Organelles are crucial compartments in living cells for carrying out biological events, and cells normally employ compartmentalization to spatially manage their cellular material transport, signaling, and metabolic processes. Engineering biomimetic nanoreactors to replicate biological processes has attracted a lot of interest in recent years. pH-responsive and photo-crosslinked polymersomes, for example, as synthetic vesicles, have tuable membrane permeability and mechanical stability, and may be utilized to build artificial organelles by encapsulating bioactive molecules in their cavity. Most existing reports of stimuli-responsive polymersomes for enzymatic cascade reactions are based on a simple mix of two types of polymersomes loaded with different enzymes, whereas cells process multi-enzyme catalytic systems in which intracellular biological reactions are carried out by combining two or more enzymes in the same organelle. In fact, the most of sophisticated biological functions and features of cells are based on self-organization, the coordination and connection between their cell organelles determines their key functions. Therefore, spatially ordered and controllable self-assembly of polymersomes to construct clusters to simulate complex intracellular biological functions has attracted widespread attention. Here, a simple one-step copper-free click strategy is present to crosslink nanoscale pH-responsive and photo-crosslinked polymersomes (less than 100 nm) to micron-level clusters (more than 90% in 0.5-2 µm range). Various influencing factors in the clustering process and subsequent purification methods were studied to obtain optimal clustered polymeric vesicles. Even if co-clustering the separately loaded polymeric vesicles with different enzymes (glucose oxidase and myoglobin), the overall permeability of the clusters can still be regulated through tuning the pH values on demand. Compared with the conventional enzyme cascade reaction through simple blending polymersomes, the rate of enzymatic cascade reaction increased significantly due to the interconnected complex microstructure established. The connection of catalytic nano-compartments into clusters confining different enzymes of a cascade reaction provide an excellent platform for the development of artificial systems mimicking natural organelles or cells. Although pH-responsive polymersomes present a good membrane permeability in response to alternate pH values and good stability in swelling/shrinking behavior owing to the photo-crosslinked membrane, they are still insufficient to simulate more complex biological activity. The intrinsic pH values for molecules transport are always acidic, whereas the majority of cellular action occurs at physiological pH levels. Due to the closed membrane, the enzyme reaction cannot be carried out efficiently under simulated physiological conditions (pH 7.4). To generate a permeable membrane at a physiological pH value, a new stimulus element must be introduced into existing polymersomes. To self-assemble pH- and light-responsive as well as photo-crosslinked polymersomes, a single azobenzene unit is used as a junction molecule between the hydrophilic and hydrophobic segments of block copolymer. To compare light utilization, block copolymers based on donor-acceptor-substituted azobenzene junction and ether-substituted azobenzene junction were prepared. Besides, the photo-isomerization of novel macroinitiators, block copolymers and polymersomes was also studied to get responsive wavelength ranges of light. The dye release experiments proven the hydrophobic dye on the membrane of polymersomes can release from the membrane under light irradiation. Despite the fact that blue light (400-500 nm) has a higher release efficiency than UV light (365 nm) and ether-substituted azobenzene polymersomes have a slightly higher release efficiency than donor-acceptor-substituted azobenzene polymersomes, the mechanism is still unknown due to the different power of light sources. Furthermore, based on the results of light-driven enzyme reaction, more experiments are required to confirm the light-induced membrane permeability, such as photo-oxidation of substrates and photo-induced deactivation of enzyme. But in general, photo-induced membrane disorder does squeeze the tiny cargo out of the membrane. The single azobenzene unit as the linkage between hydrophilic and hydrophobic block induced membrane pertubation proposes a novel concept in which a trace of azobenzene unit can affect cargo mobility on the membrane of polymersomes and even propagate the fluidity of water molecules to the entire membrane, thereby resulting in membrane permeability. This approach offers a unique framework for the development of biomimetic behaviors under physiological simulated conditions.:Part I Fundamentals 1 Theoretical Background 1.1 Polymersomes 1.1.1 Polymersomes Formation 1.1.2 Self-Assembly Principles of Amphiphilic Block Copolymers (BCPs) 1.1.3 Preparation Methods of Polymersomes 1.1.4 Cargo Loading in Polymersomes 1.2 Clustering Methods of Synthetic Vesicle 1.2.1 Host-Guest Interaction 1.2.2 DNA Hybridization 1.2.3 Copper-Free Click Chemistry 1.3 Stimuli-Responsive Polymersomes with Controllable Membrane Permeability 1.3.1 pH-Responsive Polymersomes 1.3.2 Light-Responsive Polymersomes 2 Motivation and Aim Part II Experiments 3 Materials and Methods 3.1 Materials 3.2 Analytical Methods 4 Clustered pH-Responsive Polymersomes for Enzymatic Cascade Reaction 4.1 Synthetic Methods and Characterization of Block Copolymer (BCP) for Self- Assembly of Polymersomes 4.1.1 Synthesis of Poly(Ethylene Glycol) (PEG) Macroinitiator 4.1.2 Synthesis of Photo-Crosslinker 4.1.3 Synthesis of BCP with Different Terminal Groups 4.1.4 Synthesis of Bis-BCN Poly(ethylene glycol) Crosslinker (BisBCN-PEG) 4.2 Formation of Empty and Loaded Psomes-N3 4.2.1 Formation and Photo-Crosslinking of Empty-Psomes-N3 4.2.2 Preparation of Cy5 Labeled BSA (BSA-Cy5) 4.2.3 Preparation of RhB Labeled Myo (Myo-RhB) 4.2.4 Preparation of Cy5 Labeled GOx (GOx-Cy5) 4.2.5 Formation and Photo-Crosslinking of Loaded Psomes-N3 4.3 Preparation and Purification of Clustered Empty-Psomes-N3 II 4.3.1 Preparation of Clustered Empty-Psomes-N3 at Different Conditions 4.3.2 Optimized Preparation of Clustered Empty-Psomes-N3 4.3.3 Purification Method of Clustered Empty-Psomes-N3 4.3.4 DLS Measurement of the Empty-Psomes-N3 in the Supernatant 4.3.5 Quantification of Removed Psomes-N3 after Centrifugal Purification 4.4 Preparation and Purification of Clustered Enzyme-Psomes-N3: Enzymatic Cascade Reaction 4.4.1 Preparation of Clustered GOx or Myo Loaded Psomes-N3 (GOx-Psomes-N3 or Myo-Psomes-N3) 4.4.2 Enzyme Activity of Myo Samples 4.4.3 Enzyme Activity of GOx Samples 4.5 Preparation and Purification of Co-Clustered Enzyme-Psomes-N3: Enzymatic Cascade Reaction 4.5.1 Preparation of Co-Clustered Myo/GOx-Psomes-N3 4.5.2 Enzyme Activity of Co-Clustered Myo/GOx-Psomes-N3 Samples 5 Light-Driven Enzyme Reaction Based on pH-Responsive Polymersomes 5.1 Synthetic Methods and Characterization of Block Copolymers with Single Azobenzene Unit 5.1.1 Synthesis of Block Copolymer with Donor-Acceptor-Substituted Azobenzene Linkage between Hydrophilic and Hydrophobic Segments (BCP-DA-Azo) 5.1.2 Synthesis of Block Copolymer with Ether Substituted Azobenzene Linkage between Hydrophilic and Hydrophobic Segments (BCP-Azo) 5.2 Photo-Isomerization of Macroinitiator and Block Copolymer with Azobenzene Linkage 5.2.1 Photo-Isomerization of PEG-DA-Azo Macroinitiator Based on Blue Light Irradiation or UV Irradiation 5.2.2 Photo-Isomerization of PEG-Azo Macroinitiator Based on Blue Light Irradiation or UV Irradiation 5.2.3 Photo-Isomerization of BCP-DA-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.2.4 Photo-Isomerization of BCP-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3 Formation and Characterization of Polymersomes with Azobenzene 5.3.1 Self-Assembly of Polymersomes with Azobenzene III 5.3.2 Photo-Isomerization of Psomes-DA-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3.3 Photo-Isomerization of Psomes-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3.4 Photo-Crosslinking of Polymersomes with Azobenzene 5.3.5 DLS Measurement of Photo-Crosslinked Polymersomes with Azobenzene through pH Titration 5.3.6 Photo-Stability of Polymersomes with Azobenzene 5.3.7 In-Situ Loaded Nile Red in Non-Photo-Crosslinked Polymersomes with Azobenzene (NR-Psomes-DA-Azo (+) or NR-Psomes-Azo (+)) 5.3.8 In-Situ Loaded Myo in Photo-Crosslinked Polymersomes with Azobenzene (Myo-Psomes-DA-Azo (+) or Myo-Psomes-Azo (+)) 5.4 Light-Induced Dye Release from Polymersomes with Azobenzene 5.4.1 Fluorescence Photobleaching of Nile Red under Blue Light or UV Irradiation 5.4.2 Nile Red Release under Blue Light or UV Irradiation 5.5 Light-Driven Enzyme Reaction Based on Polymersomes with Azobenzene Part III Results and Discussions 6 Clustered pH-Responsive Polymersomes for Enzymatic Cascade Reaction 6.1 Aim and Strategy 6.2 Photo-Crosslinked and pH-Responsive Polymersomes 6.2.1 Synthesis and Characterization of Block Copolymers (BCPs) 6.2.2 Formation and Characterization of Polymersomes 6.3 Preparation and Purification of Clustered Empty-Psomes-N3 6.3.1 Key Parameters of Clustering Process 6.3.2 Purification Methods of Clustered Empty-Psomes-N3 6.4 Preparation and Purification of Clustered Empty-Psomes-N3 and Enzyme-Psomes- N3 90 6.4.1 Formation and Characterization of Enzyme in-Situ Loaded Psomes-N3 (Enzyme- Psomes-N3) 6.4.2 Enzyme Location in Polymersomes 6.4.3 Deeper Characterization of Clustered Empty-Psomes-N3 and Clustered Enzyme- Psomes-N3 6.5 Clustered Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.5.1 Influence of Enzyme Activity on Clustering Condition IV 6.5.2 Mixed Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.5.3 Co-Clustered Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.6 Summary 7 Light-Driven Enzyme Reaction Based on pH-Responsive Polymersomes 7.1 Aim and Strategy 7.2 Preparation and Characterization of Light-Responsive Polymersomes 7.2.1 Synthesis and Characterization of BCP with Different Types of Azobenzene Unit 7.2.2 Self-Assembly and Photo-Crosslinking of Light-Responsive Polymersomes 7.2.3 Characterization of Photo-Crosslinked Light-Responsive Polymersomes 7.3 Photo-Isomerization of Azobenzene Containing Polymeric Macromolecules and Vesicles 7.3.1 Photo-Isomerization of Azobenzene Containing PEG Macroinitiators 7.3.2 Photo-Isomerization of Azobenzene Containing BCPs and Polymersomes 7.4 Light-Driven Dye Release from Polymersomes with Azobenzene at Simulated Physiological Conditions 7.4.1 Characterization of In-Situ Nile Red Loaded Polymersomes 7.4.2 Light-Driven Dye Release from Polymersomes at Simulated Physiological Conditions 7.5 Light-Induced Enzyme Reaction in Polymersomes with Azobenzene at Simulated Physiological Conditions 7.5.1 Characterization of Polymersomes in-Situ Loaded Myoglobin 7.5.2 Light-Induced Enzyme Reaction in Polymersomes at Simulated Physiological Conditions 7.6 Summary 8 Conclusion and Outlook Reference List of Figures List of Tables List of Abbreviations and Symbols Appendix Acknowledgements Versicherung

Diese Arbeit beschäftigt sich mit der Synthese neuer photoreaktiver Polymere, die bei Bestrahlung mit Licht (Hg-Dampflampe, fs-gepulster Ti:Sa-Laser) definierte Reaktionen durchlaufen. Mittels kontrolliert-radikalischer Polymerisation und anschließender polymeranaloger Reaktion wurden verschiedene Azobenzen-Derivate, Arylazosulfonat-Derivate und photolabil geschützte Aminogruppen (Nitroveratryloxycarbonyl, Nvoc) in Methacrylat-Polymere eingeführt. Diese Strukturen wurden ausführlich hinsichtlich ihrer Struktur und vor allem ihrer photochemischen Eigenschaften (Extinktionskoeffizienten, Zerfallskinetik) untersucht. Dünne Schichten (d<30nm) der so hergestellten Materialien wurden bei der Entwicklung einer neuen photolithographischen Methode, der plasmonischen Nanolithographie, eingesetzt.:INHALTSVERZEICHNIS ABBILDUNGSVERZEICHNIS TABELLENVERZEICHNIS ABKÜRZUNGS- UND SYMBOLVERZEICHNIS 1 EINLEITUNG UND ZIELSTELLUNG 2 THEORETISCHE GRUNDLAGEN 2.1 PHOTOREAKTIVE POLYMERE 2.1.1 Azobenzenhaltige Polymere und deren Anwendungen 2.1.2 Arylazosulfonathaltige Polymere und deren Anwendungen 2.1.3 Photolabil geschützte Amine in Polymeren und zur Oberflächenstrukturierung 2.2 NICHT-LINEAR OPTISCHE EFFEKTE - FREQUENZVERDOPPLUNG 2.3 OBERFLÄCHENPLASMONENRESONANZ UND SHG AN METALLISCHEN NANOPARTIKELN 2.4 PHOTOCHEMISCHE STRUKTURIERUNG DÜNNER SCHICHTEN 2.5 NANOLITHOGRAPHISCHE STRUKTURIERUNG MITTELS PLASMONISCHER EFFEKTE AN METALLNANOSTRUKTUREN 3 ERGEBNISSE UND DISKUSSION 3.1 AZOBENZENHALTIGE POLYMERE 3.1.1 Synthese niedermolekularer Azobenzen-Derivate 3.1.2 Anbindung an Polymermatrix 3.1.3 Photochemische matrixabhängige Untersuchung der Isomerisationskinetik 3.1.4 Bestrahlung in dünnen Schichten mit gepulstem Femtosekunden-Laser 3.2 ARYLAZOSULFONATHALTIGE POLYMERE 3.2.1 Kontrolliert-radikalische Polymerisation von Arylazosulfonat-Monomeren 3.2.1.1 Synthese der arylazosulfonathaltigen Monomere 3.2.1.2 RAFT-Polymerisation von (AS-Mon2) 3.2.2 Polymeranaloge Einführung von Arylazosulfonateinheiten 3.2.3 Präparation und Bestrahlung dünner Schichten 3.3 PHOTOLABIL GESCHÜTZTE AMINOPOLYMERE 3.3.1 Synthese von statistischen Nvoc-geschützten Aminocopolymeren 3.3.1.1 Anbindung des Nvoc-geschützten Amins über Esterbindung 3.3.1.2 Anbindung des Nvoc-geschützten Amins über Reaktion mit Nvoc-Cl 3.3.2 Nvoc-haltige Diblockcopolymere 3.3.2.1 Synthese von P(MMA-co-GMA)-b-P(MMA-co-NvocCMS) 3.3.2.2 Synthese von P(MMA-co-MAA)-b-P(MMA-co-NvocAEMA) 3.3.3 Präparation dünner Schichten der (Block-)Copolymere 3.3.4 Bestrahlung mit gepulstem Femtosekunden-Laser 4 ZUSAMMENFASSUNG UND AUSBLICK 5 EXPERIMENTALTEIL 5.1 ANALYTISCHE METHODEN 5.2 AUFBAU FÜR DIE FS-LASER-BESTRAHLUNG 5.3 VERWENDETE CHEMIKALIEN 5.4 SYNTHESEN 5.4.1 Azobenzen-Derivate 5.4.1.1 Niedermolekulare Verbindungen 5.4.1.2 Polymerisationen und polymeranaloge Reaktionen 5.4.2 Arylazosulfonate 5.4.2.1 Niedermolekulare Verbindungen 5.4.2.2 Freie radikalische Polymerisation 5.4.2.3 RAFT-Polymerisationenen 5.4.2.4 Polymeranaloge Reaktion 5.4.3 Nvoc-geschützte (Block-)Copolymere 5.4.3.1 Niedermolekulare Verbindungen 5.4.3.2 Polymerisationen 5.4.3.3 Polymeranaloge Reaktionen 5.5 WAFERREINIGUNG UND PRÄPARATION DÜNNER SCHICHTEN LITERATURVERZEICHNIS DANKSAGUNG VERSICHERUNG