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Cultivation of Hepatitis B virus producing cell line HepG2.2.15 on microcarrier and functional characterization of the Hepatitis B virus polymeraseLupberger, Joachim 11 May 2007 (has links)
Hepatitis B Virus (HBV) Infektionen verursachen entzündliche Erkrankungen der Leber. Insbesondere die frühen Phasen des HBV Lebenszyklus sind noch nicht geklärt, so ist z.B. der Rezeptorkomplex an den HBV bindet unbekannt. Mittlerweile stehen neue Infektionsmodelle zur Verfügung um den HBV Lebenszyklus zu untersuchen. Dies erfordert eine effiziente Zellkultur basierende Methode um große Mengen infektiöser Partikel zu generieren. Ein Ziel der Arbeit war durch Kultivierung auf Mikrocarrier die HBV Produktion der Zelllinie HepG2.2.15 zu steigern. Die Analyse von Protein und HBV Sekretion, Infektiösität und MAP Signalübertragung ergab eine 18x höhere HBV Produktion bei einer reduzierten Sekretion von subviralen Partikeln durch HepG2.2.15 die auf Mikrocarrier kultiviert wurden. Der Anstieg der Virusproduktion korreliert mit einer verstärkten Aktivierung der MAP Kinase ERK-2, die mit HBV Replikation in Verbindung steht. Ein weiterer wenig verstandener Teil des HBV Lebenszyklus ist der Kernimport des HBV Genoms. Spuren der viralen Polymerase finden sich im Zellkern von HBV infizierten Zellen. Ziel der Arbeit war, Motive in der HBV Polymerase zu finden, die in der Lage sind Zelllokalisation zu beeinflussen. Durch Sequenzvergleich wurde eine konservierte zweiseitige Kernlokalisationssequenz im Terminalen Protein der HBV Polymerase identifiziert, die eine Proteinkinase CKII Erkennungsstelle enthält. Inhibition der CKII Aktivität in HBV infizierten primären Hepatozyten sowie die Zerstörung der CKII Erkennungsstelle im Terminalen Protein inhibieren die HBV Replikation. Die Funktionalität der Kernlokalisationssequenz wurde durch Fusion an GFP bestätigt und war Abhängig von CKII Aktivität in der Zelle. Dies wurde in vitro durch Bindung des Adapterproteins Karyopherin-alpha an CKII-phosphoryliertes Terminales Protein bestätigt. Die HBV Polymerase enthält eine konservierte zweiseitige Kernlokalisationssequenz deren Funktionalität durch CKII Phosphorilierung vermittelt wird. / Hepatitis B virus (HBV) infection causes acute and chronic liver inflammation. Especially the early phase of the HBV life cycle is not clearly understood. For example the receptor complex that mediates viral entry is not known. Novel infection models to study the HBV lifecycle are described that demand for a large amount of cell culture generated infectious HBV particles. One aim was to enhance HBV production of the cell line HepG2.2.15 by cultivation on microcarrier substrate. Analysis of protein and viral particle secretion, infectivity, and cellular MAP kinase signaling revealed an up to 18x increased HBV production and a decreased subviral particle secretion by HepG2.2.15 when cultivated on microcarrier. The observed effect was due to an enhanced phospho-activation of MAP kinase ERK-2 that is tightly associated with HBV replication. Another poorly understood part of the HBV lifecycle is the mechanism that delivers the HBV genome into the nucleus. Traces of HBV polymerase can be found in HBV infected cells. The second objective was to identify motifs on the HBV polymerase that determine its subcellular localization. By sequence alignment a conserved bipartite nuclear localization signal was found in the terminal protein of the HBV polymerase encompassing a protein kinase CKII recognition site. Inhibition of CKII kinase in infected primary hepatocytes and destruction of the identified CKII recognition site in the viral polymerase impaired virus production. The functionality of the putative nuclear localization signal was confirmed by fusion to GFP. Moreover, its functionality was depended on CKII activity that was verified by in vitro binding experiments of terminal protein to the import adaptor karyopherin-alpha. This data identified a nuclear localization signal in the HBV polymerase, which functionality is mediated by CKII phosphorylation.
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Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Biomedical ApplicationsRajabasadi, Fatemeh 10 April 2024 (has links)
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
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