Recent Advances in Nanomechanical Measurements and Their Application for Pharmaceutical Crystals.

Mechanical behavior of pharmaceutical crystals directly impacts the formulation development and manufacturing of drug products. The understanding of crystal structure-mechanical behavior of pharmaceutical and molecular crystals has recently gained substantial attention among pharmaceutical and materials scientists with the advent of advanced nanomechanical testing instruments like nanoindentation. For the past few decades, instrumented nanoindentation was a popular technique for measuring the mechanical properties of thin films and small-length scale materials. More recently it is being implemented to investigate the mechanical properties of pharmaceutical crystals. Integration of correlative microscopy techniques and environmental control opened the door for advanced structure-property correlation under processing conditions. Preventing the degradation of active pharmaceutical ingredients from external factors such as humidity, temperature, or pressure is important during processing. This review deals with the recent developments in the synchronized nanomechanical measurements of pharmaceutical crystals toward the fast and effective development of high-quality pharmaceutical drug products. This review also summarizes some recent reports to intensify how one can design and control the nanomechanical properties of pharmaceutical solids. Measurement challenges and the scope for studying nanomechanical properties of pharmaceutical crystals using nanoindentation as a function of crystal structure and in turn to develop fundamental knowledge in the structure-property relationship with the implications for drug manufacturing and development are discussed in this review. This review further highlights recently developed capabilities in nanoindentation, for example, variable temperature nanoindentation testing, in situ imaging of the indented volume, and nanoindentation coupled Raman spectroscopy that can offer new quantitative details on nanomechanical behavior of crystals and will play a decisive role in the development of coherent theories for nanomechanical study of pharmaceutical crystal.

Self-Assembly of Chiral Cyclohexanohemicucurbit[n]urils with Bis(Zn Porphyrin): Size, Shape, and Time-Dependent Binding.

In order to investigate the ability of bis(zinc octaethylporphyrin) (bis-ZnOEP) to discriminate cyclohexanohemicucurbit[n]urils (cycHC[n]) of different shapes and sizes, the self-assembly of barrel-shaped chiral cycHC[n] with bis-ZnOEP was studied by various spectroscopic methods (absorption, fluorescence, circular dichroism (CD), and NMR). While the binding of 6-membered cycHC[6] induced a tweezer-like conformation followed by the formation of anti-form of bis-ZnOEP upon further addition of cycHC[6], the interaction of 8-membered cycHC[8] is more complex and proceeds through the featured syn-to-anti conformational change of bis-ZnOEP and further intermolecular self-assembly via multiple noncovalent associations between cycHC[8] and bis-ZnOEP. Whilst bis-porphyrins are known to be effective chemical sensors able to differentiate various guests based on their chirality via induced CD, their ability to sense small differences in the shape and size of relatively large macrocycles, such as chiral cycHC[6] and cycHC[8], is scarcely examined. Both studied complexes exhibited characteristic induced CD signals in the region of porphyrin absorption upon complexation.

Cyclohexanohemicucurbit[8]uril Inclusion Complexes With Heterocycles and Selective Extraction of Sulfur Compounds From Water.

Solid-phase extraction that utilizes selective macrocyclic receptors can serve as a useful tool for removal of chemical wastes. Hemicucurbiturils are known to form inclusion complexes with suitably sized anions; however, their use in selective binding of non-charged species is still very limited. In this study, we found that cyclohexanohemicucurbit[8]uril encapsulates five- and six-membered sulfur- and oxygen-containing unsubstituted heterocycles, which is investigated by single-crystal X-ray diffraction, NMR spectroscopy, isothermal titration calorimetry, and thermogravimetry. The macrocycle acts as a promising selective sorption material for the extraction of sulfur heterocycles, such as 1,3-dithiolane and α-lipoic acid, from water.

Thermodynamics of adsorption of alcohol dehydrogenase on the gold nanoparticle surface: a model based analysis versus direct measurement.

Characterization of the nanoparticle protein corona has gained tremendous importance lately. The parameters which quantitatively establish a specific nanoparticle-protein interaction need to be measured accurately since good quality data are necessary for the elucidation of the underlying mechanism and accurate molecular dynamics simulation. Here, we have employed surface sensitive second harmonic light scattering (SHLS) for investigating the adsorption of a tetrameric protein, alcohol dehydrogenase (ADH, Saccharomyces cerevisiae 147 kDa), on 16 nm, 27 nm, 41 nm, and 69 nm citrate capped gold nanoparticles (GNPs) in aqueous phosphate buffer at pH 7. We have extracted the binding constant, number of ADH bound per GNP, Gibbs free energy (ΔG°) from the decay of the second harmonic scattered signal as a function of protein concentration using a modified version of the Langmuir adsorption isotherm. The data obtained were checked with another technique, dynamic light scattering, using the same modified Langmuir model (MLM). While the binding constants measured by the two methods are in agreement, the number of ADH bound to each GNP obtained by the two methods varies a lot. In order to further probe this binding independent of a model fitting, we used an orthogonal fluorescence assay which measures the number of ADH bound to a GNP directly, and no model-fitting is necessary. We then used temperature dependent SHLS to measure the heat of adsorption (ΔH°) and entropy (ΔS°) for ADH-GNP corona formation. We found that the equilibrium binding constant (Kb) obtained from SHLS is of the order of 109 M-1 and the formation of the GNP-ADH corona is spontaneous with ΔG° ∼ -55 kJ mol-1. However, the adsorption is modestly endothermic, accompanied by a large increase in entropy. Stated differently, GNP-ADH corona formation is entropically driven. This is perhaps due to the tremendous disruption of the water structure at the negatively charged interface upon the arrival of the protein within the bonding distance to it. We believe that the SHLS technique is highly sensitive and reliable, at very low concentrations of both nanoparticles and proteins, for the quantitative estimation of the thermodynamic parameters of nanoparticle-protein corona formation, where many other techniques may fall short.

Probing Polymer Chain Folding in Solution Using Second Harmonic Light Scattering.

Periodically grafted amphiphilic copolymers (PGACs) were earlier shown by us to adopt a zigzag folded conformation in the solid state, which enabled the backbone and pendant segments to segregate and occupy alternate layers in a lamellar structure. The conformational transition from a random coil to a zigzag folded chain in solution is an interesting problem, which is largely unexplored. To examine this, an orthogonally clickable parent polyester was sequentially clicked with two types of poly(ethylene glycol) (PEG) segments: one is a simple PEG and the other is a PEG that carries a dipolar chromophore. These two hydrophilic PEG segments, installed in a periodic and alternating fashion along the hydrocarbon-rich (HC) polyester backbone, ensure that the Janus folded chains are formed upon folding and carry chromophoric dipoles oriented along the same direction, thereby generating a large net dipole. The folding-induced alignment of chromophores in solution was followed using second harmonic light scattering (SHLS), wherein the intensity of the frequency-doubled scattered light (I2ω) is measured. Folding was induced by adding a polar solvent, like methanol, to a chloroform solution of the polymer; methanol desolvates the HC backbone but solubilizes the pendant PEG segments, thus inducing folding. The second harmonic intensity (I2ω) increased initially with methanol concentration and then saturated; in contrast, I2ω remained invariant with the solvent composition in the case of an analogous model chromophore. Furthermore, in a model PGAC carrying chromophore-bearing PEG segments on every repeat unit, I2ω decreased with increasing methanol composition, revealing the formation of a centrosymmetric folded chain, wherein the chromophoric dipoles on either side cancel each other. Thus, this study clearly reveals that the zigzag chain folding of PGACs can be induced by a segment-selective solvent, resulting in the rather elusive directional ordering of chromophoric dipoles in solution.

Binding Between Cyclohexanohemicucurbit[n]urils and Polar Organic Guests.

Inherently chiral, barrel-shaped, macrocyclic hosts such as cyclohexanohemicucurbit[n]urils (cycHC[n]) bind zinc porphyrins and trifluoroacetic acid externally in halogenated solvents. In the current study, we tested a set of eighteen organic guests with various functional groups and polarity, namely, thiophenols, phenols, and carboxylic and sulfonic acids, to identify a preference toward hydrogen bond-donating molecules for homologous cycHC[6] and cycHC[8]. Guests were characterized by Hirshfeld partial charges on acidic hydrogens and their binding by 1H and 19F NMR titrations. Evaluation of association constants revealed the complexity of the system and indirectly proved an external binding with stoichiometry over 2:1 for both homologs. It was found that overall binding strength is influenced by the stoichiometry of the formed complexes, the partial atomic charge on the hydrogen atom of the hydrogen bond donor, and the bulkiness of the guest. Additionally, a study on the formation of complexes with halogen anions (Cl- and Br-) in methanol and chloroform, analyzed by 1H NMR, did not confirm complexation. The current study widens the scope of potential applications for host molecules by demonstrating the formation of hydrogen-bonded complexes with multisite hydrogen bond acceptors such as cycHC[6] and cycHC[8].

Dynamic chiral cyclohexanohemicucurbit[12]uril.

NMR spectroscopy and DFT modeling studies of chiral cyclohexanohemicucurbit[12]uril indicate that the macrocycle adopts a concave octagonal shape with two distinct conformational flexibilities in solution. Methylene bridge flipping occurs at temperatures above 265 K, while urea monomers rotate at temperatures above 308 K, resulting in the loss of confined space within the macrocycle.

Thermodynamics of adsorption of lysozyme on gold nanoparticles from second harmonic light scattering.

Gold nanoparticle (GNP) interaction with hen egg white lysozyme (Lyz) has been investigated by many groups in order to understand protein mediated aggregation of GNPs and the underlying mechanism of aggregation. In this article, we have studied the interaction of citrate-capped GNPs of 16, 28, 41, and 69 nm sizes with Lyz by the non-destructive label-free second harmonic light scattering (SHLS) technique at physiological pH in phosphate buffer. The surface sensitivity of the nonlinear optical SHLS technique is very high and we have looked at the GNP-Lyz interaction at nanomolar concentrations. We have followed the increase in the SHLS intensity of GNPs as a function of the added concentration of Lyz in small aliquots. The SH intensity profile exhibits saturation behaviour and was fitted with a modified Langmuir adsorption model which yielded the binding constant (Kb), the binding stoichiometry (nsat) at saturation and the free energy change (ΔG) in the adsorption process. The free energy change was further decomposed into changes in the enthalpy (ΔH) and entropy (ΔS) of adsorption by carrying out temperature dependent SHLS measurements in a specially designed cell. The thermodynamic quantities extracted from the measurements show that the binding is exothermic (ΔH < 0) as well as spontaneous (ΔS > 0). We find that the first step in the adsorption of Lyz on the GNP surface is nanoparticle protein corona (NP-PC) formation driven predominantly by electrostatic attraction. In the second step of adsorption, the adsorbed lysozymes on the surface form a bridge between two or more GNPs leading to the latter's aggregation, which is the main reason for the enhancement of the SH scattering signal. Although the interaction between the GNPs and Lyz is driven by strong electrostatic attraction, the thermodynamic quantities reported here indicate that the protein is physisorbed on the nanoparticle surface. We have also demonstrated that SHLS provides a new tool for full thermodynamic characterization of protein adsorption on metal nanoparticles at ultralow concentrations.

Structural analysis of elastically bent organic crystals using in situ indentation and micro-Raman spectroscopy.

The structural dynamics of two elastically bendable, halogenated N-benzylideneaniline organic crystals were studied using an in situ three-point bending test and Raman spectroscopy. This study reveals the inhomogeneous molecular distribution in the elastic crystal lattice during the bent stage and further validates the known qualitative mechanistic model of elastic bendable crystals.

Protein-Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study.

In this article, we have studied the binding of different naturally occurring hemoglobin (Hb) variants on erythrocyte skeletal protein, spectrin surface using the label free nondestructive second harmonic light scattering (SHLS) technique in aqueous buffer. Hemoglobin variants like sickle hemoglobin (HbS) and hemoglobin E (HbE) were chosen as they associate with sickle cell disease and HbEß-thalassemia, respectively, and their interaction with spectrin is compared with normal adult hemoglobin (HbA). The concentration dependent change in the second harmonic light intensity from nanomolar spectrin solution has been measured after addition of small aliquots of hemoglobins. From the second harmonic titration data, the binding constant is calculated using a modified Langmuir adsorption model of hemoglobin binding to the spectrin surface. Interestingly, it is found that the binding constant for HbE (13.8 × 108 M-1) is 1 order of magnitude higher than that of HbS (1.6 × 108 M-1) or HbA (2.1 × 108 M-1) which indicates higher affinity of HbE for spectrin compared to HbA and HbS. The number of the Hb molecules bound to the spectrin surface was estimated to be of the order of hundred's which is determined for the first time.