Bioadhesion of coated particles

Der Einsatz von Nano- und Mikropartikeln als Trägersysteme stellt einen vielversprechenden Ansatz zur biopharmazeutisch verbesserten Applikation von hochpotenten etablierten und Biotech-Wirkstoffen dar. Die Interaktion der Partikel mit Geweben des menschlichen Körpers wird abgesehen von der Partikelgröße vorwiegend durch die chemische Struktur der Partikeloberfläche bestimmt. Folglich kann durch Modi- fikation der Oberfläche mit bioadhäsiven Molekülen die Lokalisation und Verweildauer der partikulären Arzneiform im Körper grundlegend beeinflusst werden. Im Rahmen der vorliegenden Dissertation wurde untersucht, in- wiefern elektrostatische oder biorekognitive Wechselwirkungen aus- genützt werden können, um die Bindung von Nano- und Mikropar- tikeln an humane epitheliale und endotheliale Zellen zu erhöhen. Die negative Oberflächenladung von Polymerteilchen wurde durch Beschichtung mit dem kationischen Polyelektrolyt Polyethylenimin invertiert. Durch diese Ladungsumkehr konnte eine 3-fach erhöhte Bindung von Mikropartikeln und eine 5-fach erhöhte Bindung von Nanopartikeln an ein Gewebekulturmodell des humanen Dünndarms erzielt werden. Ein generelles Übertragen dieser Ergebnisse auf an- dere humane Zelltypen ist jedoch nicht uneingeschränkt möglich, da beispielsweise an primären humanen Endothelzellen keine bevorzugte Bindung von positiv geladenen Partikeln nachgewiesen werden konnte. Zur Erhöhung der spezifischen Bioadhäsivität wurden Nano- und Mikropartikel kovalent mit Weizenkeimlektin derivatisiert. Die Wech- selwirkung dieses zuckerbindenden Proteins mit Bestandteilen der zel- lulären Glykocalyx erhöhte die Bindungsraten an Caco-2 Einzelzellen 73-fach im Vergleich zu nicht modifizierten Partikeln. Dadurch kon- nte bestätigt werden, dass die Konjugation mit Lektinen ein vielver- sprechendes Konzept im Rahmen der Entwicklung bioadhäsiver Wirk- stoffträger darstellt. Standardgemäß werden Bioadhäsionsstudien unter stationären Be- dingungen durchgeführt. Sowohl nach peroraler als auch nach par- enteraler Applikation wirken jedoch Scherkräfte, die die Interaktion von Wirkstoffträgern mit dem Gewebe maßgeblich beeinflussen. Um eine Untersuchung der Bioadhäsivität von Partikeln unter annähernd physi- ologischen Bedingungen zu ermöglichen, wurde ein miniaturisiertes und dadurch potentiell parallelisierbares Flussmodell entwickelt. In diesem chipbasierten System werden mittels oberflächenakustischer Wellen hydrodynamische Kräfte erzeugt, die vergleichbar mit jenen im Darm und in den Blutgefäßen sind. Untersuchungen mit lektinmodi- fizierten Mikropartikeln zeigten, dass deutliche Diskrepanzen zwischen den zellgebundenen Partikelmengen unter stationären und dynamis- chen Bedingungen bestehen. Eine Integration von hydrodynamischen Parametern in präklinische biopharmazeutische Testmodelle erscheint demnach äußerst sinnvoll und könnte die Aussagekraft dieser Modelle deutlich erhöhen. Die vorliegenden Ergebnisse zeigen, dass sowohl das entwickelte Chipmodell als auch die erarbeiteten Techniken zur Partikelmodifika- tion und -detektion vielseitig anwendbares Potential für die biophar- mazeutische Optimierung der Applikation von Wirkstoffen bieten.The application of micro- and nanoparticles as drug carrier systems represents a highly promising approach to biopharmaceutically im- prove the administration of drugs. The interaction of particles with tissues of the human body will be determined by their size and most importantly surface characteristics. Consequently, modification of the particle surface with bioadhesive molecules could represent a potent means for controlling the residence time and localization of particles in the body. In the present thesis it was investigated to what extent ionic and biorecognitive interactions can be employed for mediating the adhesion of particles to epithelial and endothelial cells. By adsorption of the cationic polyelectrolyte poly(ethylene imine) (PEI) the zeta potential of negatively charged polymer particles was inverted. As a consequence of this surface modification a 3-fold higher binding of microparticles and 5-fold higher binding of nanoparticles to artificial intestinal epithelium was observed. However, a general transfer of these results to other cell types does not seem possible since positively charged particles were not characterized by preferential binding to primary endothelial cells. In order to enhance the specific bioadhesion of micro- and nanopar- ticles, covalent conjugation with wheat germ agglutinin (WGA) was employed. As a consequence of the interaction between the surface- bound lectin and carbohydrates in the glycocalyx, a 73-fold enhanced binding as compared to plain colloids was observed. This underlines that modification with lectins represents a promising concept for im- proving the bioadhesive properties of particulate drug carrier systems. Usually, bioadhesion studies are performed under stationary con- ditions. However, upon peroral as well as parenteral administration hydrodynamic forces will act on the particles. This might substantially influence their interaction with the tissue. In order to facilitate stud- ies on the effects of flow on bioadhesion, a miniaturized chip-based microfluidic system was developed. By controlled generation of sur- face acoustic waves (SAWs) fluids can be controllably actuated in this tissue-culture compatible device. Thereby, hydrodynamic conditions comparable with those in the gastrointestinal tract and in the circulatory system can be generated in vitro. This flow model was employed to in- vestigate the influence of shear forces on the binding of WGA-modified and plain microparticles to epithelial monolayers. Clear discrepancies between the number of cell-associated particles under stationary and flow conditions were observed. These results illustrate that an inte- gration of flow into preclinical biopharmaceutical test systems might enable an improved understanding of bioadhesion in a physiological environment. The developed tissue-chip hybrid for flow studies as well as the techniques for surface modification and particle detection will represent versatile tools for future studies dealing with the biopharmaceutical optimization of drug administration

On cell surface deformation during an action potential

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential (AP). It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (1-10 micron). The onset of cellular deformation coincides with the depolarization phase of the AP. The mechanical pulse (i) propagates with the same velocity as the electrical pulse (within experimental accuracy; 10 mm/s), (ii) is reversible, (iii) in most cases of biphasic nature (109 out of 152 experiments) and (iv) presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability

An acoustically-driven biochip - Impact of flow on the cell-association of targeted drug carriers

The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.86 {\pm} 0.95 {\mu}m) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, nonspecific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 {\pm} 307 mm-2 WGA-MP and 94 {\pm} 64 mm-2 BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 {\pm} 241 mm-2 and 104 {\pm} 44 mm-2 attached particles at shear rates of 0.2 s-1 and 1 s-1 respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.Comment: 19 page

On the Temperature Behavior of Pulse Propagation and Relaxation in Worms, Nerves and Gels

<div><p>The effect of temperature on pulse propagation in biological systems has been an important field of research. Environmental temperature not only affects a host of physiological processes <i>e.g.</i> in poikilotherms but also provides an experimental means to investigate the thermodynamic phenomenology of nerves and muscle. In the present work, the temperature dependence of blood vessel pulsation velocity and frequency was studied in the annelid <i>Lumbriculus variegatus</i>. The pulse velocity was found to vary linearily between 0°C and 30°C. In contrast, the pulse frequency increased non-linearly in the same temperature range. A heat block ultimately resulted in complete cessation of vessel pulsations at 37.2±2.7°C (lowest: 33°C, highest: 43°C). However, quick cooling of the animal led to restoration of regularly propagating pulses. This experimentally observed phenomenology of pulse propagation and frequency is interpreted without any assumptions about molecules in the excitable membrane (<i>e.g.</i> ion channels) or their temperature-dependent behaviour. By following Einstein’s approach to thermodynamics and diffusion, a relation between relaxation time τ and compressibility κ of the excitable medium is derived that can be tested experimentally (for κ_T ∼ κ_S). Without fitting parameters this theory predicts the temperature dependence of the limiting (<i>i.e.</i> highest) pulse frequency in good agreement with experimental data. The thermodynamic approach presented herein is neither limited to temperature nor to worms nor to living systems. It describes the coupling between pulse propagation and relaxation equally well in nerves and gels. The inherent consistency and universality of the concept underline its potential to explain the dependence of pulse propagation and relaxation on any thermodynamic observable.</p></div

Reversible heat-block of pulse propagation.

<p>Heating of a worm above a critical temperature (average threshold temperature: 37.2±2.7°C; min: 33°C, max: 43°C; number of worms studied = 17) led to cessation of blood vessel pulsations. Regular contractions reappeared upon quick cooling. Illustrated is a typical temperature-frequency response as obtained from a single worm. Dashed lines are guides to the eye.</p

Variation of pulse wave velocity with environmental temperature.

<p>Data was normalized to each individual worm’s pulse propagation velocity at 9.2±0.3°C (average: 0.19±0.05 mm s⁻¹). The black envelopes are guides to the eye. Each data point represents the average of at least 36 measurements. Number of worms studied = 29.</p

Variation of pulse frequency with environmental temperature.

<p>Data was normalized to each individual worm’s pulse frequency at 9.3±0.7°C (average: 4.4±0.8 beats min⁻¹). The black envelopes are guides to the eye. Each data point represents the average of six measurements. Number of worms studied = 68.</p

Study of pulse wave propagation in <i>Lumbriculus variegatus</i>.

<p>(<b>A</b>) Cross-sectional view of a <i>Lumbriculus</i> segment with intestine (I), ventral nerve chord (VNC), ventral (VBV) and dorsal blood vessel (DBV). The latter are partially connected by lateral vessels (LV). (<b>B</b>) A blackworm is aspirated into a buffer-filled glass capillary (WIC) and subsequently submersed in a temperature-controlled petri dish (TEMP). (<b>C</b>) Top view of WIC with the DBV (light-gray structure in the center) and a propagating pulse wave (arrow).</p