Polymeric vesicles or polymersomes are one of the supramolecular entities at the leading edge of synthetic biology. These small compartments have shown to be feasible candidates as nanoreactors, especially for enzymatic reactions. Once cross-linked and equipped with a pH sensitive material, the reaction can be switched off (pH 8) and on (pH 6) in accordance with the increased permeability of the polymersome membranes under acidic conditions. Thus cross-linked and pH sensitive polymersomes provide a basis for pH controlled enzymatic reactions where no integrated transmembrane protein is needed for regulating the uptake and release of educts and products in the polymersome lumen. This pH-tunable working tool was further used to investigate their use in sequential enzymatic reactions (glucose oxidase and myoglobin) where enzymes are loaded in one common polymersome or in two different polymersomes. Crossing membranes and overcoming the space distance between polymersomes were shown successfully, meaning that educts and products can be exchanged between enzyme compartments for successful enzymatic cascade reactions. Moreover the stabilizing effect of polymersomes is also observable by single enzymatic reactions as well as a sequence. This study is directed to establish robust and controllable polymersome nanoreactors for enzymatic reactions, describing a switch between an off (pH 8) and on (pH 6) state of polymersome membrane permeability with no transmembrane protein needed for transmembrane exchange.

Polymersomes are at the leading edge of biomedical and nanoparticle research. In order to get closer insights into their mechanical properties, the bilayer forming them needs to be studied thoroughly. Here, we report on the bilayer formation, swelling behaviour, rigidity and fluidity of our membranes derived from pH sensitive and photo-cross-linkable polymersomes.

Die Übertragung der amphiphilen Grundbausteine der Liposome in die Welt der Polymere führte zu Blockcopolymeren, welche sogenannte Polymersome bilden können. Die synthetische Herkunft der Polymere ermöglicht es, eine Vielzahl von verschiedenen chemischen Funktionalitäten einzubringen. Anwendungen von Polymersomen werden vor allem als Wirkstoffträgersystem oder im Bereich der synthetischen Biologie als Nanoreaktoren oder künstliche Zellorganellen ausgemacht. In vielen Fällen wird dabei eine Schaltbarkeit oder Responsivität der Vesikel gegenüber äußerer Stimuli benötigt. Als nächste Stufe der Komplexizität können die responsiven, »smarten« Polymersome innerhalb ihrer Membran quervernetzt werden, wodurch es möglich wird, den Durchmesser und die Membranpermeabilität der Vesikel reversibel hin- und herzuschalten. Diese Arbeit baut dabei auf pH-responsiven Polymersomen auf, welche durch photochemische Reaktionen vernetzt werden. Dabei soll zunächst der Frage nachgegangen werden, an welchem pH Wert genau der Übergang von kollabierten zu gequollenen Vesikeln erfolgt und wie sich dieser »kritischer pH« (pH*) verändern und einstellen lässt. Neben der Herstellung von maßgeschneiderten Polymersomen ist aber auch die detaillierte Charakterisierung ihrer Membraneigenschaften unabdingbar, wofür die Titration mit Fluoreszenz-Sonden eingesetzt wurde. Darüber hinaus wurden Enzyme in die Vesikel eingekapselt wobei die neuartige Methode der post-Verkapselung untersucht wurde.

As an inspiration from nature, polymeric vesicles can be formed from amphiphilic block-copolymers. These vesicles are called polymersomes and have applications in drug delivery and as nanoreactors. Within this thesis, photo cross-linked and pH sensitive polymersomes were synthesized, characterized and applied on cells as well as bionanoreactors. The stability due to the crosslinking yielded polymersomes which show a distinct and reproducible swelling upon repeated pH changes. If the non cross-linked vesicles were exposed to a plasma-cleaned surface, they formed a tethered singly and multiple bilayers. Upon studying these membranes, they turned out to harden upon crosslinking and showed a completely non-fluid behaviour. Additionaly, the polymersome-cell interactions were studied and yielded a high influence of the crosslinking conditions on cellular toxicity. If crosslinked for a long time in a phosphate-free enviroment, the polymersomes proved to be least toxic. Finally, an enzyme was incorporated into the polymersomes to create bionanoreactors. Due to the pH sensitivity and swelling, the vesicles created yielded a pH controlled nanoreactor with enzymatic activity and a swollen, e.g. acidic, state only.

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

Amphiphilic block copolymers are unique macro-molecules capable of self-assembling into bilayers analogous to naturally occurring lipid membranes. When combined with lipids, these copolymers form hybrid membranes with unique and sometimes unpredictable properties, including increased chemical and mechanical stability. These synthetically enhanced biological structures represent a versatile platform suitable for a wide range of applications, from advanced biosensing devices to drug delivery systems. The realization of these advancements necessitates a deep understanding of material properties, including the ability to predict and control interfacial behaviors. It has been shown that in the case of pure lipid membranes, interfacial behaviors are dominated by electrostatic forces. The following work will demonstrate that, electrostatic forces also represent a major driving force behind hybrid vesicle adhesion events, such as the formation of supported bilayers or interactions with biological tissues. These electrostatic forces can be manipulated to a limited degree by adjusting suspension buffer pH which primarily modulates the substrate zeta potential. Protonation of silanol groups, in the case of silicate surfaces at low pH, results in slightly positive surface zeta potential. Unfortunately, hybrid vesicles containing BdxEOy polymers exhibit a slight negative zeta potential independent of buffer pH conditions. Therefore, pH mediation can only result in supported bilayer formation in limited cases and may be insufficiently robust for many demands of application. Furthermore, the zeta potential of hybrid vesicles is surprisingly difficult to predict and control, likely due to screening and steric effects of the PEO block. This investigation provides a model to tune and control the zeta potential of such vesicles, independent of other tunable properties. This technique, in combination with pH mediation, proves to be especially effective in controlling vesicle-substrate interaction. Furthermore, translating this understanding to interactions with tissues, could facilitate more targeted drug delivery, potentially avoiding sensitive tissues, thus reducing off-target effects. In summary, this work deepens our understanding of the complex relationship between surface-potential, pH conditions, and vesicle behavior, paving the way for novel applications in bio-sensing, drug delivery, and nanotechnology.

The field of biomedical sciences has been expanded through the introduction of a novel cohort of soft and intelligent microrobots that can be remotely operated and controlled through the use of external stimuli, such as ultrasound, magnetic fields, or electric fields, or internal stimuli, such as chemotaxis. The distinguishing factor of these microrobots lies in their propulsion system, which may encompass chemical, physical, or biohybrid mechanisms. Particularly, microrobots propelled by motile cells or microorganisms have found extensive usage because they combine the control/steerability and image-enhancement capabilities of the synthetic microstructures with the taxis and cell-interaction capabilities of the biological components. Spermatozoa (sperms), among other types of motile microorganisms and cells, are promising biological materials for building biohybrid microrobots because they are inherently designed to swim through complex fluids and organs, like those in the reproductive system, without triggering negative immune responses. Sperms are suitable for a variety of gynecological healthcare applications due to their drug encapsulating capability and high drug-carrying stability, in addition to their natural role of fertilization. One objective of this project is to help sperms reach the site of fertilization in vivo where the sperm count is low (20 million sperm per mL), a condition known as oligospermia. In order to reach this goal, we are developing alternative strategies for transporting a significant number of sperms, as well as improving the functionality of sperm-hybrid microcarriers. Here, we use a thermoresponsive hydrogel made of poly(N-isopropylacrylamide) (PNIPAM) and a non-stimuli-responsive polymer (IPS photoresist) to create four dimensional (4D)-printed sperm-hybrid microcarriers via two-photon polymerization (TPP). We present a multifunctional microcarrier that can: i) transport and deliver multiple motile sperms to increase the likelihood of fertilization, ii) capacitate/hyperactivate the sperms in situ through the local release of heparin, and iii) assist the degradation of the hyaluronic acid (HA), present in extracellular matrix (ECM) of oocyte-cumulus surrounded the Egg. HA degradation occurs through the local action of hyaluronidase-loaded polymersomes (HYAL-Psomes) that have been immobilized on the microcarrier's surface. Dual ultrasonic (US)/photoacoustic (PA) imaging technology can also be used to visualize a swarm of microcarriers, making them ideal candidates for upcoming in vivo applications. In addition, as a second objective, we demonstrate that similar sperm-hybrid microcarriers can be utilized to deliver targeted enzymes and medication for the treatment of gynecological cancer. As proof of concept, we show that combined therapy using enzymes and anti-cancer drugs is an appealing strategy for disrupting the tumor tissue microenvironment and inducing cell apoptosis, thereby offering a more effective cancer therapy. To achieve this, we functionalize the microcarriers with polymersomes loaded with enzymes (such as hyaluronidase and collagenase) and anti-cancer drugs (such as curcumin), respectively, and demonstrate their cargo-release capability, enzyme function, and therapeutic effect for targeting cervical cancer cells in vitro.:Abstract iv 1 Introduction 1 1.1 Motivation 1 1.2 Objectives 3 1.3 Structure of this dissertation 4 2 Background 5 2.1 Introduction on additive manufacturing technology 5 2.2 Direct laser writing (DLW) based on two-photon polymerization 6 2.2.1 Writing principles of two-photon lithography 8 2.2.2 Available materials for two-photon lithography 9 2.2.3 Engineering (Preprogrammed designs) 12 2.3 4D Lithography 13 2.3.1 Biodegradable microrobot 13 2.3.2 Stimuli-responsive micromotors 15 2.3.3 Other 4D-printing approaches 17 2.4 Motion at the microscale (Micromotility) 21 2.4.1 Physical propelled micromotors 23 2.4.2 Chemical propelled micromotors 32 2.4.3 Biohybrid micromotors 34 2.5 Other two-photon polymerized microrobots and their biomedical applications 35 2.5.1 Functionalized carriers 36 2.5.2 Multiple-cell carrying scaffolds 38 2.5.3 Single particle and cell transporters 39 2.6 Comparison of 3D and 4D-lithography with other fabrication methods 42 3 Materials and methods 44 3.1 Synthesis and fabrication 44 3.1.1 Synthesis of PNIPAM 44 3.1.2 Fabrication of microcarrier 44 3.1.3 Preparation of sperm medium and sperm solution 45 3.1.4 Preparation and composition of different body fluids 45 3.1.5 Fluidics channels 46 3.1.6 In situ preparation of microcarriers and sperms 46 3.1.7 Loading of microcarriers with heparin 46 3.1.8 Synthesis of block copolymers (BCPs) 47 3.1.9 Fabrication of Empty-Psomes A and D 48 3.1.10 Preparation of Curcumin complex CU(βCD)2 and calibration curve 49 3.1.11 Fabrication of cargo-loaded Psomes with enzymes and antitumoral drug 50 3.2 Characterization 51 3.2.1 MTS-Assay 51 3.2.2 Toluidine blue assay 52 3.2.3 Characterization of Empty-Psomes A and D: pH cycles and pH titration by dynamic light scattering (DLS) 53 3.2.4 Characterization of cargo-loaded Psomes with enzymes and antitumoral drug 54 3.2.5 Loading efficiency of HYAL-Psomes 55 3.2.6 Loading efficiency of MMPsomes 56 3.2.7 Loading efficiency, stability and release study of CU(βCD)2-Psomes 57 3.2.8 Size and polydispersity analysis of cargo-loaded Psomes in different simulated body fluids by DLS 58 3.2.9 Conformation and stability study of cargo-loaded Psomes in different simulated body fluids by asymmetric flow field flow fractionation (AF4) 59 3.2.10 Immobilization of the cargo-loaded Psomes on the surfaces 61 3.2.11 Enzymatic assay of HYAL for enzyme activity measurement 62 3.2.12 Enzymes assay in different simulated body fluids 64 3.2.13 Stability study of RhB-HYAL-Psomes in different pH 65 3.2.14 Calculation of the magnetic field flux of an external hand-held magnet 66 3.3 Temperature actuation and imaging 67 3.3.1 Temperature actuation test of PNIPAM and video recording 67 3.3.2 Hybrid ultrasound (US) and photoacoustic (PA) Imaging 67 3.4 Other useful information 68 3.4.1 pH and temperature through the female reproductive tract 68 3.4.2 Calculation of the light-to-heat conversion during imaging process 69 4 Multifunctional 4D-printed sperm-hybrid microcarriers for assisted reproduction 72 4.1 Background 72 4.2 Concept and fabrication of the 4D-printed microcarriers 74 4.3 Sperm coupling and geometrical optimization of microcarrier 77 4.4 Characterization of the 4D-printed streamlined microcarriers 78 4.5 Microcarrier loaded with heparin for in situ sperm capacitation 82 4.6 Microcarriers decorated with HYAL-Psomes for in situ degradation of the HA-cumulus complex 86 4.6.1 Immobilization of HYAL-Psomes on the microcarrier’s surface 89 4.6.2 Qualitative study of cumulus cell removal 90 4.7 Sperm-microcarrier motion performance in oviduct-mimicking fluids 91 4.7.1 Capture, transport, and release of sperms 92 4.7.2 Sperm-microcarrier motion performance on ex vivo oviduct tissue 93 4.8 Tracking of a swarm of microcarriers with a dual ultrasound (US) and photoacoustic (PA) imaging system 95 4.9 Summary 96 5 Polymersomes-decorated micromotors with multiple cargos for gynecological cancer therapy 98 5.1 Background 98 5.2 Characterization and size quantification of Psomes before and after loading of cargoes by DLS, and Cryo-TEM 103 5.3 Characterization and size quantification of cargo-loaded Psomes by DLS, and Cryo-TEM in different simulated bodily fluids 104 5.4 Immobilization and characterization of cargo-loaded Psomes on the microcarrier’s surface 106 5.5 Immobilization and characterization of dual cargo-loaded Psomes on the microcarrier’s surface 108 5.6 Investigation of ECM degradation and antitumoral effect of cargo-loaded Psomes 110 5.7 Magnetic and bio-hybrid guidance of microcarriers toward targeted cargo delivery 115 5.8 Summary 117 6 Conclusion and Outlook 119 6.1 Achievements 119 6.2 Outlook 121 Bibliography I List of Figures and Tables XXI Acknowledgements and funding XXIV Scientific publications and contributions XXVI Curriculum Vitae XXVII

L’objectif de cette thèse était de mettre au point une analyse critique de vecteurs polymères utilisés pour la thérapie photodynamique (PDT) et de faire le lien avec l’efficacité thérapeutique observée. Pour cela, une analyse complète des vecteurs a été réalisée par des techniques classiques comme la diffusion dynamique de la lumière ou la microscopie électronique, mais aussi grâce au fractionnement flux-force, technique peu utilisée jusqu’à présent dans le domaine des auto-assemblages polymères. Dans un deuxième temps, les auto-assemblages ont été utilisés comme vecteurs d’un photosensibilisateur, le Phéophorbide a, et l’efficacité thérapeutique évaluée en travaillant sur culture cellulaire 2D et 3D de lignées HCT116 (cancer du colon) ou FaDu (cancer tête et cou). Différents vecteurs polymères simples ont tout d’abord été examinés, à savoir des micelles ou des polymersomes à base de copolymères diblocs amphiphiles comme le poly(oxyde d’éthylène-b--caprolactone), le poly(oxyde d’éthylène-b-lactide) ou le poly(oxyde d’éthylène-b-styrène). Ceci a permis d’obtenir des vecteurs présentant des tailles et des morphologies variables. Les résultats en PDT ont montré des comportements différents et une meilleure efficacité en 3D pour les systèmes à base de PEO-PDLLA. La technique de fractionnement flux-force asymétrique (AsFlFFF) a particulièrement été utilisée pour ces vecteurs afin de démontrer la pureté des auto-assemblages. Les connaissances acquises dans cette première partie ont permis de caractériser des vecteurs faits à base de mélanges d’auto-assemblages micelles/vésicules. Ceux-ci ont révélé des phénomènes d’antagonisme ou de synergie dans l’efficacité en PDT, démontrant l’existence de processus complexes au niveau de la réponse cellulaire.Des auto-assemblages figés par réticulation ont aussi été développés, caractérisés et examinés en PDT. Ils se sont avérés extrêmement intéressants pour la PDT sur les cultures cellulaires en 3D, démontrant une efficacité accrue comparée aux systèmes simples. La comparaison de ces résultats avec ceux obtenus en culture 2D pour les mêmes objets a de plus permis de mettre en évidence la différence entre ces deux modèles biologiques. Enfin, des auto-assemblages à base de complexes poly-ioniques ont aussi été formés et caractérisés. Le fractionnement flux-force s’est là encore avéré efficace, mais a nécessité l’utilisation d’une injection spéciale par Frit-inlet. Leur efficacité en PDT s’est avérée faible. / The objective of this study was to critically analyze different polymer self-assemblies used for photodynamic therapy (PDT) and to link this analysis to their therapeutic efficiency. To do that, a thorough characterization of the vectors has been performed by classical techniques such as Dynamic Light Scattering or electron Microscopy, but also using flow fractionation, which has been seldomly used so far for polymeric self-assemblies. In a second step, these have been used as vectors of a photosensitizer, namely Phéophorbide a, and the therapeutic efficiency assessed on both 2D and 3D cell cultures of HCT 116 (colon cancer) and FaDu (head and neck cancer) cells. Different simple polymer vectors have first been evaluated, namely micelles and polymersomes based on diblock amphiphilic copolymers such as poly(ethylene-oxide-b--caprolactone), poly(ethylene-oxide-b-lactide) or poly(ethylene-oxide-b-styrene). This enabled obtaining vectors exhibiting various sizes and morphologies. Results in PDT showed different behaviours and a better efficiency in 3D for PEO-PDLLA. The Asymmetric Flow Field Flow Fractionation was particularly used for these systems to demonstrate their purity. The acquired expertise on this part enabled us to also characterize vectors made of known mixtures of micelles and polymersomes. These revealed antagonism and synergy effects in PDT, demonstrating the presence of complex processes for the cell response. Other self-assemblies consisting of crosslinked systems have also been developed and characterized. These were observed to be particularly efficient for PDT on 3D cell cultures. The comparison of these results with those for the 2D cell culture enabled to highlight the difference between those two biological systems. Finally, self-assemblies based on Polyion Complexes were also formed and characterized. Field Flow Fractionation was once again used as a powerful technique for this, although this implied the use of a special injection device called Frit Inlet. Their PDT efficiency however proved to be low.