Nanoscale drug delivery systems have a great impact in current medical field. These carriers have the potential to improve the efficacy and reduce the toxicity of various medicinal products. A broad variety of different lipid based carriers had been developed and used as delivery systems in the past decades. This dissertation focused on the development of solid lipid nanoparticles (SLN) as delivery systems for a chemotherapeutic agent, docetaxel, and the use of liposomes as a carrier for recombinant protein vaccines. Docetaxel is a potent anticancer drug. However, there continues to be a need for alternative docetaxel delivery systems to improve its efficacy. Docetaxel nanoparticles comprised of lecithin as the main component were engineered using two methods, the emulsion precursor method and the solvent emulsification/evaporation method. Docetaxel in nanoparticles were more effective in killing tumor cells in culture than docetaxel solution. The intravenously injected docetaxel-nanoparticles increased the accumulation of docetaxel in tumors in mice. When administered by intravenous injection or oral routes, docetaxel-nanoparticles showed antitumor activity in tumor-bearing mice. The lecithin-based nanoparticles have the potential to be a novel biocompatible and efficacious delivery system for docetaxel. Liposomes, a well-known lipid based carrier, have been investigated extensively as a vaccine delivery system. The adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when simply admixed with protein antigens. Immunization study in mice after subcutaneously injection of different net charged liposomes showed different antibody responses, depending on the protein antigens. Antigens (OVA, PA) admixed with the negatively charged liposomes prepared with phospholipid, DOPA, induced a strong and functional antibody response comparable to the positively charged liposomes prepared with DOTAP lipid. The negatively charged DOPA liposomes admixed with OVA also induced OVA-specific CD8��� cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA melanoma in a mouse model. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Sept. 6, 2011 - Sept. 6, 2012

<p>The objective of this research is to develop the concept of "genetic memory", the storage of information into genetic material, through the demonstration of the feasibility and benefits of recording the time-history of one or more environmental state variables into genetic material. First, the amount of information that could be stored into non-coding DNA using a lossy mechanism such as regulated diffusion is determined by developing a mathematical model. Next, a mechanism through which this concept of sensing, recording, and storing information on the nanoscale could be accomplished is proposed. </p><p>In conjunction with DNA, which is the actual means of storing information, the proposed approach for the realization of genetic memory also uses thermosensitive liposomes as a means of sensing state variables and acting as a valve to transport and release the DNA in response to the appropriate stimuli. Each variant of thermally sensitive liposomes contains a unique DNA sequence that serves as an identifier. Upon release, the DNA encounters ligase, ATP, and other cofactors and binds - preserving a record of the stimulus experienced by the liposomes. Liposomes, through careful design, can be developed to have unique transition temperatures at which the permeability rate of the contents encapsulated by the liposome increases. It is only at or above this transition temperature that a liposome will become porous. </p><p>Through modeling and experimentation, the feasibility of the liposome/DNA system as a mechanism for information storage is successfully demonstrated.</p> / Dissertation

<p>Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.</p><p> Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions. </p><p>The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.</p><p> The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.</p> / Dissertation

Reproduction de : Thèse doctorat : Pharmacie. Biologie cellulaire et moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliographie f.108-124.

Thèse de doctorat : Physique : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 129-134.

Στόχος της παρούσας εργασίας είναι η ακινητοποίηση λιποσωμάτων με εγκλωβισμένη Tobramycin σε επιχρυσωμένες μεταλλικές επιφάνειες, με σκοπό την παρασκευή αντιμικροβιακών ενδοπροθέσεων του ουροποιητικού, που αποδεσμεύουν φάρμακο με ελεγχόμενο ρυθμό. / The aim of this study is to covalently link liposomes on metallic surfaces, as a method to prepare antimicrobial controlled (release) drug-eluting stents, using Tobramycin (TOB) as an anti-microbial drug. As a preliminary step for immobilization of TOB, different types of liposomes were constructed and evaluated for TOB loading efficiency, size distribution and ζ-potential. TOB concentrations were measured by a chemiluminescence immunoassay (ADVIA Centaur, Siemens), after modulating the technique as required for the specific samples. Results show that extruded DRV liposomes with similar sizes (mean diameter) with that of SUV liposomes (~95 nm), have 4 times higher drug loading efficiency.

Ο μυϊκός νικοτινικός υποδοχέας ακετυλοχολίνης (nAChR), είναι ένα πενταμερές κανάλι ιόντων νατρίου, το οποίο εδράζεται στους σκελετικούς μύες στη νευρομυϊκή σύναψη και μετατρέπει τις νευρικές ώσεις σε μυϊκές συσπάσεις. Το κανάλι αυτό γίνεται στόχος του ανοσοποιητικού συστήματος, που παράγει στην περίπτωση της βαριάς μυασθένειας αντισώματα εναντίον του. Η συχνότητα της ασθένειας αυτής στο γενικό πληθυσμό είναι 125-400 περιπτώσεις/εκατομμύριο, με αναλογία ανδρών/γυναικών ασθενών 1:2. Τα πρώτα συμπτώματα της νόσου είναι διπλωπία και βλεφαρόπτωση που εξελίσσονται σε γενική μυϊκή αδυναμία και εύκολη κόπωση έως παράλυση των άκρων ή ακόμα και των αναπνευστικών μυών. Οι καθιερωμένες σήμερα θεραπείες της βαριάς μυασθένειας περιλαμβάνουν χορήγηση αντιχολινεστερασικών, ανοσοκατασταλτικών φαρμάκων, θυμεκτομή, πλασμαφαίρεση, καθώς και ενδοφλέβια χορήγηση ανθρωπίνων ανοσοσφαιρινών σε μεγάλες δόσεις. Όμως κοινός παρονομαστής όλων αυτών των θεραπειών είναι η έλλειψη εκλεκτικότητας καθώς και οι παρενέργειες. Τα παραπάνω προβλήματα έχουν κάνει φανερή την ανάγκη για εξεύρεση νέων και πιο εξειδικευμένων θεραπειών, όπως θεραπείες στις οποίες τα παθολογικά αυτοαντισώματα θα αφαιρούνται εκλεκτικά από την κυκλοφορία. Η μελέτη που έγινε στοχεύει στην εκλεκτική αφαίρεση των παθολογικών αντισωμάτων από το αίμα μυασθενών, με μυϊκούς nAChRs ή τμήματα αυτών, που είτε θα χορηγούνται ενδοφλεβίως, είτε θα βρίσκονται ομοιοπολικά προσδεμένα σε στήλες χρωματογραφίας από όπου θα περνά ο ορός. Στο πρώτο μέρος της μελέτης δημιουργήθηκαν παρασκευάσματα που περιείχαν το μυϊκού τύπου nAChR από τα ηλεκτρικά όργανα του ψαριού Torpedo californica (Τ. nAChR). Συγκεκριμένα, παρασκευάστηκε Τ. nAChR ο οποίος ήταν διαλυμένος σε ρυθμιστικό διάλυμα φωσφορικών (PBS), σε PBS/Χολικό νάτριο 2% (PBS/Χ.Ν.2%) καθώς επίσης και ενσωματωμένος σε λιποσώματα επικαλυμμένα με αλυσίδες πολυαιθυλενογλυκόλης (PEG), με στόχο την παράταση του χρόνου ημιζωής στον οργανισμό και τη μείωση της αντιγονικότητας. Τα παρασκευάσματα συγκρίθηκαν μεταξύ τους ως προς την ικανότητά τους να δεσμεύουν το μονοκλωνικό αντίσωμα mAb35 (ένα μονοκλωνικό αντίσωμα που προσδένεται σε μυϊκούς nAChRs) καθώς και I125-α-μπουγκαροτοξίνη (I125-α-Bgt) (πρωτεΐνη-συστατικό του δηλητηρίου του φιδιού Bungarus multicinctus και ανταγωνιστής ακετυλοχολίνης, ραδιοσημασμένη με I125), σε διάφορα χρονικά διαστήματα από την παρασκευή τους. Τα αποτελέσματα έδειξαν ότι τη μεγαλύτερη ικανότητα δέσμευσης έχει το παρασκεύασμα στο οποίο ο Τ. nAChR είναι διαλυμένος σε PBS/Χ.Ν.2%. Επίσης προσδιορίστηκαν και συγκρίθηκαν τα ποσοστά του mAb35, τα οποία δεσμεύτηκαν in vivo σε αρουραίους από τα ίδια παρασκευάσματα με Τ. nAChR. Τα πειράματα αυτά έδειξαν ότι μόνο ο Τ. nAChR που ήταν διαλυμένος σε PBS/Χ.Ν.2% μπόρεσε να δεσμεύσει ποσοτικά το mAb35. Τέλος έγιναν πειράματα βιοκατανομών, του Τ. nAChR ραδιοσημασμένου με I125-α-Bgt, στα παραπάνω παρασκευάσματα, σε αρουραίους. Τα αποτελέσματα έδειξαν ότι μέγιστο χρόνο ζωής in vivo έχει ο ενσωματωμένος υποδοχέας σε λιποσώματα επικαλυμμένα με PEG. Στο δεύτερο μέρος της μελέτης χρησιμοποιήθηκαν εξωκυτταρικά τμήματα του ανθρωπίνου μυϊκού nAChR (ECDs), εκφρασμένα στο ζυμομύκητα Pichia pastoris και σε κύτταρα εντόμων High Five (από τις ωοθήκες του εντόμου Trichoplusia ni). Τα Pichia pastoris και High Five α1 ECDs συγκρίθηκαν μεταξύ τους ως προς την ικανότητα να δεσμεύουν αντισώματα από ορούς μυασθενών χρησιμοποιώντας τη ραδιοανοσολογική μέθοδο. Τα αποτελέσματα των πειραμάτων με ορούς 5 ασθενών, έδειξαν ότι το High Five α1 ECD δεσμεύει υπερδιπλάσια ποσοστά αυτοαντισωμάτων σε σχέση με το Pichia pastoris α1 ECD. Από τα πειράματα αυτά φαίνεται πως το High Five α1 ECD πλεονεκτεί του Pichia pastoris α1 ECD ποιοτικά. Συνολικά από τη μελέτη που έγινε, προέκυψε μια σειρά από συμπεράσματα. Κατ’ αρχήν αποδείχθηκε ότι nAChRs, όπως ο Τ. nAChR, μπορούν να ενσωματωθούν σε πολλά διαφορετικά παρασκευάσματα όπως σε PBS, PBS/Χ.Ν.2% και λιποσώματα, τα οποία επηρεάζουν τις ιδιότητές τους, όπως την ικανότητα δέσμευσης mAb35 και I125-α-Bgt και το χρόνο ημιζωής στον οργανισμό. Όπως αποδείχθηκε ο Τ. nAChR μπορεί να δεσμεύει mAb35 σε οποιαδήποτε από τα παραπάνω παρασκευάσματα in vitro, ενώ in vivo μπορεί να το δεσμεύει όταν είναι διαλυμένος σε PBS/Χ.Ν.2%. Επίσης φάνηκε ότι μεγαλύτερο χρόνο ημιζωής in vivo έχει ο Τ. nAChR ενσωματωμένος σε λιποσώματα επικαλυμμένα με PEG. Τέλος αποδείχθηκε ότι ανασυνδυασμένα ECDs της ανθρώπινης α1 υπομονάδας του μυϊκού nAChR, δεσμεύουν αυτοαντισώματα από τον ορό μυασθενών και ότι η ικανότητα δέσμευσης των τμημάτων αυτών, επηρεάζεται από το σύστημα στο οποίο εκφράζονται. Η πληροφορία αυτή πρέπει να ληφθεί υπόψη στη κατασκευή ανοσοπροσροφητικών στηλών για τη θεραπεία της βαριάς μυασθένειας. / The muscle nicotinic acetylcholine receptor (nAChR) is a pentameric cation channel, which is located at the neuromuscular synapse and converts neuronal impulses into muscle contractions. In myasthenia gravis patients, this particular channel is targeted by the immune system, which produces antibodies against it. The incidence of the disease is about 125-400 cases per million. The first symptoms of the disease are diplopia and blepharoptosis, which may shift to general weakness and fatigability. The established therapies for myasthenia gravis, include administration of acetylcholinesterase inhibitors, immunosuppresive drugs, thymectomy, plasmaphaeresis and administration of intravenous immunoglobulins (IVIGs). Unfortunately all these therapies are not specific and have many serious side effects. This has made clear the need for more specific therapies, in which pathological autoantibodies will not be produced or will be removed selectively. This project aims at the specific aphaeresis of pathological autoantibodies from the patients’ blood by using the muscle nAChR or extracellular domains (ECDs) of its subunits. These molecules will either be administered i.v. or will be covalently conjugated on chromatography beads, forming a column by which the serum could pass and be cleaned from autoantibodies. In the first part of this project, formulations of nAChR, from the electric organs of fish Torpedo californica (T. nAChR), were prepared. Particularly T. nAChR was diluted in PBS and PBS/Sodium Cholate 2% (PBS/S.Ch.2%) and incorporated into liposomes coated by polyethylenoglycol (PEG) chains in different percentages, in order to achieve longer half-life in vivo. Their ability to bind mAb35 (a monoclonal antibody that binds muscle nAChRs) and I125-α- bungarotoxin (I125-labeled toxin from the poison of the snake Bungarus multicinctus, a well studied nAChR antagonist) were compared. The results showed that T. nAChR has the best binding capacity in vitro in the PBS/S.Ch.2%. Apart from the in vitro mAb35 binding measurements, in vivo binding experiments were also performed, using Lewis rats. Results indicate that only T. nAChR in the PBS/S.Ch.2% formulation binds mAb35 quantitatively in vivo. Finally, the biodistribution of T. nAChR in several formulations was also studied in rats. T. nAChR formulations were radiolabelled using I125-α-bungarotoxin and administered i.v. The results indicate that T. nAChR has the longest half-life time when incorporated into PEG-coated liposomes. In the second part of this project, ECDs of human muscle nAChR α1 subunit, expressed in the yeast Pichia pastoris and High Five insect cells were used. Pichia pastoris and High Five α1 ECDs were measured and compared for their ability to bind autoantibodies from myasthenic patients’ sera using radioimmunoassay. The results from 5 patients’ sera indicate that High Five α1 ECD binds more than twice more autoantibodies than Pichia pastoris α1 ECD. In conclusion, it was proved that nAChRs can be incorporated to many different formulations, like PBS, PBS/S.Ch.2% and liposomes, which affect its binding capacity and half-life time. It was shown that Τ. nAChR, when incorporated into any of the above formulations, can bind mAb35 in vitro, while in vivo only in PBS/S.Ch.2% formulation. In addition, it was proved that Τ. nAChR has longer half-life time in vivo when incorporated in PEG-coated liposomes. In the last part of the project, it was shown that ECDs of human nAChR α1 subunit binds autoantibodies from myasthenia gravis patient’s serum and their binding capacity is strongly affected by the expression system.

Η λυοφιλοποίηση θεωρείται ιδανική μέθοδος για βραχύχρονη ή/ και μακρόχρονη αποθήκευση λιποσωμάτων. Στόχος αυτής της μελέτης ήταν να ευρεθούν οι ποσότητες κρυοπροστατευτικού που μπορεί να αυξήσουν τη συγκράτηση πρωτεϊνικών μορίων σε λιποσώματα τα οποία είναι ασταθή (δηλ. χάνουν μεγάλο τμήμα της εγκλωβισμένης πρωτεΐνης) μετά από λυοφιλοποίηση. Η μέθοδος παρασκευής των λιποσωμάτων που χρησιμοποιήθηκε ήταν η DRV μέθοδος. Είναι η πλέον κατάλληλη στην περίπτωση εγκλωβισμού πρωτεϊνικών μορίων καθώς επιτυγχάνει υψηλά ποσοστά εγκλωβισμού και επίσης χρησιμοποιεί ήπιες συνθήκες οπότε προστατεύεται η τριτοταγής δομή των πρωτεϊνών. Η διαδικασία βασίζεται στην αφυδάτωση μίγματος “άδειων” SUV (μικρών μονοστοιβαδιακών) λιποσωμάτων παρουσία του διαλύματος πρωτεΐνης που πρόκειται να εγκλωβιστεί. Μετά από ελεγχόμενη επανενυδάτωση της ξηρής κόνης σχηματίζονται τα DRV λιποσώματα. (από τις λέξεις dried-rehydrated vesicles που μεταφράζονται ως αφυδατωμένα-επανενυδατωμένα σωματίδια). Ως υλικά για την παρασκευή των λιποσωμάτων χρησιμοποιήθηκαν τα εξής: Φωσφατιδυλοχολίνη αυγού (egg PC), διστεαροϋλο-γλυκεροφωσφατιδυλοχολίνη (DSPC), διπαλμιτοϋλο-γλυκεροφωσφατιδυλοχολίνη (DPPC), διμυριστοϋλο-γλυκεροφωσφατιδυλοχολίνη (DΜPC), φωσφατιδυλογλυκερόλη (PG) και χοληστερόλη (CHOL). Παρασκευάστηκαν με τη μέθοδο DRV λιποσώματα διαφόρων λιποσωμικών συστάσεων προκειμένου να μελετηθεί η ικανότητα εγκλωβισμού για κάθε λιποσωμική σύσταση και να επιλεγούν αυτές που είναι πλέον ασταθείς για περαιτέρω μελέτη. Ως πρωτεΐνη μοντέλο χρησιμοποιήθηκε η αλβουμίνη βόειου ορού (BSA). Μελετήθηκε η επίδραση της λυοφιλοποίησης σε διάφορα DRV λιποσώματα παρουσία ή/ και απουσία κρυοπροστατευτικού παράγοντα (τρεαλόζη). Σε κάποιες περιπτώσεις προστέθηκε τρεαλόζη εντός των λιποσωμάτων κατά την αρχική παρασκευή των DRV (στο στάδιο της ανασύστασης). Επίσης, προκειμένου να αποκτήσουμε περαιτέρω πληροφορίες για τα λιποσώματα που μελετούσαμε, πραγματοποιήθηκε προσδιορισμός του μεγέθους των ορισμένων λιποσωμικών σειρών και μορφολογική μελέτη με ηλεκτρονικό μικροσκόπιο σάρωσης. Στη συνέχεια, με βάση τα αρχικά αποτελέσματα με την αλβουμίνη, επιλέχθηκαν ορισμένες λιποσωμικές συστάσεις προκειμένου να μελετηθεί η σταθερότητα λιποσωμάτων που εγκλωβίζουν ένα βιοδραστικό μόριο, το t-PA (ιστικού τύπου ενεργοποιητή πλασμινογόνου) σε ξηρή μορφή. Η μελέτη πραγματοποιήθηκε με δείκτη τη διατήρηση της ενεργότητας του μορίου μετά από FD και προσθήκη ή όχι τρεαλόζης. Απώτερος σκοπός αυτής της εργασίας είναι η δυνατότητα παρασκευής σταθερών t-PA- λιποσωμικών μορφών σε ξηρή μορφή προκειμένου να μελετηθεί η δράση τους στη θεραπεία θρομβώσεων του αμφιβληστροειδούς. Τα βασικά συμπεράσματα της μελέτης είναι τα εξής: Σχετικά με αποτελέσματα πειραμάτων με BSA: * Τα PC:PG:CHOL DRV λιποσώματα σταθεροποιούνται ικανοποιητικά αν πριν τη λυοφιλοποίηση προσθέσουμε αναλογία treh/lipid=5/1 στη λιποσωμική διασπορά. * Η καλύτερη σταθεροποίηση επιτυγχάνεται με παρουσία σακχάρου στο εσωτερικό και εξωτερικό των λιποσωμάτων * Πιθανή αλληλεπίδραση λαμβάνει χώρα μεταξύ των PC λιποσωμάτων και της αλβουμίνης, δίνοντας ψευδή υψηλά τελικά ποσοστά συγκράτησης μετά τη λυοφιλοποίηση Σχετικά με αποτελέσματα πειραμάτων με t-PA * Τα αποτελούμενα από DSPC DRV λιποσώματα παραμένουν πολύ σταθερά διατηρώντας την ενεργότητα του t-PA μετά τη λυοφιλοποίηση και χωρίς την προσθήκη κρυοπροστατευτικού. * Τα ασταθή PC:PG:CHOL και PC λιποσώματα σταθεροποιούνται ικανοποιητικά με προσθήκη τρεαλόζης πριν το FD. / Lyophilization is considered to be an ideal technique for both short-term and long-term storage of liposomes. The objective of this study was to investigate the stability of liposomes encapsulating protein molecules and the effect of freeze-drying (FD) on the stability of dehydrated-rehydrated vesicles (DRV) with and without cryoprotective agents (CPA). Τhe liposomes were prepared by the DRV method. This is considered to be the most appropriate in the cases that entrapment of proteins is desired, since it gives liposomes with high encapsulation efficiencies and by using mild conditions it protects the tertiary structure of proteins retaining thus their biological activity. The procedure followed here was based on dehydration of a mixture of “empty” SUV liposomes and the protein solution (BSA) which is to be entrapped, followed by controlled rehydration of the dry powder. The materials used for the preparation of the liposomes were: Egg-PC (phosphatidylcholine), distearoyloglycerophosphatidylcholine(DSPC), (DPPC) dipalmitoylglycerophosphatidylcholine, (DΜPC) dimyristoylglycerophosphatidylcholine, PG (phosphatidylglycerol) and CHOL (cholesterol). Liposomes of various lipid compositions were prepared so that the most unstable liposome compositions are found and available for further study. BSA was used as a protein model in these experiments. Furthermore, the influence of lyophilization for liposomes of various compositions was studied using trehalose as a cryoprotective agent. In some experiments, the dissacharide was added during the reconstitution of DRV. In order to have some more information about the liposomes under investigation, we conducted particle size determination (zetasizer) and morphological study by SEM (scanning electron microscopy. Eventually, based on the primary results with albumin, the stability of liposomes bearing a bioactive protein was studied. The protein used here was t-PA (tissue type plasminogen activator). We investigated the influence of FD on the stability of liposomes with or without the addition of a cryoprotective agent (trehalose) as a function of bioactivity retention. A long-term objective of our studies is to prepare stable after FD liposomal- tPA-formulations that may be active for therapy of ocular thrombosis cases. The mail conclusions of this study were: Concerning the results obtained with BSA: * PC:PG:CHOL DRV liposomes are adequately stabilized when trehalose in mass ratio treh/PC=5/1 is added in the liposomal dispersion * Maximum retention of BSA is achieved when trehalose is added on both sides of liposomal membrane. * There is a possible interaction between BSA and PC liposomes, which results in false estimation of BSA retention after FD. Concerning the results obtained with t-PA: * DSPC DRV liposomes are very stable after FD and they preserve t-PA activity even in absence of cryoprotectant. * Unstable PC:PG:CHOL and PC liposomes can be adequately stabilized by trehalose.

The pulmonary infection with Pseudomonas aeruginosa is considered as one of the main causes of health deterioration in cystic fibrosis patients (CF). Efficient management of P. aeruginosa in CF remains difficult mainly with the emergence of multidrug-resistant strains leading ultimately to death. There is a pressing need for new approaches to control these Pseudomonal infections. Current studies on the antimicrobial efficacy of liposomal antibiotics have shown conflicting results. We sought to assess whether the incorporation of clarithromycin into liposomes could improve its antibacterial activity against clinical isolate of Pseudomonas aeruginosa from CF patients. Different formulations of liposomal clarithromycin were prepared, characterized and their antibacterial activities against resistant strains of P. aeruginosa were investigated. These formulations reduced the biofilm formation, the virulence factors production and the bacterial motilities compared to free drug. The therapeutic importance of liposome containing macrolides in the management of experimental pseudomonal lung infection in animals is warranted.

<p>Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.</p><p>The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model. </p><p>Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment. </p><p>In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro. </p><p>Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug. </p><p>In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.</p> / Dissertation