Molecularly Imprinted Polymers (MIPs) are potential generic alternatives to antibodies in diagnostics and separations. To compete with biomolecules in these technological niches, MIPs need to share the characteristics of antibodies (solubility, size, specificity and affinity) whilst maintaining the advantages of MIPs (low cost, short development time and high stability). For this reason the interest in preparing MIPs as nanoparticles (MIP NPs) has increased exponentially in the last decade. Cont/d.

Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.