Nanoparticules de magnétite fonctionnalisées pour l’imagerie bimodale IRM/TEPLes nanoparticules d’oxydes de fer superparamagnétiques (SPIONs en anglais) font l’objet de recherches intenses dans le domaine biomédical, notamment comme nanomédicament et agent de contraste T2 en imagerie par résonance magnétique (IRM). Au cours de cette étude, des nanoparticules de magnétite (Fe3O4) à destination de l’imagerie IRM/TEP (Tomographie par Emission de Positons) ont été développées. Dans un premier temps, des SPIONs modifiées en surface, stables et superparamagnétiques ont été synthétisées via un dispositif hydrothermal en continu. A leur surface ont été greffées, durant la synthèse, des molécules hydrophiles : l’acide citrique, la LDOPA, le DHCA et le PHA. La fonctionnalisation des nanoparticules a été optimisée en modifiant des paramètres de synthèse tels que la température et le lieu de mélange, occasionnant des modifications de morphologie, taille et phase. Dans un second temps, pour améliorer leur stabilité et furtivité, des polymères de type PolyEthylène Glycol (PEG) ont été greffés à leur surface, deux longueurs de chaîne ont été évaluées. Pour une application en TEP, des macrocycles, complexant le radionucléide 64Cu, tels que le MANOTA, le NODAGA et le DOTA ont été couplés à ces SPIONs. Les essais de radiomarquage sont concluants. Ces nanohybrides, pleinement caractérisés (MET, XPS, IR, DLS, potentiels zêta, ATG, Raman) sont très prometteurs pour le diagnostic via l’imagerie bimodale IRM/TEM, notamment le composé Fe3O4-LDOPA-NODAGA (øDLS = 85±1 nm, r2 = 197±7 mM.s-1, 87% 64Cu). Des études préliminaires de cytotoxicité et génotoxicité de SPIONs modifiés par de l'APTES ont également été réalisées via des biotests très sensibles et novateurs. / Functionalized magnetite nanoparticles for bimodal MRI/PET imagingSuperParamagnetic Iron Oxide Nanoparticles (SPIONs) have been widely studied in the biomedical field due to their promising application as nanodrugs and MRI (Magnetic Resonance Imaging) contrast agents (T2). In this study, magnetite (Fe3O4) nanoparticles have been developed for use as contrast agents for MRI/PET (Positron emission tomography) double imaging. First, functionalized stable superparamagnetic SPIONs have been synthesized in a continuous hydrothermal reactor. During the synthesis, hydrophilic agents (citric acid, LDOPA, DHCA and PHA) have been grafted on the surface of the nanoparticles. The functionalization of the nanoparticles has been optimized by modifying various synthesis parameters such as the temperature and the addition sequence of the organic molecules. The morphology, the size and the structure of the nanoparticles have been shown to depend on these different parameters. Then PolyEthylene Glycol (PEG) polymers have been grafted on their surface to make them stealth and biocompatible. Two different lengths have been considered. For PET imagery, macrocycles which are chelating agents of the 64Cu radionuclide such as MANOTA, NODAGA and DOTA have been grafted on these SPIONs. The radiochemical purities are very conclusive. These nanohybrids have been extensively characterized (TEM, XPS, IR, DLS, ?-potential, TGA, Raman) and are very promising as a diagnostic tool for bimodal imaging MRI/PET in particular the Fe3O4-LDOPA-NODAGA nanoplatform (øDLS = 85±1 nm, r2 = 197±7 mM.s-1, 87% 64Cu). Preliminary cytotoxicity and genotoxicity studies on SPIONs modified by APTES have also been performed via very sensitive and innovative biotests.

This dissertation is focused on the controlled release of drugs from a biodegradable amphiphilic hydrogel based on hydrophobic poly(lactic acid), poly(glycolic acid) and hydrophilic poly(ethylene glycol) (PLGA-PEG-PLGA, ABA) and its modification with itaconic anhydride (ITA). The resulting ,-itaconyl(PLGA-PEG-PLGA) copolymer is referred to as ITA/PLGA-PEG-PLGA/ITA or ITA/ABA/ITA. Itaconic acid provides reactive double bonds and a functional carboxyl group at the ends of the PLGA-PEG-PLGA copolymer chain, thereby rendering the modified ITA/ABA/ITA copolymer less hydrophobic and offering the possibility of forming a carrier for hydrophilic drug substances. These functional copolymers are thermosensitive and change in the external environment (e.g. temperature) causes a sol-gel phase transition due to the formation of micellar structure. The bioactive substances can thus be mixed with a copolymer which is in a low viscous phase (sol phase) and subsequently the mixture can be injected into patient's body at the target site where it forms a gel at 37 °C. This hydrogel becomes a drug depot, which gradually releases the active substance. Prediction of the substance’s release profile from the hydrogel is an effective tool to determine the frequency of administration, potentially enhancing efficacy, and assessment of side effects associated with dosing. The analgesic paracetamol and the sulfonamide antibiotic sulfathiazole were used as model drugs, representing hydrophilic and hydrophobic substances, respectively. The active substances had a significant effect on the resulting hydrogel stiffness. Type of solvent, incubation medium and nanohydroxyapatite also influenced on the gel stiffness and subsequent stability of the hydrogel-drug system. Controlled release of drugs took place in simulated conditions of the human body. Verification of Korsmeyer-Peppas (KP) drug-release model is also discussed in this thesis. The KP model was found suitable for simulating the release of sulfathiazole from ABA and ITA/ABA/ITA hydrogels. On the contrary, the performance of KP model was not suitable for describing the release of paracetamol from the ABA hydrogels. Therefore, a new regression model suitable for both buffered simulated media and water has been proposed. The proposed model fitted better the release of both sulfathiazole and paracetamol from composite material prepared from ABA hydrogel and nanohydroxyapatite.

The theoretical part summarizes information on the isolation and purification of plasmid DNA and nucleic acids as. Plasmid DNA is often used in gene engineering as a vector for the transfer of a particular gene. Its insulation and transportation in sufficient quality is crucial for other processes associated with it. Isolation and survival of pDNA using magnetic carriers of different concentrations of PEG 8000 in combination with 1M NaCl was investigated in experimental parts. Furthermore, the isolation of pDNA using commercial kits was examined.

No description available.

Blood vessels including the arteries, veins, and capillaries are a critical and indispensable component of various organisms. Some studies estimate that if all the blood vessels present in our body are arranged in line, they would amount to a total length of approximately 60,000 miles. This distance is enough to circle the world two and a half times! In addition to being all pervasive, blood vessels perform certain key functions such as delivery of oxygen and nutrients to various tissues in the body. They also assist in the spread of diseases such as cancer. Therefore, it is important to study vessels from the point of view of tissue engineering applications. In this study, I have adapted the design of an open-source 3D printed device to create channels in Poly (ethylene glycol) Maleimide (PEG-Mal) hydrogels using the subtractive technique. The PEG-Mal hydrogels can be formed in various formulations to mimic the biophysical and biochemical properties of various tissues such as bone marrow, brain, and lung. These channels created within hydrogels can be easily perfused with physiologically relevant flow rates found in blood vessels and capillaries. Additionally, I have also optimized the hydrogel formulations to improve channel reproducibility. It was found that the number of arms of PEG-Mal contributed the most to channel reproducibility with higher success rates of channel formation in 8-arm gels when compared to 4-arm gels. Therefore, this project delineates the formation of simple in vitro channels in hydrogels which combines properties of the tissue specific extracellular matrix with hemodynamics. It is expected that such a system will find potential use in various tissue engineering and disease modeling studies.

Gradients of proteins play a prominent role in many biological processes, from development of multicellular organisms to chemical signaling in the immune system. Deposition of surface gradients is a way to permanently modifying a surface’s properties, resulting in the creation of novel materials which have widespread applications in biologically relevant fields, such as directed cell growth, production of biocompatible implantable materials, and creation of functional biosensors. These types of surfaces can also be used as an ex vivo tool to help understand many biological processes by mimicking the environment in gradient-related phenomena in a controllable way. However, despite the large number of applications for chemically graded surfaces, creating them remains a challenge. In this work, a novel diffusion-based patterning mechanism is presented that relies on a 3D micropatterned poly(ethylene glycol) diacrylate (PEG-DA) ‘stamps’ for the controlled deposition of fluorescently-tagged protein ‘ink’ onto pre-treated glass slides. By controlling the contact time and mechanical deformation of the PEG hydrogel on the glass surfaces, it is possible to control local concentration of protein deposition.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2008. / This dissertation can be broken into two parts comprising different strategies to synthesise novel poly-amino acid based polymers. The use of recently developed nickel(0) and cobalt(0) metal catalysts for the living polymerization of α-amino acid-N-carboxyanhydrides (NCAs) to synthesise novel poly-amino acid polymers, comprising a polar, hydrophilic block and a neutral hydrophobic block, were investigated in the first part of this study. The hydrophilic block was made up of a random sequence of arginine (Arg, R), glycine (Gly, G) and aspartic acid (Asp, D) - poly-RGD. This was followed by a polyleucine (Leu, L) hydrophobic block. Success was limited with this system due to polymer precipitation during the polymerization reaction. Because of this precipitation, the amino acid composition of the hydrophilic block was changed to a random sequence of glutamic acid (Glu, E), cystein (Cys, C) and aspartic acid – poly-ECD. Here also, the success was limited because of polymer precipitation. A novel approach to the synthesis of hybrid poly-amino acid – synthetic polymer materials constitutes the second part of this study. The final polymeric structure can be described as a carboxylic acid functionalized polyethylene glycol (PEG) sheathed polylysine polymer. The technology involves the synthesis of a lysine NCA functionalized at the ε-amino group with an α,ω-bis(carboxymethyl) ether PEG. The distal carboxylic acid group was protected as a benzyl ester during synthesis and subsequent polymerization of the PEG-lysine-NCA macro-monomer. The polymerization was successfully initiated using n-butyl amine to form short homopolymer strands. Copolymerization with lysine-NCA was also achieved as well as the successful initiation using a generation 1.0 dendritic amine initiator, N,N,N’,N’-tetrakis(3-aminopropyl)-1,4-butanediamine (DAB-Am-4). These polymers were characterized by 1H NMR.

Η συσχέτιση της πρωτεΐνης PSP94 με τον καρκίνο του προστάτη είναι γνωστή, καθώς μειωμένα επίπεδα της PSP94 σχετίζονται με τα προχωρημένα, μεταστατικά στάδια καρκίνου του προστάτη, και υποτίθεται ότι η πλήρης έκφρασή της συμβάλλει στην αναχαίτιση της καρκινικής ανάπτυξης. Το PCK3145, ένα συνθετικό δεκαπενταμερές πεπτίδιο, του οποίου η αλληλουχία αμινοξέων είναι ταυτόσημο με την αλληλουχία των αμινοξέων 31 έως 45 της PSP94, επιλέχτηκε μεταξύ 12 διαφορετικών πεπτιδίων που προέκυψαν από την PSP94 καθώς εδείχθη οτι ανακεφαλαιώνει in vitro και in vivo τις λειτουργίες και ιδιότητες της PSP94, και άρα είναι ένα μόριο που μπορεί να χρησιμοποιηθεί στην καταστολή της ανάπτυξης του καρκίνου του προστάτη. Ωστόσο, φαρμακοκινητικές μελέτες σε ζώα και ασθενείς με καρκίνο του προστάτη, κατέδειξαν ταχεία κάθαρση και μικρό χρόνο ημιζωής. Προκειμένου να ενισχυθεί το φαρμακοκινητικό προφίλ του πεπτιδίου PCK3145 και ως εκ τούτου να βελτιωθεί η φαρμακοδυναμική του, υπέστη χημική τροποποίηση με προσθήκη πολυαιθυλενικής γλυκόλης (PEG). Η πειραματική εργασία παρουσιάζει τα αποτελέσματα μιας συστηματικής μελέτης σχετικά με την επίδραση της πεγκυλίωσης στο PCK3145 συγκρίνοντας την πρωτεολυτική σταθερότητα και βιολογική δράση δύο πεγκυλιωμένων συζευγμάτων του με την καθαρή μορφή του. Για την ποσοτικοποίηση και για τα πειράματα σταθερότητας χρησιμοποιήθηκε ένα σύστημα υγρής χρωματογραφίας υψηλής απόδοσης HPLC αποτελούμενο από μια αντλία Ultimate (Dionex). Ο χρωματογραφικός διαχωρισμός επιτεύχθηκε με μια στήλη ανάστροφης φάσης C18. Επετεύχθη για πρώτη φορά η ανάπτυξη αναλυτικής μεθοδολογίας HPLC για τον προσδιορισμό των πεγκυλιωμένων αναλόγων με υψηλού βαθμού γραμμικότητα, ειδικότητα, επαναληψιμότητα και αναπαραγωγιμότητα. Η πρωτεολυτική σταθερότητα των συζευγμάτων PEG σε ανθρώπινο πλάσμα προσδιορίστηκε με χρήση υγρής χρωματογραφίας HPLC και αποκαλύφθηκε μείωση του ποσοστού αποδόμησης και ενίσχυσξ της σταθερότητας του πεπτιδικών μορίων σε ανθρώπινο πλάσμα. H μελέτη επιβίωσης της κυτταρικής σειράς καρκίνου του προστάτη PC3 μετά την προσθήκη των πεπτιδικών μορίων υπό μελέτη υπολογίστηκε με τις μεθόδους ΜΤΤ και Trypan blue. 50% αναστολή της κυτταρικής αύξησης/μεταβολισμού επετεύχθη με τις συγκεντρώσεις των 10μg/ml και για τα τρία πεπτίδια. Ωστόσο, τα πεγκυλιωμένα ανάλογα επέφεραν ισχυρότερη ανασταλτική επίδραση στην κυτταρική αύξηση σε μικρότερες δόσεις. Παρόμοια ενθαρρυντικά αποτελέσματα υπήρξαν και για την δράση των μορίων στη καρκινική σειρά του μαστού MCF-7. Τα αποτελέσματα της μελέτης συνοψίζονται στην ικανότητα της πεγκυλίωσης να βελτιώνει τη σταθερότητα του PCK3145 και έτσι να ενισχύει τη βιολογική του δράση. Μελλοντικά πειράματα θα αποσαφηνίσουν περισσότερο τόσο την δράση των μορίων σε καρκινικές σειρές πέραν του προστάτη αλλά και την φαρμακοκινητική συμπεριφορά των πεπτιδικών αναλόγων μέσω πειραμάτων in vivo της βιοκατανομής και μεταβολισμού τους. / PSP94, protein is known to have specific implications with prostate cancer where a down-regulation of PSP94 levels is associated with advanced metastatic prostate cancer and it is supposed that the full expression contributes to the inhibition of tumor growth. PCK3145 is a synthetic 15-mer peptide that matches the natural sequence of amino acids 31 to 45 of PSP94 and was selected from 12 other peptides derived from PSP94 as it exhibited the best in vitro and animal in vivo properties similar to PSP94. Thus, it is a molecule hat can be used to suppress the growth of prostate cancer.However pharmacokinetics studies in animals and in patients with prostate cancer showed rapid clearance and a short half-life. In order to alter the pharmacokinetic profile of the peptide, and thereby to improve its pharmacodynamic potential PCK3145 was chemically modified with polyethylene glycol (PEG).This experimental work presents the results of a systematic study on the influence of the PEGylation of PCK3145 peptides on the proteolytic stability and biological activity of these conjugates compared to the wild-type peptide. For the quantification and stability assays an HPLC system was used consisted of an Ultimate 3000 Pump (Dionex). Chromatographic separation was achieved on RP, C18 column. An HPLC method development for the determination of the PEG-peptide conjugates was assessed for the first time. A high degree of linearity, specificity as well as repeatability and reproducibility were also achieved. The proteolytic stability of the PEG-peptide conjugates in human plasma was assessed by HPLC chromatography, which revealed a significant decrease in the degradation. Proliferation of PC3 cancer cell line was measured using the MTT and Trypan blue test. A 50% inhibition of the cell metabolism/growth was achieved by the concentrations of 10 μg/ml of the three peptides. However, at lower concentrations stronger growth inhibitory effect was observed for the PEG-peptides. Similar encouraging results have also seen for the action of molecules in cancer cell lines MCF-7.The results of this study emphasize the ability of PEGylation to improve the stability of PCK3145 and thus to enhance the biological activity. Future experiments willi clarify more the action of molecules in cancer cell lines beyond the prostate and the pharmacokinetic behavour of peptide analogs through in vivo experiments of biodistribution and metabolism.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

<p>Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with numerous cell-cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with associated changes in NP cell phenotype, morphology and proteoglycan synthesis that may contribute to IVD degeneration. Studies demonstrate healthy, juvenile NP cells exhibit potential for preservation of multi-cell clusters and NP cell phenotype when cultured upon soft, laminin-containing substrates; however, the mechanisms that regulate metabolism and phenotype of these NP cells are not understood. N-cadherin is a cell adhesion molecule that is present in juvenile NP cells, but disappears with age. The goal of this dissertation was to reveal the role of N-cadherin for NP cells in multi-cell clusters that contribute to the maintenance of the juvenile NP cell morphology and phenotype in vitro, and to evaluate the potential for laminin- functionalized poly(ethylene glycol) (PEG-LM) hydrogels to promote human NP cells towards a juvenile NP cell phenotype. </p><p>In this dissertation, juvenile porcine IVD cells were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype on soft, laminin-rich hydrogels. In the first part of this dissertation, preservation of the porcine juvenile NP cell phenotype and presence of N-cadherin was analyzed by culturing porcine NP cells on soft, laminin-rich or PEG-LM hydrogels. Secondly, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Finally, human IVD cells were cultured on PEG-LM hydrogels to investigate the potential to revert degenerate, human NP cells toward a juvenile NP cell phenotype and morphology. </p><p>Findings reveal soft (<500 Pa), laminin-rich substrates promote NP cell clustering, a key feature of the juvenile NP cell that is associated with N-cadherin positive expression. Additionally, N-cadherin-mediated cell-clustering regulates NP cell matrix production and gene expression of NP-specific and NP-matrix related markers. Inhibition of N-cadherin-mediated contacts resulted in decreased expression of juvenile NP cell features. Finally, juvenile human NP cells are also able to form N-cadherin positive cell clusters on soft, PEG-LM hydrogels with higher expression of juvenile NP cell features compared to culturing on stiff PEG-LM hydrogels. Some degenerate, human NP cells are also able to form N-cadherin positive cell clusters with some features of the juvenile NP cell. </p><p>The studies presented in this dissertation support the proposed hypothesis and establish the importance of soft, laminin-rich substrates in promoting NP cell clustering behaviors with associated features of a juvenile cell phenotype and morphology. Additionally, these studies establish a regulatory role for N-cadherin in juvenile NP cells and suggest that preservation of N-cadherin-mediated cell-cell contacts is important for preserving the juvenile NP cell phenotype and morphology. Furthermore, findings from this dissertation reveal the ability to promote degenerate, mature human NP cells towards a juvenile NP cell phenotype, demonstrating the potential to use PEG-LM hydrogels as a means for autologous cell delivery for the restoration of healthy IVD.</p> / Dissertation