Label-free analysis of drug delivery systems and cellular interaction studies using confocal Raman microscopy

In pharmaceutical development many questions still remain unsolved despite the availability of many analytical techniques. Consequently, the need of novel analytical approaches is not yet satisfied. In this thesis, confocal Raman microscopy is utilized to fill the scientific gap. In fact, the benefit of this non-destructive, label-free visualization technique for profound analysis in complex pharmaceutical applications is successfully demonstrated. The impact of drying on drug distribution is proven by localizing the drug in wet-extruded pellets with Raman imaging. Additionally to this finding, the correlation between drug distribution and release is successfully elucidated. For the first time, confocal Raman microscopy is combined with optical profilometry. Thus, the limitations of the confocal microscope are overcome and all-encompassing component visualization in complex drug delivery systems exhibiting challenging structured surfaces is realized. During development of a lipid-based drug permeation model, the successive formation of the permeation barrier during coating is finally described using Raman analysis. Investigations benefit tremendously from a combination of chemical imaging in lateral and vertical planes to depict barrier integrity and stability. Finally, human cells as well as the uptake of different nanoparticles are analyzed label-free in aqueous environment, utilizing linear and coherent Raman techniques.In der pharmazeutischen Entwicklung bleiben trotz der Verfügbarkeit diverser analytischer Techniken viele Fragestellungen unbeantwortet. Daher ist die Etablierung neuer analytischer Ansätze notwendig. Diese Arbeit beschäftigt sich mit konfokaler Raman-Mikroskopie als Möglichkeit, die Lücke zu schließen. Die Eignung dieser zerstörungs- und label-frei arbeitenden Technik zur differenzierten Probenvisualisierung für diverse pharmazeutische Fragestellungen wird erfolgreich demonstriert. Der Einfluss vom Trocknungsprozess auf die Wirkstoffverteilung im Pellet wird durch Lokalisation des Wirkstoffs mittels Raman Imaging belegt. Dadurch wird auch die Korrelation zwischen Wirkstoffverteilung und Freisetzung aufgeklärt. Erstmals wird konfokale Raman-Mikroskopie mit optischer Profilometrie kombiniert. Es werden komplexe Arzneistoffträgersysteme mit strukturierten Oberflächen detailliert dargestellt, ohne durch das konfokale Mikroskop eingeschränkt zu sein. Durch Abbildung der einzelnen Coatingschritte in chemisch selektiven Raman-Bildern in der Entwicklungsphase eines lipidbasierten Permeationsmodells wird der sukzessive Aufbau der Permeationsbarriere erstmalig nachvollzogen. Die prozessbedingte ungleiche Dicke der Barriere sowie deren Integrität werden erfolgreich durch die Aufnahme von virtuellen Querschnittsbildern abgebildet. Darüber hinaus werden humane Zellen und die Aufnahme von Nanopartikeln label-frei mit linearen und kohärenten Raman-Techniken in wässrigem Medium untersucht

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

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.\ud \ud 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 give

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

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

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

Single Hepatitis-B Virus Core Capsid Binding to Individual Nuclear Pore Complexes in HeLa Cells

We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 ± 9 nm radial distance from the central axis