Intracellular Delivery of Poorly Soluble Polyphenols: Elucidating the Interplay of Self-Assembling Nanocarriers and Human Chondrocytes.

Increased molecular understanding of multifactorial diseases paves the way for novel therapeutic approaches requiring sophisticated carriers for intracellular delivery of actives. We designed and characterized self-assembling lipid-core nanocapsules for coencapsulation of two poorly soluble natural polyphenols curcumin and resveratrol. The polyphenols were identified as high-potential therapeutic candidates intervening in the intracellular inflammation cascade of chondrocytes during the progress of osteoarthritis. To elucidate the interplay between chondrocytes and nanocapsules and their therapeutic effect, we pursued a complementary analytical approach combining label-free visualization with biological assays. Primary human chondrocytes did not show any adverse effects upon nanocapsule application and coherent anti-Stokes Raman scattering images visualized their intracellular uptake. Further, by systematically blocking different uptake mechanisms, an energy independent uptake into the cells could be identified. Additionally, we tested the therapeutic effect of the polyphenol-loaded carriers on inflamed chondrocytes. Treatment with nanocapsules resulted in a major reduction of nitric oxide levels, a well-known apoptosis trigger during the course of osteoarthritis. For a more profound examination of this protective effect on joint cells, we pursued studies with atomic force microscopy investigations. Significant changes in the cell cytoskeleton as well as prominent dents in the cell membrane upon induced apoptosis were revealed. Interestingly, these effects could not be detected for chondrocytes which were pretreated with the nanocapsules. Overall, besides presenting a sophisticated carrier system for joint application, these results highlight the necessity of establishing combinatorial analytical approaches to elucidate cellular uptake, the interplay of codelivered drugs and their therapeutic effect on the subcellular level.

Skin penetration behavior of lipid-core nanocapsules for simultaneous delivery of resveratrol and curcumin.

Polyphenols, which are secondary plant metabolites, gain increasing research interest due to their therapeutic potential. Among them, resveratrol and curcumin are two agents showing antioxidant, anti-inflammatory, antimicrobial as well as anticarcinogenic effects. In addition to their individual therapeutic effect, increased activity was reported upon co-delivery of the two compounds. However, due to the poor water solubility of resveratrol and curcumin, their clinical application is currently limited. In this context, lipid-core nanocapsules (LNC) composed of an oily core surrounded by a polymeric shell were introduced as drug carrier systems with the potential to overcome this obstacle. Furthermore, the encapsulation of polyphenols into LNC can increase their photostability. As the attributes of the polyphenols make them excellent candidates for skin treatment, the aim of this study was to investigate the effect of co-delivery of resveratrol and curcumin by LNC upon topical application on excised human skin. In contrast to the formulation with one polyphenol, resveratrol penetrated into deeper skin layers when the co-formulation was applied. Based on vibrational spectroscopy analysis, these effects are most likely due to interactions of curcumin and the stratum corneum, facilitating the skin absorption of the co-administered resveratrol. Furthermore, the interaction of LNC with primary human skin cells was analyzed encountering a cellular uptake within 24h potentially leading to intracellular effects of the polyphenols. Thus, the simultaneous delivery of resveratrol and curcumin by LNC provides an intelligent way for immediate and sustained polyphenol delivery for skin disease treatment.

Raman microscopy for cellular investigations--From single cell imaging to drug carrier uptake visualization.

Progress in advanced therapeutic concepts requires the development of appropriate carrier systems for intracellular drug delivery. Consequently, analysis of interaction between carriers, drugs and cells as well as their uptake and intracellular fate is a current focus of research interest. In this context, Raman spectroscopy recently became an emerging analytical technique, due to its non-destructive, chemically selective and label-free working principle. In this review, we briefly present the state-of-the-art technologies for cell visualization and drug internalization. Against this background, Raman microscopy is introduced as a versatile analytical technique. An overview of various Raman spectroscopy investigations in this field is given including interactions of cells with drug molecules, carrier systems and other nanomaterials. Further, Raman instrumentations and sample preparation methods are discussed. Finally, as the analytical limit is not reached yet, a future perspective for Raman microscopy in pharmaceutical and biomedical research on the single cell level is given.

Characterization and evaluation of a modified PVPA barrier in comparison to Caco-2 cell monolayers for combined dissolution and permeation testing.

Aim of this study was to implement a modified phospholipid vesicle-based permeation assay (PVPA) barrier as alternative to Caco-2 cell monolayers in a combined dissolution and permeation system for testing of solid dosage forms. Commercially available Transwell® inserts were coated with egg phospholipids (Lipoid E 80) and characterized by confocal Raman microscopy. The modified PVPA barrier was then evaluated in permeation studies with solutions of different drugs as well as in combined dissolution and permeation studies utilizing an immediate and an extended release tablet formulation. Raman cross section images demonstrated complete filling of the membrane pores with lipids and the formation of a continuous lipid layer of increasing thickness on top of the membrane during the stepwise coating procedure. Furthermore, it could be shown that this lipid coating remains intact for at least 18h under dynamic flow conditions, significantly exceeding the viability of Caco-2 cell monolayers. Permeability data for both drug solutions as well as for a fast and slow release tablet formulation were in excellent correlation with those data obtained for Caco-2 cell monolayers. Especially under the dynamic flow conditions prevailing in such a setup, the modified PVPA barrier is more robust and easier to handle than epithelial cell monolayers and can be prepared rather easily at a fraction of costs and time. The modified PVPA barrier may therefore represent a valuable alternative to Caco-2 cell monolayers in such context.

Solid dispersion prepared by continuous cogrinding in an air jet mill.

Embedding a poorly water-soluble drug as a solid dispersion in a hydrophilic carrier by cogrinding is a possible strategy for enhancing the drug dissolution rate. Although general interest in continuous processes for manufacturing drug formulations has increased, many publications still focus on batch processes. The jet mill used in this study is a promising tool for continuous cogrinding. Investigation of different drug-to-carrier ratios (griseofulvin/mannitol) demonstrated that a drug load of 10% is best suited to investigate the enhanced dissolution behavior. To gain deeper insight into the underlying mechanisms, the coground dispersion is compared with different physical mixtures in terms of physicochemical properties and dissolution behavior. Differential scanning calorimetry and X-ray powder diffraction were used to verify the crystalline structure of the coground formulation. On the basis of the Hixson-Crowell model, particle size reduction was ruled out as the main reason for dissolution enhancement. An increase of surface free energies because of grinding is shown with contact angle measurements. Confocal Raman microscopy investigations revealed the drug's bulk dispersity in the coground formulation as an additional factor for the increased dissolution rate. In conclusion, the continuous cogrinding approach is a promising technique to prepare the drug in a rapidly dissolving, yet crystalline, form.

Microstructure of calcium stearate matrix pellets: a function of the drying process.

Drying is a common pharmaceutical process, whose potential to modify the final drug and/or dosage form properties is often underestimated. In the present study, pellets consisting of the matrix former calcium stearate (CaSt) incorporating the active pharmaceutical ingredient ibuprofen were prepared via wet extrusion and spheronization. Subsequent drying was performed by either desiccation, fluid-bed drying, or lyophilization, and the final pellets were compared with respect to their microstructure. To minimize the effect of solute ibuprofen molecules on the shrinking behavior of the CaSt, low ibuprofen loadings were used, as ibuprofen is soluble in the granulation liquid. Pellet porosity and specific surface area increased during desiccation, fluid-bed drying, and lyophilization. The inlet-air temperature during fluid-bed drying affected the specific surface area, which increased at lower inlet-air temperatures rather than the pellet porosity. The in vitro dissolution profiles were found to be a nonlinear function of the specific surface area. Overall, the microstructure, including porosity, pore size, and specific surface area, of CaSt pellets was a strong function of the drying conditions.

Chemical imaging of drug delivery systems with structured surfaces-a combined analytical approach of confocal raman microscopy and optical profilometry.

Confocal Raman microscopy is an analytical technique with a steadily increasing impact in the field of pharmaceutics as the instrumental setup allows for nondestructive visualization of component distribution within drug delivery systems. Here, the attention is mainly focused on classic solid carrier systems like tablets, pellets, or extrudates. Due to the opacity of these systems, Raman analysis is restricted either to exterior surfaces or cross sections. As Raman spectra are only recorded from one focal plane at a time, the sample is usually altered to create a smooth and even surface. However, this manipulation can lead to misinterpretation of the analytical results. Here, we present a trendsetting approach to overcome these analytical pitfalls with a combination of confocal Raman microscopy and optical profilometry. By acquiring a topography profile of the sample area of interest prior to Raman spectroscopy, the profile height information allowed to level the focal plane to the sample surface for each spectrum acquisition. We first demonstrated the basic principle of this complementary approach in a case study using a tilted silica wafer. In a second step, we successfully adapted the two techniques to investigate an extrudate and a lyophilisate as two exemplary solid drug carrier systems. Component distribution analysis with the novel analytical approach was neither hampered by the curvature of the cylindrical extrudate nor the highly structured surface of the lyophilisate. Therefore, the combined analytical approach bears a great potential to be implemented in diversified fields of pharmaceutical sciences.