Functionalization of glass surfaces with SAMs: the effect of synthesis conditions and the application to pharmaceutical crystallization

Surfaces can alter the outcome of crystallization processes occurring via heterogenous nucleation. In a pharmaceutical scenario, the promotion of specific polymorphs or crystalline habits, as well as the alteration of nucleation kinetics, are compelling issues. Surfaces with controlled physico-chemical features represent a valuable tool for the study of drug crystallization by heterogeneous nucleation. For this purpose, the functionalization of glass with Self-Assembled Monolayers (SAMs) via silane chemistry was investigated. SAMs carrying thiol, amino, glycidyloxy and methacrylate end-groups will be presented. Different sets of synthesis conditions strongly affected the quality of SAMs. In this perspective, the reaction medium and the reaction time were identified as key parameters for getting controlled surface functionalization. Typical surface roughness was approx. 130 pm and SAM thickness was below 1 nm. SAM chemistry was investigated with XPS to confirm the presence of characteristic groups on the surface of glass. Finally, the application of SAMs to the crystallization of aspirin will be presented, discussing the impact of several surface chemistries on the nucleation kinetics

Agarose, the gel to tailor your protein crystals

The growth of protein crystals in gel has proved to date to be the cheapest means to produce protein crystals of high quality similar to those obtained under microgravity conditions (Gavira et al., 2020; Robert & Lefaucheux, 1988; Snell & Helliwell, 2005). Gels create a stable environment for crystals to grow in convection-free conditions avoiding sedimentation and the formation of aggregates and increasing crystals uniformity. The use of agarose has allowed progress in the limitations of crystal size and quality and even to obtain protein crystals inaccessible by other techniques (Sica et al., 1994). In this work we have exploited the nucleation inducing ability of agarose gels in diffusiondominated environments. Crystal size was successfully tuned in a wide range of agarose, protein and precipitant concentrations. The impact of gel content on crystal size resulted to be independent of the specific protein, allowing the mathematical prediction of crystals size and pointing out the exclusivity of the physical interactions between the gel and the protein to explain the observed behaviour. The versatility of the technique and the fine-tuning of the nucleation flux was demonstrated by crystallizing five different model proteins using two different techniques, batch and counter-diffusion. In addition, the potential of agarose to be used as a growth and delivery medium for serial crystallography applications has been proven by preparing unidimensional micro-crystals slurries in 0.1 % (w/v) gel

Protein crystallisation in agarose gel, a cheap and versatile technique

Crystallization in hydrogels is not a frequent practice in bio-crystallography, although the benefits are multiple: prevents convection and crystal sedimentation, acts as impurity filter, etc., and have been proven to be the cheapest means to produce protein crystals of high quality similar to those obtained under microgravity conditions. Moreover, gel grown protein crystals are excellent candidates as seeds to produce crystals of bigger size for neutron diffraction or as media for crystals delivery in serial femtosecond crystallography. Hydrogel should also be considered to exert control over the nucleation and growth processes. In this work we will present our most recent studies on the influence of agarose over the nucleation and growth of protein crystals. Crystal number and size was successfully tuned in a wide range of agarose concentration while keeping constant other conditions. Using five model proteins we demonstrate that the influence of gel content is independent of the protein nature, allowing the mathematical prediction of crystals flux and size with little experimental effort. The convection free environment obtained even at low agarose concentration permits the obtention of high homogeneous micro-crystals slurries that could be used for serial crystallography application or for the mass production of enzyme crystals for industrial application. Last, we will also show how it allows to explore the phase diagram under a kinetic regime that may facilitate the growth of different polymorphs

Surface-induced nucleation strategies: seeking symmetries between self-assembly of heteronucleants and crystals

The present paper presents the application of surfaces having defined and controlled attributes as heteronucleants for the crystallization of a model pharmaceutical molecule. The synthesis of the substrate was optimized in order to relate surface features to the crystallization outcome. Extremely flat and topographically uniform glass supports bearing amino and thiol head groups were successfully synthesized and characterized by means of contact angle, AFM and XPS analyses. Such surfaces were then used as supports for aspirin (ASA) crystallization in order to investigate their influence on nucleation kinetics. Compared to untreated glass, amino-functionalized glass was dramatically nucleation-active, whereas thiol-functionalized supports strongly repressed ASA heterogeneous nucleation. The promoting or inhibiting action towards the stabilization of ASA nuclei on a functionalized surface and their successive growth into crystals was therefore related to the chemistry of exposed head groups

A general and adaptive synthesis protocol for high-quality organosilane self‐assembled monolayers as tunable surface chemistry platforms for biochemical applications

The controlled modification of surface properties represents a pervasive requirement to be fulfilled when developing new technologies. In this paper, we propose an easy-to-implement protocol for the functionalization of glass with Self-Assembled Monolayers (SAMs). The adaptivity of the synthesis route was demonstrated by the controlled anchoring of thiol, amino, glycidyloxy, and methacrylate groups onto the glass surface. The optimization of the synthetic pathway was mirrored by extremely smooth SAMs (approx. 150 pm roughness), layer thickness comparable to the theoretical molecule length, absence of silane islands along the surface, quasi-unitary degree of packing, and tailored wettability and charge. The functionalization kinetics of two model silanes, 3-mercapto- and 3-amino-propyltrimethoxysilane, was determined by cross-comparing X-Ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) data. Our SAMs with tailored physico-chemical attributes will be implemented as supports for the crystallization of pharmaceuticals and biomolecules in upcoming studies. Here, the application to a small molecule drug model, namely aspirin, was discussed as proof of concept

Surface analysis of functionalized substrates for the nucleation and crystallization of pharmaceutical molecules

Nucleation represents the core of a variety of natural processes, ranging from ice crystal formation to protein aggregation, which can occur homogeneously or heterogeneously, depending on whether aggregation mechanism involves the presence of single or multiple phases. 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.1 Surface functionalization using SAMs has been largely investigated and applied to different substrates and constitutes one of the most robust methods available to obtain well controlled functionalized surfaces.2,3,,4 In the present study, the use of glass substrates functionalized with self-assembly of trimethoxysilanes differing for their head group chemistry for the nucleation and crystallization of small pharmaceutical molecules was investigated. Silane anchoring was achieved via wet chemistry-based route whilst systematic characterisation of morphology and chemistry was carried on in order to determine the influence of different parameters (Figure 1). Assessment of effective functionalization was carried out by means of contact angle and surface Z-potential analyses, whilst the surface chemistry of functionalized glass was probed using XPS and ToF SIMS. Lastly, AFM was adopted for the characterization of surface topography. High-quality monolayers carrying thiol, methacrylate and glycidyloxy exposed groups were successfully synthesized whereas in the case of amino-terminated silanes surface roughness dramatically increased and correlation between ideal and experimental elemental ratios characterizing the monolayer was not achieved. Nucleation and crystallization of biopharmaceutical molecules was also carried out by studying aspirin and paracetamol crystallization out in a thin-film solution deposited onto SAMs. Crystallization outcome was studied according to kinetic and thermodynamic aspects by optical microscopy, whilst crystal orientation and form were evaluated by means of X-Ray Diffractometry (XRD). Finally, preliminary results on nanostructurated surfaces will be also presented

Development of freeze-drying cycle for a peptide-based drug in trays

Freeze-drying of a peptide solution is here presented, from the early identification of critical product temperature to the design of an appropriate cycle. Synergy between experimental characterisation techniques, mathematical modelling and process simulation has been implemented to develop a robust lyophilisation process and guarantee the removal of solvents impurities. Process has been carried out in bulk using a tray equipped with an anisotropic membrane, enabling unidirectional vapour flow

Freeze-drying of pharmaceuticals in vials nested in a rack system

The distribution of biopharmaceuticals often requires lyophilisation. The drug product is first frozen and potentially exposed to stress conditions that can be detrimental to its quality. These stresses are also encountered when a drug product has to be distributed under ultra-cold conditions. Adjusting the formulation and/or freezing conditions allows for limiting the impact of these stresses on the final product. This paper investigates two loading configurations, vials directly resting on the shelf and nested in a rack system, and their impact on the freezing and drying behaviour of a sucrose-based formulation. First, two key freezing parameters, i.e., ice nucleation temperature and cooling rate, were studied as they can affect the product behaviour during drying. The product freezing rate and the ice nucleation temperature distribution were affected by the loading configuration, resulting in larger ice crystals in the case of vials nested in a rack system. The analysis was also extended to the drying phase, showing that the loading configuration impacted the product temperature during drying and the overall heat transfer coefficient between the equipment and the product

Lignin: a sustainable antiviral coating material

Transmission of viruses through contact with contaminated surfaces is an important pathway for the spread of infections. Antiviral surface coatings are useful to minimize such risks. Current state-of-the-art approaches toward antiviral surface coatings either involve metal-based materials or complex synthetic polymers. These approaches, however, even if successful, will have to face great challenges when it comes to large-scale applications and their environmental sustainability. Here, an antiviral surface coating was prepared by spin-coating lignin, a natural biomass residue of the paper production industry. We show effective inactivation of herpes simplex virus type 2 (>99% after 30 min) on a surface coating that is low-cost and environmentally sustainable. The antiviral mechanism of the lignin surface was investigated and is attributed to reactive oxygen species generated upon oxidation of lignin phenols. This mechanism does not consume the surface coating (as opposed to the release of a specific antiviral agent) and does not require regeneration. The coating is stable in ambient conditions, as demonstrated in a 6 month aging study that did not reveal any decrease in antiviral activity. This research suggests that natural compounds may be used for the development of affordable and sustainable antiviral coatings