Small-Angle X-ray Scattering (SAXS) Used for the Identification of Nicomorphine Polymorphic Changes at the Early Stage to Avoid Varied Stability and Possible Side Effects.

In this paper, we present the identification of polymorphisms at an early stage, identified by applying non-standard methods such as SAXS. We provide an analytical approach to polymorphism in the quality/purity of an active pharmaceutical ingredient (API), supplied to a generic company by two different suppliers (i.e., manufacturers). Changes in thermodynamic polymorphism firstly become visible in traces in the larger crystal lattices, which are visible on the SAXS spectrum only using the logarithmic scale, as shown in the result figures. Hence, we are here on the trail of the beginning of a new polymorph in nicomorphine, whose crystal waviness at the early stage is visible only in the additional symmetrical peaks identified and calculated using SAXS, while the chemical analyses excluded all kinds of chemical impurities. The chemical and structural properties were studied using the following techniques: SAXS, WAXS, DSC, dissolution, Raman spectroscopy, and FTIR. Only the SAXS technique could identify crucial differences and calculate the additional signals related to giant crystals, whilst a standard method such as WAXS showed none, and nor did the chemical analyses, such as Raman spectroscopy and FT-IR. This means that due to water in crystallization (known in nicomorphine) or thermodynamic waviness, the formation of the new polymorph starts first in traces, which become visible at larger distances from the crystal lattice, detectible only in the SAXS range. This is a very important premise and hypothesis for further research, and we believe that this work lays a new stone in understanding the origin of new unknown polymorphs and their mixtures. Therefore, the aim of this work is to show that the use of non-standard methods (i.e., SAXS) can be of great benefit to API analysis and the identification of polymorphic changes in the early phase, which can cause varied stability, solubility and bioavailability and thus different therapeutic effects or side effects.

KIT-5-Assisted Synthesis of Mesoporous SnO2 for High-Performance Humidity Sensors with a Swift Response/Recovery Speed.

Developing highly efficient semiconductor metal oxide (SMOX) sensors capable of accurate and fast responses to environmental humidity is still a challenging task. In addition to a not so pronounced sensitivity to relative humidity change, most of the SMOXs cannot meet the criteria of real-time humidity sensing due to their long response/recovery time. The way to tackle this problem is to control adsorption/desorption processes, i.e., water-vapor molecular dynamics, over the sensor's active layer through the powder and pore morphology design. With this in mind, a KIT-5-mediated synthesis was used to achieve mesoporous tin (IV) oxide replica (SnO2-R) with controlled pore size and ordering through template inversion and compared with a sol-gel synthesized powder (SnO2-SG). Unlike SnO2-SG, SnO2-R possessed a high specific surface area and quite an open pore structure, similar to the KIT-5, as observed by TEM, BET and SWAXS analyses. According to TEM, SnO2-R consisted of fine-grained globular particles and some percent of exaggerated, grown twinned crystals. The distinctive morphology of the SnO2-R-based sensor, with its specific pore structure and an increased number of oxygen-related defects associated with the powder preparation process and detected at the sensor surface by XPS analysis, contributed to excellent humidity sensing performances at room temperature, comprised of a low hysteresis error (3.7%), sensitivity of 406.8 kΩ/RH% and swift response/recovery speed (4 s/6 s).

Surface Morphology of Textured Transparent Conductive Oxide Thin Film Seen by Various Probes: Visible Light, X-rays, Electron Scattering and Contact Probe.

Fluorine-doped tin oxide thin films (SnO2:F) are widely used as transparent conductive oxide electrodes in thin-film solar cells because of their appropriate electrical and optical properties. The surface morphology of these films influences their optical properties and therefore plays an important role in the overall efficiencies of the solar cells in which they are implemented. At rough surfaces light is diffusely scattered, extending the optical path of light inside the active layer of the solar cell, which in term improves light absorption and solar cell conversion efficiency. In this work, we investigated the surface morphology of undoped and doped SnO2 thin films and their influence on the optical properties of the films. We have compared and analysed the results obtained by several complementary methods for thin-film surface morphology investigation: atomic force microscopy (AFM), transmission electron microscopy (TEM), and grazing-incidence small-angle X-ray scattering (GISAXS). Based on the AFM and TEM results we propose a theoretical model that reproduces well the GISAXS scattering patterns.

Biocompatibility of mesoporous SBA-16/hydroxyapatite nanocomposite and dentin demineralized particles on human dental pulp stem cells.

In the present work, a biomaterial (SBA-16/HA) based on the growth of hydroxyapatite (HA) particles within an organized silica structure SBA-16 (Santa Barbara Amorphous-16) was developed to evaluate its application to act as a porous microenvironment promoting attachment and viability of human dental pulp stem cells of healthy deciduous teeth (SHED). First, SHED were isolated and their phenotypes were evaluated by flow cytometry. The samples of SBA-16/HA were characterized by X-ray diffraction (XRD), small and wide angle X-ray scattering (SWAXS), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) equipped with energy dispersive spectra detector (EDS). Afterward, cells were cultured in the eluates of the above-mentioned biomaterial aged for 24 hr, 7. and 14 days. Bio-Oss® and dentin particles are involved for comparison and cells are cultured in the eluates of these two materials also. Thiazolyl Blue Tetrazolium bromide assay-MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay) was used for the determination of cell viability. The results obtained by all aforementioned characterization methods of SBA-16/HA, revealed a uniform spherical mesoporous structure, an intrinsic characteristic of this material. This material displayed excellent biocompatibility on SHEDs, and even proliferative potential, indicating that SBA-16/HA could potentially serve as a suitable substrate for bone regeneration. Contrary to SBA-16/HA, dentin particles showed low cytotoxicity at all time points, compared to control and Bio-Oss®groups. Our results substantiate the idea that SBA-16/HA has a beneficial effect on SHEDs, thus paving the way toward developing new material for bone replacement.

Origin of Mangetotransport Properties in APCVD Deposited Tin Oxide Thin Films.

Transparent conducting oxides (TCO) with high electrical conductivity and at the same time high transparency in the visible spectrum are an important class of materials widely used in many devices requiring a transparent contact such as light-emitting diodes, solar cells and display screens. Since the improvement of electrical conductivity usually leads to degradation of optical transparency, a fine-tuning sample preparation process and a better understanding of the correlation between structural and transport properties is necessary for optimizing the properties of TCO for use in such devices. Here we report a structural and magnetotransport study of tin oxide (SnO2), a well-known and commonly used TCO, prepared by a simple and relatively cheap Atmospheric Pressure Chemical Vapour Deposition (APCVD) method in the form of thin films deposited on soda-lime glass substrates. The thin films were deposited at two different temperatures (which were previously found to be close to optimum for our setup), 590 °C and 610 °C, and with (doped) or without (undoped) the addition of fluorine dopants. Scanning Electron Microscopy (SEM) and Grazing Incidence X-ray Diffraction (GIXRD) revealed the presence of inhomogeneity in the samples, on a bigger scale in form of grains (80-200 nm), and on a smaller scale in form of crystallites (10-25 nm). Charge carrier density and mobility extracted from DC resistivity and Hall effect measurements were in the ranges 1-3 × 1020 cm-3 and 10-20 cm2/Vs, which are typical values for SnO2 films, and show a negligible temperature dependence from room temperature down to -269 °C. Such behaviour is ascribed to grain boundary scattering, with the interior of the grains degenerately doped (i.e., the Fermi level is situated well above the conduction band minimum) and with negligible electrostatic barriers at the grain boundaries (due to high dopant concentration). The observed difference for factor 2 in mobility among the thin-film SnO2 samples most likely arises due to the difference in the preferred orientation of crystallites (texture coefficient).

Unravelling the growth mechanism of the co-precipitation of iron oxide nanoparticles with the aid of synchrotron X-Ray diffraction in solution.

Co-precipitation is the most ubiquitous method for forming iron oxide nanoparticles. For a typical co-precipitation synthesis, the pH of a ferrous and/or ferric ion solution is increased via the addition of a base. The latter can be added either slowly (a steady addition over either minutes or hours) or fast (a one-time addition) resulting in an abrupt increase in the pH. However, understanding the mechanism of particle formation is still lacking, which limits the reproducibility of the co-precipitation reaction due to intermediate phases still being present in the final product. In this work, we study in detail a co-precipitation synthesis with an abrupt increase in pH via the addition of sodium carbonate. Fast and reproducible mixing at defined precursor and base solution temperatures was achieved utilising a flow reactor. Transmission electron microscopy, electron diffraction and room temperature 57Fe Mössbauer spectroscopy showed a distinct transition from an amorphous ferrihydrite phase to a mixture of magnetite-maghemite (Fe3O4/γ-Fe2O3). Synchrotron X-ray diffraction revealed the initial formation of crystalline iron hydroxide carbonate (green rust) plates occurring before the Fe3O4/γ-Fe2O3 appeared. The ferrihydrite particles increase in size over time as the proportion of iron hydroxide carbonate plates are re-dissolved into solution, until the ferrihydrite particles crystallise into Fe3O4/γ-Fe2O3.

Comprehensive characterization of nanostructured lipid carriers using laboratory and synchrotron X-ray scattering and diffraction.

The development of lipid nanoparticles requires knowledge on the crystalline structure, polymorphic transitions and lipid-drug interactions. This study aimed at introducing advanced techniques to characterize nanostructured lipid carriers (NLC) comprising palmitic acid, oleic acid, stabilizer and Domperidone. Crystallinity of single components and mixtures was investigated by laboratory Small Angle X-ray Scattering (SAXS). NLC were studied with laboratory Small and Wide Angle X-ray Scattering (SWAXS). Photon Correlation Spectroscopy and Freeze Fracture Transmission Electron Microscopy were used to monitor particle size, zeta potential and shape. Stability of NLC was investigated using synchrotron X-ray Diffraction (XRD) and SAXS and laboratory SAXS. Palmitic acid showed a lamellar structure (polymorph C), which was still present after particle preparation. Spherical 300 nm-sized particles with zeta potential values above -30 mV were obtained and Domperidone was incorporated in its amorphous form. During storage, no differences in synchrotron XRD spectra were seen. However, laboratory SAXS measurements showed a second lamellar structure, identified as polymorph B. Synchrotron SAXS temperature scans confirmed that polymorph B did not affect the morphology of the encapsulated drug or the shape of NLC. These results highlight the unique capabilities of laboratory and synchrotron X-ray Scattering and Diffraction for improved structural characterization of lipid nanoparticles.

Monitoring of Pentoxifylline Thermal Behavior by Novel Simultaneous Laboratory Small and Wide X-Ray Scattering (SWAXS) and Differential Scanning Calorimetry (DSC).

The thermal and structural evolutions associated to active pharmaceutical ingredient (API) purity are monitored using a laboratory instrument (S3-MicroCaliX) allowing simultaneous time-resolved X-ray scattering at both wide and small angles (SWAXS) as a function of temperature. This is performed simultaneously with differential scanning calorimetric (DSC) that is carried out in the same apparatus at scanning rate of 2 K/min on the same sample in the range from 20° to 200°C. We have studied simultaneous thermal and structural properties of pentoxifylline, as an active pharmaceutical ingredient (API), for its purity quality control. We have found a satisfying API purity, due to obtained melting temperature and enthalpy values, which are in a well agreement with literature. We have also found that the combination of these techniques allows the thermal monitoring of scanning rates of 2 K/min, continuously without the need for static thermal equilibration, particularly for X-ray spectra. Hence, DSC and SWAXS allowing better identification of the structural thermal events recorded by following of the phase transitions simultaneously. This interpretation is much better possible when X-ray scattering at small and wide angles is coupled with DSC from the same sample. Hence, as a laboratory tool, the method presents a reproducible thermal and crystallographic API purity quality control of non-complex samples, as crucial information for pharmaceutical technology.

Condensation reaction between carbohydrazide and salicylaldehyde: in-line vibrational spectroscopy monitoring and characterization of the reaction products in solution and solid state.

The condensation reaction between carbohydrazide and salicylaldehyde was monitored in-line by using vibrational NIR and Raman spectroscopies and statistical methods. Prior to in-line data analysis the reaction products were fully characterized in solution and solid state in order to check the potential of the in-line approach as a tool for in-process Schiff bases reaction control. It was demonstrated that a combination of vibrational spectroscopy and principal component analysis made it possible to detect and identify the reaction products, e.g. mono(salicylidene)carbohydrazide (1) and bis(salicylidene)carbohydrazide (2) in different solvents, and to determine the reaction end points in real time. Owing to complexity of the reaction mixtures and band overlapping, it was not possible to determine the relative ratio of the reaction products in-line. The off-line analysis showed that 1 was predominant in methanol while the highest portion of 2 was obtained in ethanol.

Fast real-time monitoring of entacapone crystallization and characterization of polymorphs via Raman spectroscopy, statistics and SWAXS.

Crystallization of the drug entacapone from binary solvent mixtures was monitored in situ using a Raman optical probe. The recorded Raman spectra and statistical analysis, which included the principal components method and indirect hard modeling made it possible to estimate the starting point of crystallization, to assess crystallization temperatures and to provide information on the polymorphic content of the mixture. It was established that crystallization temperatures were proportional to the volume content of the solvent in mixtures. The samples were also evaluated off-line via Raman spectroscopy and SWAXS. The collected data showed the presence of forms b and g in all solvent mixtures. In a toluene/methanol 30:70 mixture, in addition to forms b and g, at least one of the forms A, D or a was also indicated by SWAXS. The results have shown that the presence of a particular polymorph is strongly dependent on the nature and portion of the solvent in the binary solvent mixture.

Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers.

Drug-membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution ¹H and ¹³C nuclear magnetic resonance (NMR) and solid state ¹³C and ³¹P (NMR) spectroscopies. In addition, ³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT1 receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.

Ibuprofen-loaded calcium stearate pellets: drying-induced variations in dosage form properties.

Pellets intended for oral dosing are frequently produced via extrusion/spheronization followed by drying. Typically, the last active process step, i.e., drying, is assumed to have little effect on the final dosage form properties (e.g., dissolution characteristics). Thus, there exist only a few studies of this subject. In the present study, calcium stearate/ibuprofen pellets were used as model system to investigate the impact of the drying conditions. Lipophilic calcium stearate matrix pellets containing 20% ibuprofen were prepared via wet extrusion/spheronization. Subsequently, desiccation, fluid-bed drying, and lyophilization were applied for granulation liquid removal. The impact of these drying techniques on the final pellet properties was evaluated. The in vitro dissolution behavior was dramatically altered by the drying techniques that were considered. The investigated pellets showed drug release rates that varied as much as 100%. As no polymorphic transitions occurred during drying, we focused on two possible explanations: (a) a change in the drug distribution within the pellets and (b) a change in pellet micro-structure (porosity, pore size). The ibuprofen distribution proved to be homogeneous regardless of the drying conditions. Pellet porosity and pore sizes, however, were modified by the drying process. Our results clearly demonstrate that a single process step, such as drying, can play a crucial role in achieving desired pellet properties and release profiles.

Development of sustained-release lipophilic calcium stearate pellets via hot melt extrusion.

The objective of this study was the development of retarded release pellets using vegetable calcium stearate (CaSt) as a thermoplastic excipient. The matrix carrier was hot melt extruded and pelletized with a hot-strand cutter in a one step continuous process. Vegetable CaSt was extruded at temperatures between 100 and 130°C, since at these temperatures cutable extrudates with a suitable melt viscosity may be obtained. Pellets with a drug loading of 20% paracetamol released 11.54% of the drug after 8h due to the great densification of the pellets. As expected, the drug release was influenced by the pellet size and the drug loading. To increase the release rate, functional additives were necessary. Therefore, two plasticizers including glyceryl monostearate (GMS) and tributyl citrate (TBC) were investigated for plasticization efficiency and impact on the in vitro drug release. GMS increased the release rate due to the formation of pores at the surface (after dissolution) and showed no influence on the process parameters. The addition of TBC increased the drug release to a higher extent. After dissolving, the pellets exhibited pores at the surface and in the inner layer. Small- and Wide-Angle X-ray Scattering (SWAXS) revealed no major change in crystalline peaks. The results demonstrated that (nearly) spherical CaSt pellets could be successfully prepared by hot melt extrusion using a hot-strand cutter as downstreaming system. Paracetamol did not melt during the process indicating a solid suspension. Due to the addition of plasticizers, the in vitro release rate could be tailored as desired.

Conformational and hydrational properties during the L(beta)- to L(alpha)- and L(alpha)- to H(II)-phase transition in phosphatidylethanolamine.

Differential scanning calorimetry (DSC) measurements have been carried out simultaneously with small- and wide-angle X-ray scattering recordings on liposomal dispersions of stearoyl-oleoyl-phosphatidylethanolamine (PE) in a temperature range from 20 to 80 degrees C. The main transition temperature, T(m), was determined at 30.9 degrees C with an enthalpy of 28.5 kJ/mol and the lamellar-to-inverse hexagonal phase transition temperature, T(hex), at 61.6 degrees C with an enthalpy of 3.8 kJ/mol. Additionally highly resolved small angle X-ray diffraction experiments performed at equilibrium conditions allowed a reliable decomposition of the lattice spacings into hydrophobic and hydrophilic structure elements as well as the determination of the lipid interface area of the lamellar gel-phase (L(beta)), the fluid lamellar phase (L(alpha)) and of the inverse hexagonal phase (H(II)). The rearrangement of the lipid matrix and the coincident change of free water per lipid is illustrated for both transitions. Last, possible transition mechanisms are discussed on a molecular level.

Differential modulation of membrane structure and fluctuations by plant sterols and cholesterol.

We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C(17) alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.

Rigidification of neutral lipid bilayers in the presence of salts.

We studied the influence of sodium and calcium chloride on the global and local membrane properties of fluid palmitoyl-oleoyl phosphatidylcholine bilayers, applying synchrotron small-angle x-ray diffraction, spin-labeling electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, as well as simultaneous density and acoustic measurements. The salt concentration was varied over a wide range from 0 to 5 M. We found that NaCl leads to a continuous swelling of the bilayers, whereas the behavior of the bilayer separation dW in the presence of CaCl2 is more complex, showing an initial large dW value, which decreased upon further addition of salt and finally increased again in the high concentration regime. This can be understood by a change of balance between electrostatic and van der Waals interactions. We were further able to show that both salts lead to a significant increase of order within the lipid bilayer, leading to a decrease of bilayer elasticity and shift of main phase transition temperature. This effect is more pronounced for Ca2+, and occurs mainly in the high salt-concentration regime. Thus, we were able to reconcile previous controversies between molecular dynamics simulations and x-ray diffraction experiments regarding the effect of salts on neutral lipid bilayers.