Αμφίφιλα Συμπολυμερή Κατά Συστάδες πολυ(Μεθακρυλικού Εστέρα της Διμεθυλοαμινοαιθανόλης) –b–πολυ(Μεθακρυλικού Εστέρα της Δωδεκανόλης): Σύνθεση, Χημική Τροποποίηση, Χαρακτηρισμός, Αυτοοργάνωση σε Διαλύματα και Σύμπλεξη με DNA

Η παρούσα ερευνητική εργασία αφορά τη σύνθεση καινοτόμων πολυμερικών συστημάτων με εφαρμογή στο τομέα της νανοβιοϊατρικής. Η ικανότητα των πολυμερών να αυτό-οργανώνονται σε μικκυλιακές δομές και άλλες μορφολογίες, όταν εισαχθούν σε κατάλληλο διαλύτη, τα καθιστά ιδιαίτερα χρήσιμα ως φορείς φαρμάκων και γονιδίων με σκοπό τη στοχευμένη θεραπεία. Η μελέτη της εργασίας αφορά τη σύνθεση αμφίφιλων δισυσταδικών συμπολυμερών πολυ(μεθακρυλικού εστέρα της διμεθυλοαμινοαιθανόλης) –b–πολυ(μεθακρυλικού εστέρα της δωδεκανόλης) ([poly(2-dimethylamino ethyl methacrylate))-b- (poly (lauryl methacrylate)], PDMAEMA-b-PLMA) μέσω της τεχνικής πολυμερισμού RAFT αντιστρεπτής προσθήκης-μεταφοράς αλυσίδας με απόσπαση (RAFT Polymerization) και την χημική τους τροποποίηση προς δημιουργία παράγωγων πολυηλεκτρολυτικών συμπολυμερών με νέες ιδιότητες. Επιπροσθέτως, γίνεται μελέτη ως προς το φυσικοχημικό και μοριακό τους χαρακτηρισμό όσο και της αυτοοργάνωσης τους σε υδατικό μέσο. Έγινε χρήση τεχνικών χαρακτηρισμού που περιλαμβάνει φασματοσκοπικές τεχνικές χαρακτηρισμού, τεχνικές σκέδασης φωτός, χρωματογραφία αποκλεισμού μεγεθών και ηλεκτρονική μικροσκοπία διέλευσης. Μελετάται η συμπεριφορά και οι ιδιότητες των μικκυλίων υπό την επίδραση μια σειράς φυσικοχημικών παραμέτρων όπως pH, θερμοκρασία και ιοντική ισχύς. Η απόκριση των συστημάτων σε αυτές τις παραμέτρους τους προσδίδει νέες ιδιότητες για τη χρήση τους ως νανοφορείς. Στη συνέχεια, πραγματοποιείται η σύμπλεξη των πολυηλεκτρολυτικών πολυμερών με DNA. Διερευνάται η διαδικασία της σύμπλεξης, οι ιδιότητες και η συμπεριφορά των σχηματιζόμενων συμπλόκων συμπολυμερούς-DNA.This research concerns the synthesis of novel block copolymer systems with application in the field of nanobiomedicine. Their property to self- assemble into micelle structures and other morphologies, when diluted in a proper solvent, make them particularly useful as drug and gene delivery systems for targeted therapy. The aim of this research is the synthesis of amphiphilic diblock copolymers of poly(2-dimethylaminoethylmethacrylate)-b-poly(lauryl methacrylate), PDMAEMA-b-PLMA, using the polymerization technique of reversible addition-fragmentation chain transfer (RAFT polymerization) and their chemical modification in a way to create derivative polyelectrolyte block copolymers with new properties. Furthermore, their physicochemical and molecular characterization is performed in order to elucidate their self-assembly in aqueous media. The characterization of the block copolymers was performed by using characterization techniques such as spectroscopic characterization techniques, light scattering techniques, size exclusion chromatography and transmission electron microscopy. We studied the behavior and properties of the micelles under the effect of physical and chemical parameters such as pH, temperature and ionic strength. These stimuli-responsive block copolymers respond to changes of these parameters giving showing interesting new properties for their use as nanocarriers. Subsequently, we studied the complexation of the polyelectrolyte block copolymers with DNA. We focus on the investigation of the complexation process, the properties and the behavior of the formed block copolymer-DNA complexes

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells

Hydrophilic Random Cationic Copolymers as Polyplex-Formation Vectors for DNA

Research on the improvement and fabrication of polymeric systems as non-viral gene delivery carriers is required for their implementation in gene therapy. Random copolymers have not been extensively utilized for these purposes. In this regard, double hydrophilic poly[(2-(dimethylamino) ethyl methacrylate)-co-(oligo(ethylene glycol) methyl ether methacrylate] [P(DMAEMA-co-OEGMA)] random copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymers were further modified by quaternization of DMAEMA tertiary amine, producing the cationic P(QDMAEMA-co-OEGMA) derivatives. Fluorescence and ultraviolet–visible (UV–vis) spectroscopy revealed the efficient interaction of copolymers aggregates with linear DNAs of different lengths, forming polyplexes, with the quaternized copolymer aggregates exhibiting stronger binding affinity. Light scattering techniques evidenced the formation of polyplexes whose size, molar mass, and surface charge strongly depend on the N/P ratio (nitrogen (N) of the amine group of DMAEMA/QDMAEMA over phosphate (P) groups of DNA), DNA length, and length of the OEGMA chain. Polyplexes presented colloidal stability under physiological ionic strength as shown by dynamic light scattering. In vitro cytotoxicity of the empty nanocarriers was evaluated on HEK293 as a control cell line. P(DMAEMA-co-OEGMA) copolymer aggregates were further assessed for their biocompatibility on 4T1, MDA-MB-231, MCF-7, and T47D breast cancer cell lines presenting high cell viability rates

Amphiphilic Copolymer-Lipid Chimeric Nanosystems as DNA Vectors

Lipid-polymer chimeric (hybrid) nanosystems are promising platforms for the design of effective gene delivery vectors. In this regard, we developed DNA nanocarriers comprised of a novel poly[(stearyl methacrylate-co-oligo(ethylene glycol) methyl ether methacrylate] [P(SMA-co-OEGMA)] amphiphilic random copolymer, the cationic 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP), and the zwitterionic L-α-phosphatidylcholine, hydrogenated soybean (soy) (HSPC) lipids. Chimeric HSPC:DOTAP:P[(SMA-co-OEGMA)] nanosystems, and pure lipid nanosystems as reference, were prepared in several molar ratios of the components. The colloidal dispersions obtained presented well-defined physicochemical characteristics and were further utilized for the formation of lipoplexes with a model DNA of linear topology containing 113 base pairs. Nanosized complexes were formed through the electrostatic interaction of the cationic lipid and phosphate groups of DNA, as observed by dynamic, static, and electrophoretic light scattering techniques. Ultraviolet–visible (UV–Vis) and fluorescence spectroscopy disclosed the strong binding affinity of the chimeric and also the pure lipid nanosystems to DNA. Colloidally stable chimeric/lipid complexes were formed, whose physicochemical characteristics depend on the N/P ratio and on the molar ratio of the building components. Cryogenic transmission electron microscopy (Cryo-TEM) revealed the formation of nanosystems with vesicular morphology. The results suggest the successful fabrication of these novel chimeric nanosystems with well-defined physicochemical characteristics, which can form stable lipoplexes

Aqueous Heat Method for the Preparation of Hybrid Lipid–Polymer Structures: From Preformulation Studies to Protein Delivery

Liposomes with adjuvant properties are utilized to carry biomolecules, such as proteins, that are often sensitive to the stressful conditions of liposomal preparation processes. The aim of the present study is to use the aqueous heat method for the preparation of polymer-grafted hybrid liposomes without any additional technique for size reduction. Towards this scope, liposomes were prepared through the combination of two different lipids with adjuvant properties, namely dimethyldioctadecylammonium (DDA) and D-(+)-trehalose 6,6′-dibehenate (TDB) and the amphiphilic block copolymer poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) (PLMA-b-PDMAEMA). For comparison purposes, PAMAM dendrimer generation 4 (PAMAM G4) was also used. Preformulation studies were carried out by differential scanning calorimetry (DSC). The physicochemical characteristics of the prepared hybrid liposomes were evaluated by light scattering and their morphology was evaluated by cryo-TEM. Subsequently, in vitro nanotoxicity studies were performed. Protein-loading studies with bovine serum albumin were carried out to evaluate their encapsulation efficiency. According to the results, PDMAEMA-b-PLMA was successfully incorporated in the lipid bilayer, providing improved physicochemical and morphological characteristics and the ability to carry higher cargos of protein, compared to pure DDA:TDB liposomes, without affecting the biocompatibility profile. In conclusion, the aqueous heat method can be applied in polymer-grafted hybrid liposomes for protein delivery without further size-reduction processes