Dissertation committee:
Κωνσταντίνος Δεμέτζος, Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Αστέριος Πίσπας, Διευθυντής Ερευνών, Ινστιτούτο Θεωρητικής και Φυσικής Χημείας, Εθνικό Ίδρυμα Ερευνών
Νεκτάριος Ταβερναράκης, Καθηγητής, Ιατρική Σχολή, Πανεπιστήμιο Κρήτης & Διευθυντής Ερευνών, Ινστιτούτο Μοριακής Βιολογίας και Βιοτεχνολογίας, Ίδρυμα Τεχνολογίας και Έρευνας
Μαριλένα Βλάχου, Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Νικόλαος Δρακούλης, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ερμής Ιατρού, Καθηγητής, Τμήμα Χημείας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Barbara Trzebicka, Professor, Centre of Polymer and Carbon Materials, Polish Academy of Sciences
Summary:
Lyotropic liquid crystals are resulting by the self-assembly process of amphiphilic molecules, such as lipids, into water, being organized in different mesophases. The non-lamellar formed mesophases, such as bicontinuous cubic and inverse hexagonal, attract great scientific interest, because they can be further exploited upon the formation of liquid crystalline nanoparticles, that can be utilized as nanosystems intended for the delivery of a wide range of active pharmaceutical ingredients, exhibiting major advantages, including the ability of controlled release and high grade of internal organization. Since the targets of many therapeutic agents against several severe disorders are localized in subcellular compartments, such as lysosomes, mitochondria and nucleus, the ability of targeting the subcellular organelles remains a critical challenge in pharmaceutical nanotechnology, in order to enhanced therapeutic efficiency be achieved and possible toxic side effects be avoided. Τhe nanocarriers being capable of subcellular targeting are also referred as the last generation of nanosystems. Thus, the combination of the advantageous category of lyotropic liquid crystalline nanosystems with smart biomaterials, such as innovative copolymers, in order to subcellular targeting get achieved, would result in advanced nanocarriers with upgrade functionality and increased therapeutic efficacy.
The aim of the present PhD Thesis is the development and evaluation of non-lamellar lyotropic lipidic liquid crystalline nanoparticles, intended for the delivery of active pharmaceutical agents that are able to perform subcellular targeting. Towards this scope, liquid crystalline nanoparticles were prepared from two different lipids and got stabilized by a variety of polymers. The polymers were chosen according to their high biocompatibility, promising stealth properties and their functionality due to presence of stimuli-responsive blocks and cationic moieties. The physicochemical (size, size distribution and ζ-potential) and morphological characteristics (Rg/Rh ratio and fractal dimension) of the resultant, new developed, liquid crystalline nanosystems were obtained from a gamut of light scattering techniques (dynamic, electrophoretic and static), while their microenvironmental parameters (micropolarity and microfluidity) from fluorescence spectroscopy. Their colloidal stability upon time was monitored. The effect of the environmental parameters on their physicochemical behavior, namely of the pH, temperature, serum proteins and ionic strength, was investigated. Their internal and external morphology was assessed by cryogenic-Transmission Electron Microscopy (cryo-TEM), while their thermal behavior was evaluated by microcalorimetry. Subsequently, the active pharmaceutical agent resveratrol was loaded into the nanosystems, the entrapment efficiency was estimated and in vitro release studies were carried out. The biosafety of the nanosystems was documented by in vitro toxicity studies in cells, while their ability to target subcellular organelles was monitored by confocal laser scanning microscopy. Finally, the nanoparticles were administrated in vivo in the nematode Caenorhabditis elegans and further nanotoxicity studies were conducted.
According to the results, the type of the lipid and the polymeric stabilizer, as well as their ratio, were found to strictly affect the physicochemical and morphological behavior of the nanosystems, their interaction with the environmental parameters, as well as their release profile. Within the present research, a variety of advanced liquid crystalline nanoparticulate formulations was developed, resulting finally to nanosystems exhibiting biosafety along with multi-functional properties, namely dual stimuli-responsive (pH- and thermo-) physicochemical and morphological behavior, facilitating stimuli-triggered controlled drug release, as well as subcellular targeting ability. To the best of the author knowledge, it is the first time that the nanosystems, which were developed in the present PhD Thesis, along with some extra characterization techniques that have not been applied to liquid crystalline nanoparticles before, are referred in the literature.
In conclusion, the present PhD Thesis highlights the significance of the rational design that should be carried out during the development process of nanosystems being in liquid crystalline state, as an effort to upgrade the current technological platforms and thus the therapeutic strategies. The careful selection of appropriate biomaterials and the comprehensive study of all the different interactions that maybe take place among the consisting biomaterials or the effects from external parameters, such as the environment, as well as the application of robust characterization techniques are absolutely necessary in order to innovative, safe and effective medicines can be created.
Keywords:
lyotropic liquid crystals, non-lamellar mesophases, active pharmaceutical ingredients delivery nanosystems, lipid-polymer nanoparticles, resveratrol, subcellular organelles targeting, stimuli-responsive nanosystems, light scattering, cryo-TEM
