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Vectorization of oligonucleotides with cell-penetrating peptides : Characterization of uptake mechanisms and cytotoxicityEL Andaloussi, Samir January 2007 (has links)
The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis. Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism. Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.
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Neutrophil Extracellular Trap (NET) Formation: From Fundamental Biophysics to Delivery of NanosensorsMeyer, Daniel 26 June 2019 (has links)
No description available.
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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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Systèmes modèles de membranes et potentiel de pénétration de polypeptides / Model lipid systems and their interactions with polypeptidesWeinberger, Andreas 30 September 2013 (has links)
Les vésicules géantes unilamellaires (GUV) permettent d’étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l’attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l’auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d’un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées. / Giant Unilamellar Vesicles (GUVs) are a valuable tool to study lipid bilayer-biomolecule interactions in simplified cell-like model systems. In this work, a new method to efficiently form GUVs within minutes from more complex systems was developed. This method is based on swelling of a PVA-film under a lipid bilayer and minimizes damage of involved molecules during GUV formation. It also opens up many interesting perspectives for the formation of GUVs composed from new classes of synthetic lipids, such as glycolipids and amide-bearing phospholipids, where the traditional methods fail to efficiently produce “undamaged” vesicles. GUVs were also used for studying lipid-peptide interactions of a new class of elastin-like polypeptides functionalized with arginine-rich residues. It is shown that attractive interactions between lipids and peptides are suppressed by cargo-attachment and can be tuned by self-assembly into micelles and the arginine-amount of the cell penetrating residue.
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