nucleation. In this study, self-assembled monolayers (SAMs) have been selected as ideal candidates to probe superficial chemical effects on nuclei formation and successive crystal growth. An amorphous substrate was selected for supporting monolayer assembly in order to exclude epitaxial interaction between the final heteronucleant and solute molecules. In this way, we were able to exclude complex ordering phenomena rising from lattice matching between surface and growing nuclei. In addition, such an approach enables the development of crystallization supports which are structurally inert, but chemically active. By carefully tailoring synthesis conditions, we synthesized high-quality SAMs on amorphous substrates. Thorough characterization of the final product has been carried out in order to confirm functionalization effectiveness and gain a structured overview of surface properties.

SAMs were made of silane molecules carrying a propyl chain with a terminal functionality. The latter represented the only difference among our monolayers, being thiol, amino, methacrylate or glycidyloxy terminated. The portfolio of elected end groups permits the deep investigation of secondary interactions between surface and solute molecules and their impact on nuclei formation. As a matter of fact, our final goal is to design and develop a versatile benchmark for crystallization studies assessing extent of specific and non-specific interactions. This is achieved by only changing substrate, while all crystallization conditions, e.g. supersaturation, solvent, temperature, are not to be modified.

Trimethoxysilanes were chosen because of their high reactivity and degree of packing when organized in monolayers. These molecules have been anchored to pre-activated glass substrates by means of condensation reactions between surface hydroxyl groups and hydrolyzed methoxy groups of silane. Synthesis conditions have been optimized in terms of reaction solvent, functionalizing agent, reaction time and temperature in order to ensure the formation of reproducible and single layers of organic matter on glass. As a matter of fact, for crystallization studies devoted to the mechanistic understanding of surface effects on nucleation, it is crucial to deal with extremely uniform substrates both as concerns the sample itself, but also intra and inter-batch homogeneity. This need is direct consequence of the intrinsic stochastic nature of nucleation, thus requiring a high number of experiments involving same experimental conditions to get statistically significant data.

Surfaces were characterized in terms of a variety of techniques to get an as wide as possible panorama of information regarding superficial properties. Contact angle analyses were firstly carried out in order to assess the effectiveness of functionalization step and surface uniformity. Moreover, using different probing liquids, surface energy and relative components were calculated for our four types of monolayers. In this way, the extent of dispersive and polar (acid-base) components of surface energy were determined and could be then related to nucleation behavior. In a parallel fashion, surface zeta potential was evaluated by means of particle scattering techniques as it provides useful information to probe functionalization and electrostatic interaction between the surface and crystallization environment. Surface morphology and topography was evaluated by means of Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Absence of local deposits of polymerized silanes was confirmed by SEM when comparing samples prepared by optimized synthesis protocol to previous ones. Extremely regular functionalization was achieved, as highlighted by AFM investigation, and some interesting insights into surface roughness and profile height were collected. It turned out that the presence of a monolayer on the surface only altered surface mean roughness (R_q) to a minor extent, whereas, when uncontrolled functionalization was carried out, major effects on this parameter were detected. More specifically, untreated glass represented an extremely flat surface, having R_q less than 1 nm. Then, covalent bonding of an organic monolayer on top led to average roughness values of approximately 1 nm for all the functionalities. Microscopy investigations also pointed out the continuity of the silane layer over the surface: a high degree of packing was reached in optimized conditions, leading to the absence of voids or not functionalized areas. These features represent a particular appealing aspect of the application of high-quality SAMs to heterogeneous nucleation studies since, as topography is not altered by the functionalization process, the effect of different superficial chemistries may be isolated from morphological interactions between substrate and solute aggregates. The effectivity of functionalization was corroborated by means of X-Ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Second Ions Mass Spectrometry (ToF SIMS). In particular, XPS analyses were carried out for different purposes: the main goal was the confirmation of the presence of characteristic elements of SAMs on glass and the elucidation of elements chemical state. Nevertheless, starting from XPS data, we were able to calculate the thickness of organic layer thanks to angle-resolved analyses. By comparing the theoretical SAM thickness linked to the silane molecule length to the experimental ones calculated by means of XPS and ellipsometry, we confirmed that functionalization occurred in monolayer. Further confirmation derived from the comparison of characteristic elements ratios in the ideal and real case, as revealed by XPS analyses, and the identification of silane typical fragments, as highlighted by ToF SIMS.

Complete characterization of the SAMs led us to confirm the suitability of these surfaces to be used as heteronucleants in crystallization studies. Extremely flat surfaces were selected in order to deconvolute, as much as physically possible, the contribution of surface chemistry and topography to nucleation phenomena. We designed and implemented a high-throughput crystallization platform in order to perform a large number of crystallization experiments simultaneously. Aspirin crystallization was carried out in a thin-film solution deposited onto SAMs. Crystallization outcome was studied according to kinetic and thermodynamic aspects, involving nucleation induction time and crystalline form and habit selection. The first aspect was studied by means of optical microscopy coupled to motorized stage and cooling system, enabling time-lapse multiple-positions acquisition and ensuring precise supersaturation conditions until the nuclei formation. Nucleation kinetics was evaluated by calculation of detectable induction time, corresponding to the time when detectable crystals could be evinced inside each crystallizer. Crystal orientation and form were evaluated by means of X-Ray Diffractometry (XRD) and served for probing if stable or metastable crystal forms had been obtained according to surface chemistry.

In conclusion, an optimized method for the synthesis of self-assembled monolayers is here presented, along with a wide set of characterization techniques for the evaluation of surface quality and features. Such a reference platform is to be linked to crystallization behavior of heteronucleants. Self-assembly was therefore exploited at two different levels: firstly, for the synthesis of heteronucleants with defined properties and, then, for the study of the interplay between surfaces and self-assembly of solute molecules in solution leading to crystals.