Abstract by Gizem Bor

The last three decades have witnessed an explosive growth in research focused on developing cancer nanomedicines. These nanomedicines are not only attractive due to their ability to efficiently transport loaded therapeutic cargos, but also have the potential to bypass biological barriers such as the blood–brain barrier (BBB). In particular, there has recently been growing interest in the development of nanomedicines for the treatment of brain cancers, which are considered as lethal and life-threatening diseases.

In addition to liposomes, non-lamellar lyotropic liquid crystalline (LLC) nanoparticles (NPs) based on biologically relevant amphiphilic lipids have received particular attention due to their biocompatibility, ability to load therapeutic agents and imaging probes and potential use for targeting brain tumors. These NPs, known in the literature as ISAsomes (internally self-assembled somes), have unique well-defined inner architectural arrangements that allow encapsulation of amphiphilic, hydrophilic, and poorly water-soluble drugs. Despite their attractiveness, the number of studies on in vitro and in vivo evaluation is still limited. In particular, their in vivo behaviour after administration to model animals and involved mechanisms, remain poorly understood.

We aimed in this PhD project at introducing a family of stabilizer-free lamellar and non-lamellar LLC nano-self-assemblies containing omega-3 polyunsaturated fatty acids (ω-3 PUFAs), which may find potential applications as injectable nanocarriers for delivery of ω-3 PUFAs alone or in combination with therapeutic agents. The structural features of these nano-self-assemblies, as well as their overall morphology and hydrodynamic size characteristics, were thoroughly investigated by following a pan-integrated approach that involves synchrotron small-angle scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and nanoparticle tracking analysis (NTA). These biophysical investigations were further combined with relevant in vitro and in vivo studies. It was also our interest to gain further insight into the dynamic biophysical alterations that occur on exposure of these nano-self-assemblies to biologically relevant fluids (e.g., blood serum/plasma or cell media), and may modulate their cellular responses in patient-derived xenograft glioblastoma (GBM T10) and human monocytic (THP-1) cell lines. Such alterations may also affect the behaviour of these nano-self-assemblies after administration. Here, we aimed at gaining important information on their fate after intravenous (I.V.) administration to healthy and xenograft GBM tumor-bearing mice models. 

The first report focuses on a simple-by-design approach for the production of a stabilizer-free library of lamellar and non-lamellar LLC NPs at different lipid compositions. In the absence of any organic solvent, these nano-self-assemblies were produced by means of ultrasonication through emulsification of binary mixtures of the negatively charged phospholipid, phosphatidylglycerol (DOPG), and three ω-3 PUFAs. These fatty acids are docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA). We showed for the first time, to our knowledge, how the inclusion of ω-3 PUFAs to liposomes in a concentration- and pH-dependent manners led to direct liposome-hexosome and liposome-micellar cubosome colloidal transformations: structural transitions in the NPs’ interiors from a lamellar (L_α) phase to inverse hexagonal (H₂) and discontinuous (micellar) cubic Fd3m phases, respectively. We also reported on the effect of pH on the structural features of these nanodispersions and suggested possible pathways for the detected lamellar-non-lamellar phase transitions. Taking into account the growing interest in the development of pH-responsive drug nanocarriers, as well as the therapeutic benefits of ω-3 PUFAs, this new structurally tunable, colloidally stable, and pH-responsive family of lamellar and non-lamellar LLC NPs, comprising vesicles, hexosomes, and micellar cubosomes, are attractive for the delivery of ω-3 PUFAs alone or in combination with other therapeutic agents.

As alterations in the integrity of internal nanostructure, morphology, and size characteristics of the produced nano-self-assemblies may modulate their cellular uptake behaviour, intracellular responses, and overall therapeutic efficacy, it was also our interest to conduct a set of biophysical investigations prior to the performed in vitro studies. We aimed at evaluating the time-dependent effects of two relevant cell incubation media on a selected DOPG/DHA (3:2) nanodispersion (hexosomes at pH 6.0). Here, we showed dynamic alterations in NP size distribution, number, and morphology in both cell media. Using GBM T10 and THP-1 cell lines, we further discussed how these dynamic biophysical transformations can affect the kinetics of NP-cell interactions and internalization mechanisms of these nano-self-assemblies. Our findings indicate the complexity of cellular uptake data interpretation, arising from pH- and cell medium-dependent dynamic fluctuations in biophysical characteristics (including NP size distribution, number, and morphology) of LLC NPs.

Considering the potential modulatory role of DHA in the prevention and inhibition of brain cancer, and for evaluating the suitability of the produced NPs as injectable nanocarriers in the development of brain cancer nanomedicinesto gain insight into the in vivo biodistribution and behaviour of a few selected nanodispersions. Here, three non-PEGylated (hexosomes) and PEGylated 3:2 DOPG/DHA nanodispersions were tested. PEGylation was achieved through inclusion of DSPE-mPEG₂₀₀₀ (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol)₂₀₀₀), which is typically used in liposome preparations, or TPGS-mPEG₂₀₀₀ (D-a-tocopheryl succinate poly(ethylene glycol)₂₀₀₀). The latter was used as an immune-safe mPEG-lipid as recently reported. It was also our interest to report for first time, to our knowledge, on the influence of direct PEGylation-triggered hexosome-liposome colloidal transformations on the biodistribution of these nano-self-assemblies after I.V. administration to healthy and xenograft GBM tumor-bearing mice models.

In the second report, we used an in vivo imaging system (IVIS), to evaluate the in vivo behaviour of a selected set of naked (non-PEGylated) and PEGylated nanodispersions after I.V. administration to both healthy and xenograft GBM tumor-bearing mice models. We showed that the detected PEGylation-triggered colloidal hexosome-liposome transformations are associated with significant changes in the in vivo biodistribution and blood clearance profiles of the intravenously injected NPs. The results also shed light on pH- and plasma-dependent dynamic alterations in NP size, nanostructural features, and morphology. These dynamic changes may play an important role in dictating the in vivo behaviour of these nano-self-assemblies after administration.

In summary, our findings provide a fundamental understanding and guidance for the development of stabilizer-free ω-3 PUFA nanocarriers. Clearly, the in vitro and in vivo behaviour of this new family of nano-self-assemblies may have implications for the future design of multifunctional and pH-responsive nanocarriers for co-delivering of ω-3 PUFAs with drugs. Through an in-depth understanding of the interactions of these nano-self-assemblies with biological fluids, these investigations may also contribute to the identification of the necessary attributes for the successful pharmaceutical development of ISAsomes as injectable nanocarriers.