Nonlinear electric response of the diffuse double layer to an abrupt charge displacement inside a biological membrane.

In order to elucidate the old, still unsolved problem of how the diffuse electric double layer responds to an abrupt, intramolecular charge displacement inside a biological membrane, we investigated the fastest components of the light-induced electric signals of bacteriorhodopsin and its mutants, in numerous ionic and buffer solutions. The obtained data for temperature and solute concentration dependence were interpreted as a consequence of changes in the capacity of the diffuse double layer surrounding the purple membrane. The possible physiological consequences of this so far not demonstrated phenomenon are discussed.

Directing Crystallization Outcomes of Conformationally Flexible Molecules: Polymorphs, Solvates, and Desolvation Pathways of Fluconazole.

Control over polymorphism and solvatomorphism in API assisted by structural information, e.g., molecular conformation or associations via hydrogen bonds, is crucial for the industrial development of new drugs, as the crystallization products differ in solubility, dissolution profile, compressibility, or melting temperature. The stability of the final formulation and technological factors of the pharmaceutical powders further emphasize the importance of precise crystallization protocols. This is particularly important when working with highly flexible molecules with considerable conformational freedom and a large number of hydrogen bond donors or acceptors (e.g., fluconazole, FLU). Here, cooling and suspension crystallization were applied to access polymorphs and solvates of FLU, a widely used azole antifungal agent with high molecular flexibility and several reported polymorphs. Each of four polymorphic forms, FLU I, II, III, or IV, can be obtained from the same set of alcohols (MeOH, EtOH, isPrOH) and DMF via careful control of the crystallization conditions. For the first time, two types of isostructural channel solvates of FLU were obtained (nine new structures). Type I solvates were prepared by cooling crystallization in Tol, ACN, DMSO, BuOH, and BuON. Type II solvates formed in DCM, ACN, nPrOH, and BuOH during suspension experiments. We propose desolvation pathways for both types of solvates based on the structural analysis of the newly obtained solvates and their desolvation products. Type I solvates desolvate to FLU form I by hydrogen-bonded chain rearrangements. Type II solvates desolvation leads first to an isomorphic desolvate, followed by a phase transition to FLU form II through hydrogen-bonded dimer rearrangement. Combining solvent-mediated phase transformations with structural analysis and solid-state NMR, supported by periodic electronic structure calculations, allowed us to elucidate the interrelations and transformation pathways of FLU.

Self-assembling, supramolecular chemistry and pharmacology of amphotericin B: Poly-aggregates, oligomers and monomers.

Antifungal drugs such as amphotericin B (AmB) interact with lipids and phospholipids located on fungal cell membranes to disrupt them and create pores, leading to cell apoptosis and therefore efficacy. At the same time, the interaction can also take place with cell components from mammalian cells, leading to toxicity. AmB was selected as a model antifungal drug due to the complexity of its supramolecular chemical structure which can self-assemble in three different aggregation states in aqueous media: monomer, oligomer (also known as dimer) and poly-aggregate. The interplay between AmB self-assembly and its efficacy or toxicity against fungal or mammalian cells is not yet fully understood. To the best of our knowledge, this is the first report that investigates the role of excipients in the supramolecular chemistry of AmB and the impact on its biological activity and toxicity. The monomeric state was obtained by complexation with cyclodextrins resulting in the most toxic state, which was attributed to the greater production of highly reactive oxygen species upon disruption of mammalian cell membranes, a less specific mechanism of action compared to the binding to the ergosterol located in fungal cell membranes. The interaction between AmB and sodium deoxycholate resulted in the oligomeric and poly-aggregated forms which bound more selectively to the ergosterol of fungal cell membranes. NMR combined with XRD studies elucidated the interaction between drug and excipient to achieve the AmB aggregation states, and ultimately, their diffusivity across membranes. A linear correlation between particle size and the efficacy/toxicity ratio was established allowing to modulate the biological effect of the drug and hence, to improve pharmacological regimens. However, particle size is not the only factor modulating the biological response but also the equilibrium of each state which dictates the fraction of free monomeric form available. Tuning the aggregation state of AmB formulations is a promising strategy to trigger a more selective response against fungal cells and to reduce the toxicity in mammalian cells.

Chemoenzymatic Synthesis of Fluorinated Cellodextrins Identifies a New Allomorph for Cellulose-Like Materials*.

Understanding the fine details of the self-assembly of building blocks into complex hierarchical structures represents a major challenge en route to the design and preparation of soft-matter materials with specific properties. Enzymatically synthesised cellodextrins are known to have limited water solubility beyond DP9, a point at which they self-assemble into particles resembling the antiparallel cellulose II crystalline packing. We have prepared and characterised a series of site-selectively fluorinated cellodextrins with different degrees of fluorination and substitution patterns by chemoenzymatic synthesis. Bearing in mind the potential disruption of the hydrogen-bond network of cellulose II, we have prepared and characterised a multiply 6-fluorinated cellodextrin. In addition, a series of single site-selectively fluorinated cellodextrins was synthesised to assess the structural impact upon the addition of one fluorine atom per chain. The structural characterisation of these materials at different length scales, combining advanced NMR spectroscopy and microscopy methods, showed that a 6-fluorinated donor substrate yielded multiply 6-fluorinated cellodextrin chains that assembled into particles presenting morphological and crystallinity features, and intermolecular interactions, that are unprecedented for cellulose-like materials.

Automation Potential of a New, Rapid, Microscopy-Based Method for Screening Drug-Polymer Solubility.

For the pharmaceutical industry, the preformulation screening of the compatibility of drug and polymeric excipients can often be time-consuming because of the use of trial-and-error approaches. This is also the case for selecting highly effective polymeric excipients for forming molecular dispersions in order to improve the dissolution and subsequent bio-availability of a poorly soluble drug. Previously, we developed a new thermal imaging-based rapid screening method, thermal analysis by structure characterization (TASC), which can rapidly detect the melting point depression of a crystalline drug in the presence of a polymeric material. In this study, we used melting point depression as an indicator of drug solubility in a polymer and further explored the potential of using the TASC method to rapidly screen and identify polymers in which a drug is likely to have high solubility. Here, we used a data bank of 5 model drugs and 10 different pharmaceutical grade polymers to validate the screening potential of TASC. The data indicated that TASC could provide significant improvement in the screening speed and reduce the materials used without compromising the sensitivity of detection. It should be highlighted that the current method is a screening method rather than a method that provides absolute measurement of the degree of solubility of a drug in a polymer. The results of this study confirmed that the TASC results of each drug-polymer pair could be used in data matrices to indicate the presence of significant interaction and solubility of the drug in the polymer. This forms the foundation for automating the screening process using artificial intelligence.

Spectrokinetic characterization of photoactive yellow protein films for integrated optical applications.

In this paper, the photocycle of the dried photoactive yellow protein film has been investigated in different humidity environments, in order to characterize its nonlinear optical properties for possible integrated optical applications. The light-induced spectral changes of the protein films were monitored by an optical multichannel analyser set-up, while the accompanying refractive index changes were measured with the optical waveguide lightmode spectroscopy method. To determine the number and kinetics of spectral intermediates in the photocycle, the absorption kinetic data were analysed by singular value decomposition and multiexponential fitting methods, whose results were used in a subsequent step of fitting a photocycle model to the data. The absorption signals of the films were found to be in strong correlation with the measured light-induced refractive index changes, whose size and kinetics imply that photoactive yellow protein may be a good alternative for utilization as an active nonlinear optical material in future integrated optical applications.

Novel Thermal Imaging Method for Rapid Screening of Drug-Polymer Miscibility for Solid Dispersion Based Formulation Development.

This study aimed to develop a rapid, simple, and inexpensive screening method for selecting the best polymeric candidates possessing high active pharmaceutical ingredient (API) miscibility during the early stages of formulation development of solid dispersion based pharmaceutical products. A new thermal imaging based method, thermal analysis by structural characterization (TASC), was used as a thermoptometric tool in conjunction with data analysis software to detect the melting point depression and postmelting dissolution of felodipine particles screened over thin spin-coated films of ten polymers commonly used in the pharmaceutical field. On the polymeric substrates the drug showed different degrees of melting point reduction, reflecting their different levels of polymer-drug miscibility. Using TASC to detect melting point depression is significantly (20-40 times) faster than the conventional DSC method without loss of the sensitivity of detection. The quantity of the material required for the screening is less than 1/1000th of the material used in conventional DSC tests, which significantly reduce the material wastage. Isothermal TASC tests and IR imaging confirmed the occurrence of thermal dissolution of the drug in the polymer for more miscible pairs. The real-time stability tests validate the accuracy of the polymer-drug miscibility screening results. These results demonstrate TASC as a promising screening tool for rapidly selecting the polymeric excipients for pharmaceutical formulations development.

Nanocrystallization of Rare Tolbutamide Form V in Mesoporous MCM-41 Silica.

Encapsulation of pharmaceuticals inside nanoporous materials is of increasing interest due to their possible applications as new generation therapeutics, theranostic platforms, or smart devices. Mesoporous silicas are leading materials to be used as nanohosts for pharmaceuticals. Further development of new generation of nanoscale therapeutics requires complete understanding of the complex host-guest interactions of organic molecules confined in nanosized chambers at different length scales. In this context, we present results showing control over formation and phase transition of nanosize crystals of model flexible pharmaceutical molecule tolbutamide confined inside 3.2 nm pores of the MCM-41 host. Using low loading levels (up to 30 wt %), we were able to stabilize the drug in highly dynamic amorphous/disordered state or direct the crystallization of the drug into highly metastable nanocrystalline form V of tolbutamide (at loading levels of 40 and 50 wt %), providing first experimental evidence for crystallization of pharmaceuticals inside the pores as narrow as 3.2 nm.

Redox-dependent control of i-Motif DNA structure using copper cations.

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C-C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand. Herein, we present a dynamic and redox-sensitive system for conformational control of an i-motif forming DNA sequence in response to copper cations.

Towards controlling the crystallisation behaviour of fenofibrate melt: triggers of crystallisation and polymorphic transformation.

Fenofibrate (FEN) is a dyslipidemia treatment agent which is poorly soluble in water. FEN has tendency to form polymorphs and its crystallisation behaviour is difficult to predict. The nucleation process can be initiated by mechanical disruption such as ball milling or surface scratching which may result in different crystallisation behaviour to that observed in the unperturbed system. This study has obtained insights into the controllability of FEN crystallisation by means of regulating the exposed surface and growth temperatures during its crystallisation. The availability of an open top surface (OTS) during the crystallisation of the FEN melt resulted in a mixture containing FEN form I and IIa (I ≫ IIa) at room temperature, and in the range 40 to 70 °C. Covering the surface led to significant increases in the yield of form IIa at room temperature and at 40 and 50 °C. These temperatures also yielded the highest amount of form IIa in the OTS samples whilst crystallisation at 70 °C led to only FEN form I crystals regardless of the availability of the free surface. The metastable FEN form IIa transforms to the stable form I under the influence of a mechanical stress. Additionally, the introduction of OTS before the completion of crystallisation of form IIa led to a 'switch' of from IIa growth to form I. This study demonstrates that the polymorph selection of FEN can be obtained by the manipulation of the crystallisation conditions.

Insight into Flufenamic Acid Cocrystal Dissolution in the Presence of a Polymer in Solution: from Single Crystal to Powder Dissolution.

Effects of three polymers, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and copolymer of vinylpyrrolidone/vinyl acetate (PVP-VA), on the dissolution behavior of the cocrystals of flufenamic acid with theophylline (FFA-TP CO) and nicotinamide (FFA-NIC CO) were investigated at multiple length scales. At the molecular level, the interactions of crystal surfaces with a polymer were analyzed by observing etching pattern changes using atomic force microscopy. At the macroscopic scale, dissolution rates of particular faces of a single crystal were determined by measurement of the physical retreat velocities of the faces using optical light microscopy. In the bulk experiments, the FFA concentration in a dissolution medium in the absence or presence of a polymer was measured under both sink and nonsink conditions. It has been found that the dissolution mechanisms of FFA-TP CO are controlled by the defect sites of the crystal surface and by precipitation of the parent drug FFA as individual crystals in the bulk fluid. In contrast, the dissolution mechanisms of FFA-NIC CO are controlled by surface layer removal and by a surface precipitation mechanism, where the parent drug FFA precipitates directly onto the surface of the dissolving cocrystals. Through controlling the dissolution environment by predissolving a polymer, PVP or PVP-VA, which can interact with the crystal surface to alter its dissolution properties, improved solubility, and dissolution rates of FFA-TP CO and FFA-NIC CO have been demonstrated.

(19) F†NMR Spectroscopy as a Highly Sensitive Method for the Direct Monitoring of Confined Crystallization within Nanoporous Materials.

The introduction of fluorine into the structure of pharmaceuticals has been an effective strategy for tuning their pharmacodynamic properties, with more than 40 new drugs entering the market in the last 15 years. In this context, (19) F NMR spectroscopy can be viewed as a useful method for investigating the host-guest chemistry of pharmaceuticals in nanosized drug-delivery systems. Although the interest in confined crystallization, nanosized devices, and porous catalysts is gradually increasing, understanding of the complex phase behavior of organic molecules confined within nanochambers or nanoreactors is still lacking. Using (19) F magic-angle-spinning NMR spectroscopy, we obtained detailed mechanistic insight into the crystallization of flufenamic acid (FFA) in a confined environment of mesoporous silica materials with different pore diameters (3.2-29 nm), providing direct experimental evidence for the formation of a molecular-liquid-like layer besides crystalline confined FFA form I.

Electrospun Polymer Blend Nanofibers for Tunable Drug Delivery: The Role of Transformative Phase Separation on Controlling the Release Rate.

Electrospun fibrous materials have a wide range of biomedical applications, many of them involving the use of polymers as matrices for incorporation of therapeutic agents. The use of polymer blends improves the tuneability of the physicochemical and mechanical properties of the drug loaded fibers. This also benefits the development of controlled drug release formulations, for which the release rate can be modified by altering the ratio of the polymers in the blend. However, to realize these benefits, a clear understanding of the phase behavior of the processed polymer blend is essential. This study reports an in depth investigation of the impact of the electrospinning process on the phase separation of a model partially miscible polymer blend, PVP K90 and HPMCAS, in comparison to other conventional solvent evaporation based processes including film casting and spin coating. The nanoscale stretching and ultrafast solvent removal of electrospinning lead to an enhanced apparent miscibility between the polymers, with the same blends showing micronscale phase separation when processed using film casting and spin coating. Nanoscale phase separation in electrospun blend fibers was confirmed in the dry state. Rapid, layered, macroscale phase separation of the two polymers occurred during the wetting of the fibers. This led to a biphasic drug release profile from the fibers, with a burst release from PVP-rich phases and a slower, more continuous release from HPMCAS-rich phases. It was noted that the model drug, paracetamol, had more favorable partitioning into the PVP-rich phase, which is likely to be a result of greater hydrogen bonding between PVP and paracetamol. This led to higher drug contents in the PVP-rich phases than the HPMCAS-rich phases. By alternating the proportions of the PVP and HPMCAS, the drug release rate can be modulated.

Mechanistic and Kinetic Insight into Spontaneous Cocrystallization of Isoniazid and Benzoic Acid.

Solid-state cocrystallization is of contemporary interest because it offers an easy and efficient way to produce cocrystals, which are recognized as prospective pharmaceutical materials. Research explaining solid-state cocrystallization mechanisms is important but still too scarce to give a broad understanding of factors governing and limiting these reactions. Here we report an investigation of the mechanism and kinetics of isoniazid cocrystallization with benzoic acid. This reaction is spontaneous; however, its rate is greatly influenced by environmental conditions (humidity and temperature) and pretreatment (milling) of the sample. The acceleration of cocrystallization in the presence of moisture is demonstrated by kinetic studies at elevated humidity. The rate dependence on humidity stems from moisture facilitated rearrangements on the surface of isoniazid crystallites, which lead to cocrystallization in the presence of benzoic acid vapor. Furthermore, premilling the mixture of the cocrystal ingredients eliminated the induction time of the reaction and considerably increased its rate.

On the Hofmeister effect: fluctuations at the protein-water interface and the surface tension.

We performed molecular dynamics simulations on the tryptophane-cage miniprotein using a nonpolarizable force field, in order to model the effect of concentrated water solutions of neutral salts on protein conformation, which is a manifestation of Hofmeister effects. From the equilibrium values and the fluctuations of the solvent accessible surface area of the miniprotein, the salt-induced changes of the mean value of protein-water interfacial tension were determined. At 300 K, the chaotropic ClO4(-) and NO3(-) decreased the interfacial tension according to their position in the Hofmeister series (by approximately 5 and 2.7 mN/m, respectively), while the kosmotropic F(-) increased it (by 1 mN/m). These values were compared to those obtained from the Gibbs equation using the excess surface adsorption calculated from the probability distribution of the water molecules and ions around the miniprotein, and the two sets were found to be very close to each other. Our results present a direct evidence for the central role of interfacial tension and fluctuations at the protein-water interface in Hofmeister phenomena, and provide a computational method for the determination of the protein-water interfacial tension, establishing a link between the phenomenological and microscopic description of protein-water interfaces.

Efflux transport of serum amyloid P component at the blood-brain barrier.

Serum amyloid P component (SAP), a member of the innate immune system, does not penetrate the brain in physiological conditions; however, SAP is a stabilizing component of the amyloid plaques in neurodegenerative diseases. We investigated the cerebrovascular transport of human SAP in animal experiments and in culture blood-brain barrier (BBB) models. After intravenous injection, no SAP could be detected by immunohistochemistry or ELISA in healthy rat brains. Salmonella typhimurium lipopolysaccharide injection increased BBB permeability for SAP and the number of cerebral vessels labeled with fluorescein isothiocyanate (FITC)-SAP in mice. Furthermore, when SAP was injected to the rat hippocampus, a time-dependent decrease in brain concentration was seen demonstrating a rapid SAP efflux transport in vivo. A temperature-dependent bidirectional transport of FITC-SAP was observed in rat brain endothelial monolayers. The permeability coefficient for FITC-SAP was significantly higher in abluminal to luminal (brain to blood) than in the opposite direction. The luminal release of FITC-SAP from loaded endothelial cells was also significantly higher than the abluminal one. Our data indicate the presence of BBB efflux transport mechanisms protecting the brain from SAP penetration. Damaged BBB integrity due to pathological insults may increase brain SAP concentration contributing to development of neurodegenerative diseases.

High-speed integrated optical logic based on the protein bacteriorhodopsin.

The principle of all-optical logical operations utilizing the unique nonlinear optical properties of a protein was demonstrated by a logic gate constructed from an integrated optical Mach-Zehnder interferometer as a passive structure, covered by a bacteriorhodopsin (bR) adlayer as the active element. Logical operations were based on a reversible change of the refractive index of the bR adlayer over one or both arms of the interferometer. Depending on the operating point of the interferometer, we demonstrated binary and ternary logical modes of operation. Using an ultrafast transition of the bR photocycle (BR-K), we achieved high-speed (nanosecond) logical switching. This is the fastest operation of a protein-based integrated optical logic gate that has been demonstrated so far. The results are expected to have important implications for finding novel, alternative solutions in all-optical data processing research.

Identification and characterization of stoichiometric and nonstoichiometric hydrate forms of paroxetine HCl: reversible changes in crystal dimensions as a function of water absorption.

Paroxetine hydrochloride (HCl) is an antidepressant drug, reported to exist in the anhydrous form (form II) and as a stable hemihydrate (form I). In this study, we investigate the hydration behavior of paroxetine HCl form II with a view to understanding both the nature of the interaction with water and the interchange between forms II and I as a function of both temperature and water content. In particular, we present new evidence for both the structure and the interconversion process to be more complex than previously recognized. A combination of characterization techniques was used, including thermal (differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)), spectroscopic (attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)), dynamic vapor sorption (DVS) and X-ray powder diffraction (XRPD) with variable humidity, along with computational molecular modeling of the crystal structures. The total amount of water present in form II was surprisingly high (3.8% w/w, 0.8 mol of water/mol of drug), with conversion to the hemihydrate noted on heating in hermetically sealed DSC pans. XRPD, supported by ATR-FTIR and DVS, indicated changes in the unit cell dimensions as a function of water content, with clear evidence for reversible expansion and contraction as a function of relative humidity (RH). Based on these data, we suggest that paroxetine HCl form II is not an anhydrate but rather a nonstoichiometric hydrate. However, no continuous channels are present and, according to molecular modeling simulation, the water is moderately strongly bonded to the crystal, which is in itself an uncommon feature when referring to nonstoichiometric hydrates. Overall, therefore, we suggest that the anhydrous form of paroxetine HCl is not only a nonstoichiometric hydrate but also one that shows highly unusual characteristics in terms of gradual unit cell expansion and contraction despite the absence of continuous channels. These structural features in turn influence the tendency of this drug to convert to the more stable hemihydrate. The study has implications for the recognition and understanding of the behavior of pharmaceutical nonstoichiometric hydrates.