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Pluronic F127-stabilized non-lamellar liquid crystalline aqueous nanodispersions are promising injectable platforms for drug and contrast agent delivery. These nanodispersions, however, trigger complement activation in the human blood, where the extent of complement activation and opsonization processes may compromise their biological performance and safety. Here, we introduce a broad family of nanodispersions from glycerol monooleate (GMO) and oleic acid (OA) in different weight ratios, and stabilized with a plethora of nonionic methoxypoly(ethylene glycol) (mPEG)-lipids of different PEG chain length and variable lipid moiety (monounsaturated or saturated diglycerides or D-α-tocopheryl succinate). Through an integrated biophysical approach involving dynamic light scattering, synchrotron small-angle scattering, and cryo-transmission electron microscopy, we examine the impact of nonionic mPEG-lipid stabilization on size, internal self-assembled architecture, and gross morphological characteristics of nanodispersions. The results show how the nonionic mPEG-lipid type and concentration, and dependent on GMO/OA weight ratio, can variably modulate the internal architectures of nanoparticles. Assessment of complement profiling from selected nanodispersions with diverse structural heterogeneity further suggests a variable modulatory role for the lipid type of the nonionic mPEG-lipid in the extent of complement activation, which span from no activation to moderate to high levels. We comment on plausible mechanisms driving the observed complement activation variability and discuss the potential utility of these nanodispersions for future development of injectable nanopharmaceuticals.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (Lα) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H2) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development.

Microfluidic platforms have become highly attractive tools for synthesis of nanoparticles, including lipid nano-self-assemblies, owing to unique features and at least three important aspects inherent to miniaturized micro-devices. Firstly, the fluids flow under controlled conditions in the microchannels, providing well-defined flow profiles and shorter diffusion lengths that play important roles in enhancing the continuous production of lipid and polymer nanoparticles with relatively narrow size distributions. Secondly, various geometries adapted to microfluidic device designs can be utilized for enhancing the colloidal stability of nanoparticles and improving their drug loading. Thirdly, microfluidic devices are usually compatible with in situ characterization methods for real-time monitoring of processes occurring inside the microchannels. This is unlike conventional nanoparticle synthesis methods, where a final solution or withdrawn aliquots are separately analysed. These features inherent to microfluidic devices provide a tool-set allowing not only precise nanoparticle size control, but also real-time analyses for process optimization. In this review, we focus on recent advances and developments in the use of microfluidic devices for synthesis of lipid nanoparticles. We present different designs based on hydrodynamic flow focusing, droplet-based methods and controlled microvortices, and discuss integration of microfluidic platforms with synchrotron small-angle X ray scattering (SAXS) for in situ structural characterization of lipid nano-self-assemblies under continuous flow conditions, along with major challenges and future directions in this research area., INP grant from the Danish Agency for Science and Higher Education [8073-00022B], AY and OYC acknowledge the INP grant (Ref. no.: 8073-00022B) from the Danish Agency for Science and Higher Education for initiating and exploring new networking and collaboration opportunities between Danish and Turkish research institutions.