TY - THES A1 - Prokopović, Vladimir Z. T1 - Light-triggered release of bioactive compounds from HA/PLL multilayer films for stimulation of cells T1 - Licht-induzierte Freisetzung von Biomolekülen aus Hyaluronsäure-Poly-L-Lysin-Schichten zur Stimulierung von Zellen N2 - The concept of targeting cells and tissues by controlled delivery of molecules is essential in the field of biomedicine. The layer-by-layer (LbL) technology for the fabrication of polymer multilayer films is widely implemented as a powerful tool to assemble tailor-made materials for controlled drug delivery. The LbL films can as well be engineered to act as mimics of the natural cellular microenvironment. Thus, due to the myriad possibilities such as controlled cellular adhesion and drug delivery offered by LbL films, it becomes easily achievable to direct the fate of cells by growing them on the films. The aim of this work was to develop an approach for non-invasive and precise control of the presentation of bioactive molecules to cells. The strategy is based on employment of the LbL films, which function as support for cells and at the same time as reservoirs for bioactive molecules to be released in a controlled manner. UV light is used to trigger the release of the stored ATP with high spatio-temporal resolution. Both physico-chemical (competitive intermolecular interactions in the film) and biological aspects (cellular response and viability) are addressed in this study. Biopolymers hyaluronic acid (HA) and poly-L-lysine (PLL) were chosen as the building blocks for the LbL film assembly. Poor cellular adhesion to native HA/PLL films as well as significant degradation by cells within a few days were shown. However, coating the films with gold nanoparticles not only improved cellular adhesion and protected the films from degradation, but also formed a size-exclusion barrier with adjustable cut-off in the size range of a few tens of kDa. The films were shown to have high reservoir capacity for small charged molecules (reaching mM levels in the film). Furthermore, they were able to release the stored molecules in a sustained manner. The loading and release are explained by a mechanism based on interactions between charges of the stored molecules and uncompensated charges of the biopolymers in the film. Charge balance and polymer dynamics in the film play the pivotal role. Finally, the concept of light-triggered release from the films has been proven using caged ATP loaded into the films from which ATP was released on demand. ATP induces a fast cellular response, i.e. increase in intracellular [Ca2+], which was monitored in real-time. Limitations of the cellular stimulation by the proposed approach are highlighted by studying the stimulation as a function of irradiation parameters (time, distance, light power). Moreover, caging molecules bind to the film stronger than ATP does, which opens new perspectives for the use of the most diverse chemical compounds as caging molecules. Employment of HA/PLL films as a nouvelle support for cellular growth and hosting of bioactive molecules, along with the possibility to stimulate individual cells using focused light renders this approach highly efficient and unique in terms of precision and spatio-temporal resolution among those previously described. With its high potential, the concept presented herein provides the foundation for the design of new intelligent materials for single cell studies, with the focus on tissue engineering, diagnostics, and other cell-based applications. N2 - Das Konzept des Targetings von Zellen und Geweben mittels kontrollierter Wirkstoffverabreichung ist im Bereich der Biomedizin unerlässlich. Die layer-by-layer (LbL) Technologie ist eine etablierte Methode für die Herstellung von Polyelektrolyt-Multischichten, um intelligente, maßgeschneiderte Materialien für eine kontrollierte Wirkstoffabgabe aufzubauen. Die LbL Filme können das Verhalten einer natürlichen zellulären Mikroumgebung nachahmen. Wegen der unzähligen Möglichkeiten dieser Filme sind nicht nur das Wachstum und die Beeinflussung der Zellen, sondern auch die kontrollierte Wirkstoffabgabe leicht umzusetzen. Das Ziel der vorliegenden Arbeit war die Entwicklung eines nicht-invasiven Systems für die präzise Verabreichung von Biomolekülen an einzelne Zellen. Die Strategie beruht auf LbL Filmen, die einerseits als geeignete Oberfläche für das Zellwachstum dienen, andererseits aber auch die kontrollierte Abgabe von Biomolekülen ermöglichen. Die zeitlich und räumlich präzise Freisetzung von ATP wurde durch UV Bestrahlung eingeleitet/ausgelöst. Sowohl die physiko-chemischen als auch die biologischen Eigenschaften des Verfahrens wurden analysiert. Die LbL Filme wurden auf Basis der biokompatiblen Polymere Hyaluronsäure (HA) und Poly-L-Lysin (PLL) assembliert. Nach der Assemblierung waren die Filme zunächst zellabweisend und wurden von den Zellen innerhalb von ein paar Tagen abgebaut. Die Beschichtigung der Filme mit Gold-Nanopartikeln verbesserte nicht nur die Adhäsion der Zellen, sondern auch den Schutz gegen Abbau. Zudem wurde gezeigt, dass die Beschichtigung eine Molekulargewichtsausschlussgrenze mit verstellbarem cut-off in einem Bereich von nur einigen 10 kDa darstellt. Weiterhin wurde gezeigt, dass die Filme eine enorme Beladungskapazität aufweisen (bis hin zu einer Beladung im mM Bereich). Darüber hinaus konnten die Biomoleküle, die in diese Filme eingebettet wurden, langfristig und nachhaltig freigesetzt werden. Die Beladung und Freisetzung der Moleküle erfolgt durch die Interaktion zwischen eingebetteten Molekülen und freien Ladungen innerhalb des Polymerfilms. Das Ladungsverhältnis und die Polymerdynamik spielen dabei eine zentrale Rolle. Schließlich wurde gezeigt, dass die in Filmen eingebetteten caged-ATP Moleküle gezielt durch einen Laser freigesetzt werden können. ATP verursacht die schnelle zelluläre Antwort durch den Anstieg von intrazellulären Ca2+-Ionen, der in Echtzeit beobachtet werden kann. Um die Limitierungen dieses Ansatzes bei der zellulären Stimulation hervorzuheben, wurden verschiedene Bestrahlungsparameter (Zeit, Abstand, Laserleistung) analysiert. Außerdem binden viele caging-Moleküle stärker an die Polymerfilme als ATP, wodurch eine Vielzahl von chemischen Verbindungen als geeignete Moleküle für die Caging zur Verfügung stehen. Der Einsatz von HA/PLL Filmen als neue Oberfläche für Zellwachstum und als Reservoire für die Einbettung bioaktiver Moleküle samt der Möglichkeit einzelne Zellen mittels fokussierter Laserbestrahlung anzuregen, machen die Methode hoch effizient und einzigartig. Die Vorteile dieser Methode kommen durch die zeitliche und räumliche Präzision zustande. In Zukunft kann das in dieser Arbeit beschriebene Konzept für den Entwurf neuer Materialien für Untersuchungen mit einzelnen Zellen eingesetzt werden; mit dem Fokus auf Tissue Engineering, Diagnostik und anderen Zell- und biomedizinisch-basierten Applikationen. KW - multilayer films KW - light-triggered KW - stimulation of cells KW - drug release KW - Polyelektrolyt-Multischichten KW - Licht-induzierte KW - kontrollierte Freisetzung von Biomolekülen KW - Stimulierung von Zellen Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-97927 ER - TY - JOUR A1 - Prokopovic, Vladimir Z. A1 - Duschl, Claus A1 - Volodkin, Dmitry T1 - Hyaluronic Acid/Poly-l-Lysine Multilayers as Reservoirs for Storage and Release of Small Charged Molecules JF - Macromolecular bioscience N2 - Polyelectrolyte multilayer films are nowadays very attractive for bioapplications due to their tunable properties and ability to control cellular response. Here we demonstrate that multilayers made of hyaluronic acid and poly-l-lysine act as high-capacity reservoirs for small charged molecules. Strong accumulation within the film is explained by electrostatically driven binding to free charges of polyelectrolytes. Binding and release mechanisms are discussed based on charge balance and polymer dynamics in the film. Our results show that transport of molecules through the film-solution interface limits the release rate. The multilayers might serve as an effective platform for drug delivery and tissue engineering due to high potential for drug loading and controlled release. KW - diffusion KW - drug delivery KW - dye KW - release mechanism Y1 - 2015 U6 - https://doi.org/10.1002/mabi.201500093 SN - 1616-5187 SN - 1616-5195 VL - 15 IS - 10 SP - 1357 EP - 1363 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Feoktistova, Natalia A1 - Rose, Jürgen A1 - Prokopovic, Vladimir Z. A1 - Vikulina, Anna S. A1 - Skirtach, Andre A1 - Volodkin, Dmitry T1 - Controlling the Vaterite CaCO3 Crystal Pores. Design of Tailor-Made Polymer Based Microcapsules by Hard Templating JF - Langmuir N2 - The spherical vaterite CaCO3 microcrystals are nowadays widely used as sacrificial templates for fabrication of various microcarriers made of biopolymers (e.g., proteins, nucleic acids, enzymes) due to porous structure and mild template elimination conditions. Here, we demonstrated for the first time that polymer microcarriers with tuned internal nanoarchitecture can be designed by employing the CaCO3 crystals of controlled porosity. The layer-by-layer deposition has been utilized to assemble shell-like (hollow) and matrix-like (filled) polymer capsules due to restricted and free polymer diffusion through the crystal pores, respectively. The crystal pore size in the range of few tens of nanometers can be adjusted without any additives by variation of the crystal preparation temperature in the range 745 degrees C. The temperature-mediated growth mechanism is explained by the Ostwald ripening of nanocrystallites forming the crystal secondary structure. Various techniques including SEM, AFM, CLSM, Raman microscopy, nitrogen adsorptiondesorption, and XRD have been employed for crystal and microcapsule analysis. A three-dimensional model is introduced to describe the crystal internal structure and predict the pore cutoff and available surface for the pore diffusing molecules. Inherent biocompatibility of CaCO3 and a possibility to scale the porosity in the size range of typical biomacromolecules make the CaCO3 crystals extremely attractive tools for template assisted designing tailor-made biopolymer-based architectures in 2D to 3D targeted at drug delivery and other bioapplications. Y1 - 2016 U6 - https://doi.org/10.1021/acs.langmuir.6b00717 SN - 0743-7463 VL - 32 SP - 4229 EP - 4238 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Prokopovic, Vladimir Z. A1 - Vikulina, Anna S. A1 - Sustr, David A1 - Duschl, Claus A1 - Volodkin, Dmitry T1 - Biodegradation-Resistant Multilayers Coated with Gold Nanoparticles. Toward a Tailor-made Artificial Extracellular Matrix JF - Journal of colloid and interface science N2 - Polymer multicomponent coatings such as multilayers mimic an extracellular, matrix (ECM) that attracts significant attention for the use of the multilayers as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles were described. Nanoparticle coating acts as a semipermeable barrier that governs molecular transport into/from the multilayers, and makes them biodegradation-resistant. Model protein lysozyme (mimics of ECM-soluble signals) diffuses into the multilayers as fast- and, slow-diffusing populations existing in an equilibrium,. Such a. composite system may have high potential to be exploited as degradation-resistant drug-delivery platforms suitable for cell-based applications. KW - hyaluronic acid KW - polylysine KW - diffusion KW - semipermeable KW - fluorescence recovery after photobleaching KW - layer-by-layer KW - enzymatic degradation KW - cell adhesion Y1 - 2016 U6 - https://doi.org/10.1021/acsami.6b10095 SN - 1944-8244 VL - 8 SP - 24345 EP - 24349 PB - American Chemical Society CY - Washington ER -