Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.

• Keywords
• advanced crystallization, co‐crystallization, crystal soaking, crystallization protocol, microseeding,
• MeSH
• Protein Conformation MeSH
• Crystallization methods   MeSH
• Crystallography, X-Ray methods   MeSH
• Ligands MeSH
• Proteins * chemistry   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ligands MeSH
• Proteins * MeSH

The production of high-quality crystals is a key step in crystallography in general, but control of crystallization conditions is even more crucial in serial crystallography, which requires sets of crystals homogeneous in size and diffraction properties. This protocol describes the implementation of a simple and user-friendly microfluidic device that allows both the production of crystals by the counter-diffusion method and their in situ analysis by serial crystallography. As an illustration, the whole procedure is used to determine the crystal structure of three proteins from data collected at room temperature at a synchrotron radiation source.

• Keywords
• CrystalChip, crystallization, microcrystals, microfluidics, serial crystallography,
• MeSH
• Protein Conformation MeSH
• Crystallization methods   instrumentation   MeSH
• Crystallography, X-Ray methods   instrumentation   MeSH
• Proteins * chemistry   MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteins * MeSH

Spray drying and hot-melt extrusion are among the most prevalent preparation techniques used in the pharmaceutical industry to produce amorphous solid dispersions (ASDs). This study advances previous research by integrating sample production, comprehensive analytical characterization, intrinsic dissolution rate measurements, and assessments of the behavior of ASDs under elevated temperature and humidity conditions. The study focuses on indomethacin, a widely used model for poorly soluble drugs, processed with PVP K30 or HPMC E5, both commonly used polymers. The findings demonstrate that hot-melt extruded samples exhibit superior stability against recrystallization, whereas spray dried samples achieve higher intrinsic dissolution rates. Furthermore, PVP K30 significantly outperforms HPMC E5 in the co-processing of indomethacin, enhancing both the intrinsic dissolution rate and the stability.

• Keywords
• Amorphous solid dispersions, Dissolution, Hot-melt extrusion, Recrystallization, Spray drying, Stability,
• MeSH
• Chemistry, Pharmaceutical methods   MeSH
• Indomethacin * chemistry   MeSH
• Crystallization MeSH
• Methylcellulose analogs & derivatives   chemistry   MeSH
• Povidone chemistry   MeSH
• Drug Compounding methods   MeSH
• Pyrrolidines chemistry   MeSH
• Solubility MeSH
• Spray Drying MeSH
• Drug Stability MeSH
• Hot Melt Extrusion Technology * methods   MeSH
• Drug Liberation MeSH
• Humidity MeSH
• Hot Temperature MeSH
• Desiccation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indomethacin * MeSH
• Methylcellulose MeSH
• Povidone MeSH
• Pyrrolidines MeSH

Vibrational circular dichroism (VCD) spectroscopy appears as a useful method for characterizing optically active substances in the solid state. This is particularly important for active pharmaceutical ingredients. However, measurement and interpretation of the spectra bring about many difficulties. To assess the experimental and computational methodologies, we explore an anti-inflammatory drug, naproxen. Infrared (IR) and VCD spectra of the pure compound and its cocrystals with alanine and proline were recorded, and the data were interpreted by quantum chemical simulations based on a cluster model and density functional theory. Although unpolarized IR spectroscopy can already distinguish pure ingredients from cocrystals or a mixture, the VCD technique is much more sensitive. For example, the naproxen carboxyl group strongly interacts with the zwitterionic alanine in the cocrystal via two strong hydrogen bonds, which results in a rather rigid structure crystallizing in the chiral P212121 Sohncke group and its VCD is relatively strong. In contrast, the d-proline and (S)-naproxen cocrystal (P21 group) involves a single hydrogen bond between the subunits, which together with a limited motion of the proline ring gives a weaker signal. Solid-state VCD spectroscopy thus appears useful for exploring composite crystal structures and interactions within them, including studies of pharmaceutical compounds.

• Keywords
• alanine, cocrystals, density functional theory, naproxen, proline, solid state, spectra modeling, vibrational circular dichroism,
• MeSH
• Alanine chemistry   MeSH
• Anti-Inflammatory Agents, Non-Steroidal chemistry   MeSH
• Circular Dichroism * methods   MeSH
• Crystallization MeSH
• Models, Molecular MeSH
• Naproxen * chemistry   MeSH
• Proline chemistry   MeSH
• Spectrophotometry, Infrared MeSH
• Stereoisomerism MeSH
• Vibration MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alanine MeSH
• Anti-Inflammatory Agents, Non-Steroidal MeSH
• Naproxen * MeSH
• Proline MeSH

The integration of 3D printing into the pharmaceutical sciences opens new possibilities for personalized medicine. Poly(lactide) (PLA), a biodegradable and biocompatible polymer, is highly suitable for biomedical applications, particularly in the context of 3D printing. However, its processability often requires the addition of plasticizers. This study investigates the use of phase diagram modeling as a tool to guide the rational selection of plasticizers and to assess their impact on the thermodynamic and kinetic stability of PLA-based amorphous solid dispersions (ASDs) containing active pharmaceutical ingredients (APIs). Thermodynamic stability against API recrystallization was predicted based on the API solubility in PLA and Plasticizer-PLA carriers using the Conductor-like Screening Model for Real Solvents (COSMO-RS), while the kinetic stability of the ASDs was evaluated by modeling the glass transition temperatures of the mixtures. Two APIs, indomethacin (IND) and naproxen (NAP), with differing glass-forming abilities (i.e., recrystallization tendencies), and three plasticizers, triacetin (TA), triethyl citrate (TEC), and poly(L-lactide-co-caprolactone) (PLCL), were selected for investigation. The physical stability of ASD formulations containing 9 wt% API and plasticizer to PLA in two ratios, 10:81 and 20:71 w/w %, was monitored over time using differential scanning calorimetry and X-ray powder diffraction and compared with phase diagram predictions. All formulations were predicted to be thermodynamically unstable; however, those containing no plasticizer or with TEC and TA at 10 wt% were predicted to exhibit some degree of kinetic stability. Long-term physical studies corroborated these predictions. The correlation between the predicted phase behavior and long-term physical stability highlights the potential of phase diagram modeling as a tool for the rational design of ASDs in pharmaceutical 3D printing.

• Keywords
• 3D printing, Active pharmaceutical ingredient, Amorphous solid dispersion, COSMO-RS, PLA, Personalized medicine, Phase diagram, Plasticizers,
• MeSH
• Printing, Three-Dimensional * MeSH
• Citrates chemistry   MeSH
• Calorimetry, Differential Scanning methods   MeSH
• Chemistry, Pharmaceutical methods   MeSH
• Indomethacin chemistry   MeSH
• Kinetics MeSH
• Crystallization MeSH
• Naproxen chemistry   MeSH
• Polyesters * chemistry   MeSH
• Drug Compounding methods   MeSH
• Drug Design MeSH
• Solubility MeSH
• Drug Stability MeSH
• Thermodynamics MeSH
• Triacetin chemistry   MeSH
• Plasticizers chemistry   MeSH
• Phase Transition MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Citrates MeSH
• ethyl citrate MeSH Browser
• Indomethacin MeSH
• Naproxen MeSH
• poly(lactide) MeSH Browser
• Polyesters * MeSH
• Triacetin MeSH
• Plasticizers MeSH

Nanocrystalline cerium dioxide is able to protect living cells from oxidative stress under the influence of various stress factors, in particular under the one of low temperatures. This study investigates the phase-structural transformations in aqueous solutions containing CeO2 nanoparticles (NPs) and their impact on the cryopreservation process. Differential scanning calorimetry and thermomechanical analysis were used to analyse the phase transitions in aqueous suspensions of CeO2 NPs and aqueous solutions of the cryoprotectant dimethyl sulfoxide (Me2SO) with CeO2 NPs. Various concentrations of CeO2 NPs were tested to observe their effects on the crystallization and melting behaviours. The addition of CeO2 NPs significantly altered the temperatures and enthalpies of melting and crystallization in water. Low concentrations of CeO2 NPs promoted crystallization, while higher concentrations inhibited it, reducing supercooling and recrystallization during thawing. In Me2SO solutions, CeO2 NPs raised the glass transition temperature and affected the recrystallization process, with higher concentrations leading to more pronounced vitrification and reduced recrystallization. We also investigated the regularities of the effect of CeO2 NPs on phase transitions in combined cryoprotective media with Ham's F12, fetal bovine serum and Me2SO, which can be used in future to design the cryopreservation protocols. In the complex media, CeO2 NPs decreased the metastability and altered eutectic crystallization patterns, indicating potential cryoprotective effects. In conclusion, CeO2 NPs modulate the thermophysical properties of cryoprotective solutions, enhancing vitrification and reducing recrystallization, which could improve cryopreservation efficiency. Optimizing NP concentrations is crucial for practical applications in cryopreservation.

• Keywords
• Cerium dioxide nanoparticles, Cryopreservation, Cryoprotective agents, Differential scanning calorimetry, Phase transitions, Thermomechanical analysis,
• MeSH
• Cerium * chemistry   pharmacology   MeSH
• Calorimetry, Differential Scanning MeSH
• Dimethyl Sulfoxide chemistry   MeSH
• Cryopreservation * methods   MeSH
• Cryoprotective Agents * chemistry   pharmacology   MeSH
• Crystallization MeSH
• Nanoparticles * chemistry   MeSH
• Cattle MeSH
• Transition Temperature MeSH
• Vitrification MeSH
• Phase Transition MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cerium * MeSH
• ceric oxide MeSH Browser
• Dimethyl Sulfoxide MeSH
• Cryoprotective Agents * MeSH

Poor aqueous solubility of crystalline active pharmaceutical ingredients (APIs) restricts their bioavailability. Amorphous solid dispersions with biocompatible polymer excipients offer a solution to overcome this problem, potentially enabling a broader use of many drug candidate molecules. This work addresses various aspects of the in silico design of a suitable combination of an API and a polymer to form such a binary solid dispersion. Molecular interactions in such bulk systems are tracked at full atomic resolution within molecular-dynamics (MD) simulations, enabling to identify API-polymer pairs that exhibit the most beneficial interactions. Importance of these interactions is manifold: increasing the mutual miscibility, kinetic stabilization of their amorphous dispersions and impedance of the spurious recrystallization of the API component. MD tools are used to investigate the structural and cohesive properties of pure compounds and mixtures, with a special emphasis on molecular interactions, microscopic structures and internal dynamics. This analysis is then accompanied by a macroscopic image of the energetic compatibility and vitrification tendency of the mixtures in terms of their excess enthalpies and glass transition temperatures. Density-functional theory (DFT) and non-covalent interaction (NCI) analysis fortify our computational conclusions and enable us to map the intensities of particular NCI among the individual target materials and relevant molecular sites therein. Three archetypal polymer excipients and four API molecules are included in this study. The results of our computational analysis of molecular interactions in bulk systems agree with the experimentally observed trends of solubility of the given API in polymers. Our calculations confirm PVP as the most potent acceptor of hydrogen bonding among the three considered polymer excipients, whereas ibuprofen molecules are predicted to be the most efficient hydrogen bond donors among our four target APIs. Our simulations also suggest that carbamazepine does not exhibit particularly strong interactions with the considered polymer excipients. Although current MD cannot offer quantitative accuracy of many of the discussed descriptors, current computational models focusing on NCI of APIs with polymer excipients contribute to understanding of the behavior of these materials at the molecular level, and thus also to the rational design of novel efficient drug formulations.

• MeSH
• Ibuprofen chemistry   MeSH
• Kinetics MeSH
• Crystallization MeSH
• Pharmaceutical Preparations * chemistry   MeSH
• Polymers chemistry   MeSH
• Excipients chemistry   MeSH
• Solubility MeSH
• Molecular Dynamics Simulation MeSH
• Density Functional Theory MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ibuprofen MeSH
• Pharmaceutical Preparations * MeSH
• Polymers MeSH
• Excipients MeSH

Creatinine is the end product of the catabolism of creatine and creatine phosphate. Creatine phosphate serves as a reservoir of high-energy phosphate, especially in skeletal and cardiac muscle. Besides typical known changes in serum and urinary creatinine concentrations, rare cases associated with changes in serum and urinary creatine levels have been described in the literature in humans. These cases are mostly linked to an excessive intake of creatine ethyl ester or creatine monohydrate, often resulting in increased urine creatinine concentrations. In addition, it is known that at such elevated creatinine concentrations, creatinine crystallisation may occur in the urine. Analysis of crystals and urinary concrements, often of heterogenous chemical composition, may provide diagnostic and therapeutic hints to the benefit of the patient. The aim of the present work was to analyze urine crystals of unclear composition with microscopic and spectroscopic techniques. On routine microscopic analysis of urine, a preliminary suspicion of uric acid or creatinine crystals was expressed. The crystals were of a cuboid shape and showed polarization effects in microscopy. The dried urine sample was whitish-orange in colour, odourless and dissolved well in water. Protein concentration in dry weight (DW) urine was about 0.3 mg/mg. The measured zinc content in the studied sample was approximately 660 µg/g DW sample and copper content was approximately 64 µg/g DW sample. A lead signal of around 10 µg/g DW sample was also observed. UV-Vis analysis showed a maximum creatine peak around 220 nm, compatible with the spectrum of creatinine with a maximum peak of 230 nm. Using HPLC technique, an extreme high ratio of creatine to creatinine of about 38 was measured, which led to the conclusion of the occurrence of rare creatine crystals in urine.

• Keywords
• Electrochemistry, FTIR spectroscopy, High-performance liquid chromatography, Polarization microscopy analysis, UV–Vis spectroscopy, Urine analysis,
• MeSH
• Creatine * urine   MeSH
• Creatinine * urine   MeSH
• Crystallization * MeSH
• Middle Aged MeSH
• Humans MeSH
• Spectrophotometry methods   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Creatine * MeSH
• Creatinine * MeSH

Highlighting the essential role of chitosan (CS), known for its biocompatibility, biodegradability, and ability to promote cell adhesion and proliferation, this study explores its utility in modulating the biomimetic mineralization of calcium phosphate (CaP). This approach holds promise for developing biomaterials suitable for bone regeneration. However, the interactions between the CS surface and in situ precipitated CaP still require further exploration. In the theoretical section, molecular dynamics (MD) simulations demonstrate that, at an appropriate pH level during the prenucleation stage, calcium ions (Ca2+) and hydrogen phosphate ions (HPO42-) form Posner-like clusters. Additionally, the interaction between these clusters and the CS molecule enhances system stability. Together, these phenomena facilitate the transition to subsequent heterogeneous nucleation on the surface of the organic matrix, which is a more controlled process than homogeneous nucleation in solution. Dynamic simulation results suggest that CS acts as a stabilizing matrix at pH 8.0 during biomimetic mineralization. In the experimental section, the effects of pH and the molecular weight of CS were investigated, with a focus on their impact on the crystal structure of the resulting material. X-ray diffraction and scanning electron microscopy analyses reveal that, under conditions of approximately pH 8.0 and a CS molecular weight of 20 000 g/mol, and controlled ion concentration, ultrasound radiation, and temperature, the dominant CaP phases in the material are carbonate-doped hydroxyapatite (CHA) and octacalcium phosphate (OCP). These findings suggest that CS, when adjusted for molecular weight and pH, facilitates the formation of CaP crystal phases that closely resemble the natural inorganic composition of bone, highlighting its protective and regulatory roles in the growth and maturation of crystals during mineralization. The theoretical predictions and experimental outcomes confirm the crucial role of CS as a templating agent, enabling the development of a biomimetic mineralization pathway. CS's ability to guide this process may prove valuable in the design of materials for bone tissue engineering, particularly in developing effective materials for bone tissue healing and regeneration.

• Keywords
• biomimetic mineralization, carbonate-doped hydroxyapatite, chitosan, molecular dynamics simulation, octacalcium phosphate, ultrasonic radiation,
• MeSH
• Biomimetic Materials chemistry   MeSH
• Chitosan * chemistry   MeSH
• Calcium Phosphates * chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Crystallization MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• calcium phosphate MeSH Browser
• Chitosan * MeSH
• Calcium Phosphates * MeSH

X-ray diffraction is a commonly used technique in the pharmaceutical industry for the determination of the atomic and molecular structure of crystals. However, it is costly, sometimes time-consuming, and it requires a considerable degree of expertise. Vibrational circular dichroism (VCD) spectroscopy resolves these limitations, while also exhibiting substantial sensitivity to subtle modifications in the conformation and molecular packaging in the solid state. This study showcases VCD's ability to differentiate between various crystal structures of the same molecule (polymorphs, cocrystals). We examined the most effective approach for producing high-quality spectra and unveiled the intricate link between structure and spectrum via quantum-chemical computations. We rigorously assessed, using alanine as a model compound, multiple experimental conditions on the resulting VCD spectra, with the aim of proposing an optimal and efficient procedure. The proposed approach, which yields reliable, reproducible, and artifact-free results with maximal signal-to-noise ratio, was then validated using a set comprising of three amino acids (serine, alanine, tyrosine), one hydroxy acid (tartaric acid), and a monosaccharide (ribose) to mimic active pharmaceutical components. Finally, the optimized approach was applied to distinguish three polymorphs of the antiviral drug sofosbuvir and its cocrystal with piperazine. Our results indicate that solid-state VCD is a prompt, cost-effective, and easy-to-use technique to identify crystal structures, demonstrating potential for application in pharmaceuticals. We also adapted the cluster and transfer approach to calculate the spectral properties of molecules in a periodic crystal environment. Our findings demonstrate that this approach reliably produces solid-state VCD spectra of model compounds. Although for large molecules with many atoms per unit cell, such as sofosbuvir, this approach has to be simplified and provides only a qualitative match, spectral calculations, and energy analysis helped us to decipher the observed differences in the experimental spectra of sofosbuvir.

• Keywords
• Amino acids, Hydroxy acids, Polymorphs, Sofosbuvir, Solid-state, Vibrational circular dichroism,
• MeSH
• Antiviral Agents chemistry   MeSH
• Circular Dichroism * MeSH
• Crystallization * MeSH
• Models, Molecular MeSH
• Sofosbuvir * chemistry   MeSH
• Vibration MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH
• Sofosbuvir * MeSH