Investigation on smart triggers and smart lipid vesicles as new tools for drug delivery applications

The rational design of systems for controlled drug delivery is an important area of research for advancing new therapies for many diseases. Nanomaterial based controllable drug delivery platforms would overcome many of the major drawbacks in pharmacological therapy, because they would store the therapeutic molecules during transportation within the body and provide triggered and finely controlled release of the agent at the target site. In this context, lipid vesicles as liposomes attracted, since their discovery, growing interest for their potential applications as drug delivery vectors. They are now considered clinically established nanometer-scaled systems for the delivery of cytotoxic drugs or agents for biomedical applications. However, liposomes can be further engineered to improve their performance in terms of stability and controlled delivery, since the rapid degradation, due to the reticuloendothelial system (RES), and inability to achieve sustained drug delivery, over a prolonged period, limit their biological efficacy and use in pharmaceutics. Interesting results were obtained in terms of physical stability through the approach that provides the combination of different biomaterials within the same delivery lipid system. Following this concept, it was possibile to change the surface properties of the bilayer, by coating it with water-soluble polymers, like polyethylene glycol, leading to “stealth liposomes”. More recently, satisfying achievements resulted from the modification of the internal structure of liposomes, with the aim to convert the aqueous inner core into a soft and elastic hydrogel, obtaining structures able to retain the cargo for longer time, without unwanted burst release of the delivered compound. An ideal drug delivery platform should encompass not only the carrier stability, but also controllable timing, dosage and site specificity of drug release, and permit remote, noninvasive and reliable switching of the therapeutic agent, in order to prevent deleterious side effects of cytotoxic drugs toward normal and healthy tissue. Much of innovations in materials design, for drug delivery, manifest in producing “smart liposomes” that are able to respond to “smart triggers”, in order to realize on-demand processes, allowing for tailored release profiles with excellent temporal and dosage control. In principle, the ondemand drug delivery is becoming feasible through the design of stimuli-responsive systems that recognize their microenvironment and react in a dynamic way. Specifically, it is possible to engineere the liposomal structure making it capable of responding to physical, chemical or biological triggers. Among the endogenous or exsogenous stimuli that can be applied, magnetic stimulus represents a potential trigger for the remotely-on demand release, evaluating that normal biological tissues are essentially transparent to low-frequency magnetic fields. Through the encapsulation of superparamagnetic nanoparticles, giving rise to the Magneto-Liposomes (MLs), it is possibile to modulate the transmembranal drug diffusion by using an external magnetic field with intensity significantly lower that no heat generation, harmful to healthy tissues, is observed. Another challenging way to activate release from liposomes is the use of pulsed electric fields. In particular, since it was demonstrated that electric pulses of shorter duration (nanosecond) and higher intensity (in the order of MV/m) directly interact not only with cell membrane, but also with internal cell organelles of nanometer dimensions, nanosecond electric pulses are proposed as sufficient signals to generate an alteration of the liposomal transmembrane voltage, which is followed by the formation of temporary hydrophilic pores. Without implying the phenomenon of irreversible poration, the nanosecond pulses therefore can be considered useful external stimuli to trigger the simultaneous permabilization of liposomes and cells membranes, in order to let that the chemical load internalized in the vesicles to be released inside the cells. In this scenario is placed the main activity of this Ph.D. thesis, whose aim is to provide a multiscale and multidisciplinary approach to demonstrate the capability of liposomes to prove effective smart systems for the on-demand and modified drug delivery, able to minimize off-target effects and maximize programmability of therapy. Following a briefly overview of this Ph.D. thesis is given. In Chapter 3 is reported the study which highlights the utility in trapping MNPs within phospholipid vesicles, generating hybrid magneto-responsive constructs. Particularly, in this work the inclusion of hydrophilic Fe3O4 nanoparticles (MNPs) within phospholipid vesicles, characterized by different rigidity and stiffness, was investigated as novel strategy for improving stability and reactivity of these MNPs, since the integration in liposomes may prevent MNPs from aggregation and extend their potential use in the environmental remediation. The stability of these hybrid systems was indirectly investigated evaluating the ability of retaining a fluorescent marker in their structure, under both mechanical and thermal stress conditions. In particular, for the mechanical stress test, a low intensity nonthermal alternating magnetic field (AMF) was applyed to magnetic liposomes. The AMF could, in fact, cause a mechanical destabilization of the vesicle membrane, due to MNPs oscillation within the liposomes, which may induce the release of the dye. In Chapter 4, according to the results obtained in the previous work, shown in Chapter 3, is presented the research project which combines engineering skills, specifically focused on electromagnetic fields, with competences in synthesis and characterization of hybrid magnetic nanocarriers, to assess a remotely on-demand drug delivery. Specifically, here is refiled the possibility to trigger drug release from high-transition temperature magnetoliposomes (high-Tm MLs) entrapping MNPs, through a magneto-nanomechanical approach, where the mechanical actuation of the MNPs is used to enhance the membrane permeability, avoiding temperature rise. Since the AMF, as an external magnetic signal, found rare application in clinic, in this case, the ability of the non-thermal pulsed electromagnetic fields (PEMFs), that are already employed in theraphy, due to their antiinflammatory effects, was tested, in order to verify if, once applied to high-Tm MLs, PEMFs could be able to efficiently trigger the transmembranal drug diffusion. In the Chapter 5 and 6 is illustrated another sophisticated and innovative drug delivery strategy to activate an efficient on-demand release. Specifically, in Chapter 5 is reported the theoretical work and the experimental proof-of-concept of the possibility of applying ultrashort (ns) and intense (MV/m) external pulsed electric fields (nsPEFs), to remotely trigger the release from liposomes of nanometer-sizing. The nanoelectropermeabilization, that probably occurs with the formation of transient pores in the bilayer, because of the external pulsed electric field enforcement, was evaluated in relation to the diffusion across the bilayer of a probe, previously trapped in the core of liposomes. To support the experimental data, a numerical model of liposomes suspension, exposed to nsPEF by means a standard electroporation cuvette, was carried out according with the experimental conditions. In Chapter 6 was demonstrated, once again, the possibility of permeabilizing the liposomal membrane, applying the same type of pulses, described in Chapter 5, but, in this case, delivered to the nanometer-sized lipid vesicles by a coplanar exposure system. Furthermore, the electropermeabilization mechanism in liposomes membrane was investigated through the Raman Coherent anti-Stokes spectroscopy (CARS), highlighting, for the first time, the experimental proof of the role of water molecules of the interstitial phase in the electropermeabilization of vesicles bilayer. Finally, in Chapter 7 is described the project who leaded to the development of a novel hybrid lipid-polymer nanocostructs, designed to merge the beneficial properties of both polymeric drug delivery systems and liposomes in a single nanocarrier and at the same time take care of liposomes limitations, such as the physical and chemical stability issues. Starting from previous studies on the use of liposomes as template to create nanohydrogel, this research brought to the novel Gel-in-Liposome (GiL) systems, relying from the combination of lipid vesicles and the polymer polyethylene glycol-dimethacrylate (PEG-DMA) at two different molecular weight. These hybrid systems are characterized by the presence of a chemically crosslinked polymeric network within the aqueous compartment of liposomes. The effect of PEG-DMA, on the properties of the new lipid-polymer nanosystems and on the related changes of the membrane permeability and stability, against different stresses, were evaluated to understand GiL potential use as drug carriers in clinics.