A validation of discrete-element model simulations for predicting tablet coating variability.

Achieving an even coating distribution on tablets during the coating process can be challenging, not to mention the challenges of accurately measuring and quantifying inter-tablet coating variability. Computer simulations using the Discrete Element Method (DEM) provide a viable pathway towards model-predictive design of coating processes. The purpose of this study was to assess their predictivity accounting for both experimental and simulation input uncertainties. To this end, a comprehensive set of coating experiments covering various process scales, process conditions and tablet shapes were conducted. A water-soluble formulation was developed to enable rapid spectroscopic UV/VIS analysis of coating amounts on a large number of tablets. DEM predictions are found to lie within the experimentally inferred confidence intervals in all cases. A mean absolute comparison error of 0.54 % was found between model predictions of coating variability and respective sample point estimates. Among all simulation inputs the parameterization of spray area sizes is considered the most significant source for prediction errors. However, this error was found significantly smaller in magnitude compared to experimental uncertainties at larger process scales underlining the value of DEM in the design of industrial coating processes.

Towards a Better Understanding of Verapamil Release from Kollicoat SR:IR Coated Pellets Using Non-Invasive Analytical Tools.

The aim of this study was to gain deeper insight into the mass transport mechanisms controlling drug release from polymer-coated pellets using non-invasive analytical tools. Pellet starter cores loaded with verapamil HCl (10% loading, 45% lactose, 45% microcrystalline cellulose) were prepared by extrusion/spheronization and coated with 5% Kollicoat SR:IR 95:5 or 10% Kollicoat SR:IR 90:10. Drug release was measured from ensembles of pellets as well as from single pellets upon exposure to acetate buffer pH = 3.5 and phosphate buffer pH = 7.4. The swelling of single pellets was observed by optical microscopy, while dynamic changes in the pH in the pellet cores were monitored by fluorescence spectroscopy. Also, mathematical modeling using a mechanistically realistic theory as well as SEM and Raman imaging were applied to elucidate whether drug release mainly occurs by diffusion through the intact film coatings or whether crack formation in the film coatings plays a role. Interestingly, fluorescence spectroscopy revealed that the pH within the pellet cores substantially differed upon exposure to acetate buffer pH = 3.5 and phosphate buffer pH = 7.4, resulting in significant differences in drug solubility (verapamil being a weak base) and faster drug release at lower pH: from ensembles of pellets and single pellets. The monitoring of drug release from and the swelling of single pellets indicated that crack formation in the film coatings likely plays a major role, irrespective of the Kollicoat SR:IR ratio/coating level. This was confirmed by mathematical modeling, SEM and Raman imaging. Importantly, the latter technique allowed also for non-invasive measurements, reducing the risk of artifact creation associated with sample cutting with a scalpel.

Redispersible Spray-Dried Powder Containing Nanoencapsulated Curcumin: the Drying Process Does Not Affect Neuroprotection In vitro.

A redispersible spray-dried formulation containing curcumin-loaded, lipid-core nanocapsules (LNC-C) was developed for oral administration. The neuroprotective activity of curcumin after the spray-drying process was evaluated in vitro. The spray-dried powder (SD-LNC-C) was produced using a drying adjuvant composed of a blend of maltodextrin and L-leucine (90:10 w/w). Acceptable process yield (~ 70%) and drug content (6.5 ± 0.2 mg g-1) were obtained. SD-LNC-C was formed by smooth, spherical-shaped particles, and confocal Raman analysis indicated the distribution of the LNC-C on the surface of the leucine/maltodextrin agglomerates. The surface of the agglomerates was formed by a combination of LNC-C and adjuvants, and laser diffraction showed that SD-LNC-C had adequate aqueous redispersion, with no loss of controlled drug release behaviour of LNC-C. The in vitro curcumin activity against the lipopolysaccharide (LPS)-induced proinflammatory response in organotypic hippocampal slice cultures was evaluated. Both formulations (LNC-C and SD-LNC-C) reduced TNF-α to similar levels. Therefore, neuroprotection of curcumin in vitro may be improved by nanoencapsulation followed by spray-drying, with no loss of this superior performance. Hence, the redispersible spray-dried powder proposed here represents a suitable approach for the development of innovative nanomedicines containing curcumin for the prevention/treatment of neurodegenerative diseases.

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three-dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.

Multifunctional electrospun nanofibers for wound application - Novel insights into the control of drug release and antimicrobial activity.

Therapeutic management of skin wounds is still faced with major challenges associated with chronicity and bacterial infection. Consequently, there is a high clinical need for effective therapeutic approaches addressing these aspects. In this context, electrospun fibers emerged as beneficial carrier systems for local and controlled delivery of wound healing agents, additionally providing a protective barrier against bacterial invasion. However, depending on the material, such fibers were also shown to provide a potential substrate for bacterial colonization and growth. Thus, profound understanding of fiber characteristics and the respective interactions of fibers with biological systems, cells as well as bacteria, is of major importance. To address these issues, we designed drug-loaded hybrid fibers consisting of polycaprolactone (PCL) and chitosan. Pure PCL fibers provided suitable drug release kinetics for wound healing, but were colonized by Pseudomonas bacteria which were used as model pathogens. The addition of chitosan to the fiber matrix reduced the number of adherent bacteria by tenfold compared to pure PCL fibers and did not show any adverse effects to human skin cells. Further, chitosan incorporation significantly improved fiber hydrophilicity, identified as one of the key regulators of optimized cell-fiber interactions. We successfully encapsulated dexpanthenol as an established wound healing active into these hybrid fibers and solvent polarity was found to be a key factor for controlling drug release kinetics from the fibers. For the final formulation, controlled drug release over seven days with a burst release of 11.54% within 3 h could be achieved and the wound healing effect of the fiber mats could successfully be demonstrated in a cell-based wound healing assay as a proof-of-concept. Such multifunctional fibers simultaneously deliver actives and prevent bacterial growth, and consequently show a high potential for future wound therapy.

Redispersible spray-dried lipid-core nanocapsules intended for oral delivery: the influence of the particle number on redispersibility.

This study proposes a new approach to produce easily redispersible spray-dried lipid-core nanocapsules (LNC) intended for oral administration, evaluating the influence of the particle number density of the fed sample. The proposed approach to develop redispersible spray-dried LNC formulations intended for oral route is innovative, evidencing the needing of an optimization of the initial particle number density in the liquid suspension of nanocapsules. A mixture of maltodextrin and L-leucine (90:10 w/w) was used as drying adjuvant. Dynamic light scattering, turbidimetry, determination of surface area and pore size distribution, electron microscopy and confocal Raman microscopy (CRM) were used to characterize the proposed system and to better understand the differences in the redispersion behavior. An easily aqueous redispersion of the spray-dried powder composed of maltodextrin and L-leucine (90:10 w/w) was obtained, depending on the particle number density. Their surface area decreased in the presence of LNC. CRM enabled the visualization of the spatial distribution of the different compounds in the powders affording to better understand the influence of the particle number density of the fed sample on their redispersion behavior. This study shows the need for optimizing initial particle number density in the liquid formulation to develop redispersible spray-dried LNC powders.

Tracing molecular and structural changes upon mucolysis with N-acetyl cysteine in human airway mucus.

The conducting airways of the human lungs are lined by mucus, which lubricates the lung epithelium and provides a first-line protection against airborne threats. As a novel approach for visualization of the human mucus microstructure, we applied confocal Raman microscopy as a label-free and chemically selective technique. We were successfully able to chemically resolve the pulmonary surfactant from the mucus matrix and show its spatial distribution, as well as to visualize the structural changes within the freeze-dried mucus mesh upon chemical mucolysis. Subsequently, we performed rheological measurements before and after mucolysis and correlated morphology and chemical structure of the mucus with its rheological characteristics. These results do not only enrich the knowledge about the mucus microstructure, but can also, significantly contribute to rational development of future lung therapeutics.

Circulating Lipoproteins: A Trojan Horse Guiding Squalenoylated Drugs to LDL-Accumulating Cancer Cells.

Selective delivery of anticancer drugs to rapidly growing cancer cells can be achieved by taking advantage of their high receptor-mediated uptake of low-density lipoproteins (LDLs). Indeed, we have recently discovered that nanoparticles made of the squalene derivative of the anticancer agent gemcitabine (SQGem) strongly interacted with the LDLs in the human blood. In the present study, we showed both in vitro and in vivo that such interaction led to the preferential accumulation of SQGem in cancer cells (MDA-MB-231) with high LDL receptor expression. As a result, an improved pharmacological activity has been observed in MDA-MB-231 tumor-bearing mice, an experimental model with a low sensitivity to gemcitabine. Accordingly, we proved that the use of squalene moieties not only induced the gemcitabine insertion into lipoproteins, but that it could also be exploited to indirectly target cancer cells in vivo.

The bacterial cell envelope as delimiter of anti-infective bioavailability - An in vitro permeation model of the Gram-negative bacterial inner membrane.

Gram-negative bacteria possess a unique and complex cell envelope, composed of an inner and outer membrane separated by an intermediate cell wall-containing periplasm. This tripartite structure acts intrinsically as a significant biological barrier, often limiting the permeation of anti-infectives, and so preventing such drugs from reaching their target. Furthermore, identification of the specific permeation-limiting envelope component proves difficult in the case of many anti-infectives, due to the challenges associated with isolation of individual cell envelope structures in bacterial culture. The development of an in vitro permeation model of the Gram-negative inner membrane, prepared by repeated coating of physiologically-relevant phospholipids on Transwell® filter inserts, is therefore reported, as a first step in the development of an overall cell envelope model. Characterization and permeability investigations of model compounds as well as anti-infectives confirmed the suitability of the model for quantitative and kinetically-resolved permeability assessment, and additionally confirmed the importance of employing bacteria-specific base materials for more accurate mimicking of the inner membrane lipid composition - both advantages compared to the majority of existing in vitro approaches. Additional incorporation of further elements of the Gram-negative bacterial cell envelope could ultimately facilitate model application as a screening tool in anti-infective drug discovery or formulation development.

Synthesis of a deuterated probe for the confocal Raman microscopy imaging of squalenoyl nanomedicines.

The synthesis of ω-di-(trideuteromethyl)-trisnorsqualenic acid has been achieved from natural squalene. The synthesis features the use of a Shapiro reaction of acetone-d 6 trisylhydrazone as a key step to implement the terminal isopropylidene-d 6 moiety. The obtained squalenic acid-d 6 has been coupled to gemcitabine to provide the deuterated analogue of squalenoyl gemcitabine, a powerful anticancer agent endowed with self-assembling properties. The Raman spectra of both deuterated and non-deuterated squalenoyl gemcitabine nanoparticles displayed significant Raman scattering signals. They revealed no differences except from the deuterium peak patterns in the silent spectral region of cells. This paves the way for label-free intracellular trafficking studies of squalenoyl nanomedicines.

Three-dimensional hierarchical cultivation of human skin cells on bio-adaptive hybrid fibers.

The human skin comprises a complex multi-scale layered structure with hierarchical organization of different cells within the extracellular matrix (ECM). This supportive fiber-reinforced structure provides a dynamically changing microenvironment with specific topographical, mechanical and biochemical cell recognition sites to facilitate cell attachment and proliferation. Current advances in developing artificial matrices for cultivation of human cells concentrate on surface functionalizing of biocompatible materials with different biomolecules like growth factors to enhance cell attachment. However, an often neglected aspect for efficient modulation of cell-matrix interactions is posed by the mechanical characteristics of such artificial matrices. To address this issue, we fabricated biocompatible hybrid fibers simulating the complex biomechanical characteristics of native ECM in human skin. Subsequently, we analyzed interactions of such fibers with human skin cells focusing on the identification of key fiber characteristics for optimized cell-matrix interactions. We successfully identified the mediating effect of bio-adaptive elasto-plastic stiffness paired with hydrophilic surface properties as key factors for cell attachment and proliferation, thus elucidating the synergistic role of these parameters to induce cellular responses. Co-cultivation of fibroblasts and keratinocytes on such fiber mats representing the specific cells in dermis and epidermis resulted in a hierarchical organization of dermal and epidermal tissue layers. In addition, terminal differentiation of keratinocytes at the air interface was observed. These findings provide valuable new insights into cell behaviour in three-dimensional structures and cell-material interactions which can be used for rational development of bio-inspired functional materials for advanced biomedical applications.

Non-invasive insight into the release mechanisms of a poorly soluble drug from amorphous solid dispersions by confocal Raman microscopy.

In this study, we investigated the release mechanism of the poorly water soluble drug aprepitant from different amorphous solid dispersions using confocal Raman microscopy (CRM). Solid dispersions were fabricated based on either Soluplus®, as an amphiphilic copolymer and solubilizer, or on polyvinylpyrrolidone, as a hydrophilic polymer, in order to elucidate the influence of the polymer characteristics on the drug form and dissolution mechanisms. Aprepitant exhibited its amorphous form in both solid dispersions. However, the release differed depending on the polymer. The high complexation effect of Soluplus was shown to be a crucial factor for stabilization of the amorphous drug, resulting in continuous release without any recrystallization of aprepitant. In contrast, solid dispersions based on polyvinylpyrrolidone showed a different mechanism of dissolution; due to the good affinity of PVP and water, the polymer is dissolving fast, leading to phase separation and local recrystallization of the drug. The study highlights the complexity of release processes from solid dispersions and elucidates the influence of the polymer on drug release kinetics.