PEGylation of Phosphatidylglycerol/Docosahexaenoic Acid Hexosomes with d-α-Tocopheryl Succinate Poly(ethylene glycol)₂₀₀₀ Induces Morphological Transformation into Vesicles with Prolonged Circulation Times

Abstract

Considering the broad therapeutic potential of omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), here we study the effect of PEGylation of DHAincorporated hexosomes on their physicochemical characteristics and biodistribution following intravenous injection into mice. Hexosomes were formed from phosphatidylglycerol and DHA with a weight ratio of 3:2. PEGylation was achieved through the incorporation of either D-α-tocopheryl succinate poly(ethylene glycol) 2000 (TPGS-mPEG 2000 ) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol) 2000 (DSPE-mPEG 2000 ) at a concentration of 1.5 wt %. Nanoparticle tracking analysis, synchrotron small-angle scattering, and cryo-transmission electron microscopy were employed to characterize the nanodispersions. The results show that PEGylated lipids induce a structural transition from an inverse hexagonal (H 2 ) phase inside the nanoparticles (hexosomes) to a lamellar (L α ) phase (vesicles). We also followed the effect of mouse plasma on the nanodispersion size distribution, number, and morphology because changes brought by plasma constituents could regulate the in vivo performance of intravenously injected nanodispersions. For comparative biodistribution studies, fluorescently labeled nanodispersions of equivalent quantum yields were injected intravenously into healthy mice. TPGS-mPEG 2000 -induced vesicles were most effective in avoiding hepatosplenic clearance at early time points. In an orthotopic xenograft murine model of glioblastoma, TPGS-mPEG 2000 -induced vesicles also showed improved localization to the brain compared with native hexosomes. We discuss these observations and their implications for the future design of injectable lyotropic nonlamellar liquid crystalline drug delivery nanosystems for therapeutic interventions of brain and liver diseases.

Intrinsic and Dynamic Heterogeneity of Nonlamellar Lyotropic Liquid Crystalline Nanodispersions

“…LLC nanodispersion multiformity in size, shape, and thermodynamically driven inner structure can be modulated by the physicochemical characteristics of their lipid and stabilizer components and concentrations. − ,,,, Notwithstanding, LLC nanoparticles often coexist with vesicles and/or micelles (Figure and Figure ). ,,,, For instance, PEGylated stabilizers in a concentration-dependent manner, depending on PEG chain length, form normal micelles and stabilize vesicles in excess water, and this might explain the typical coexistence of these structures in LLC nanodispersions (Figure a and Figure b). ,,, Such coexistence (particularly vesicles), and the tendency of vesicular structures to adhere to the outer surfaces of the dispersed LLC nanoparticles, apparently contribute to their colloidal stabilization in excess water (Figure , Figure , and Figure ). In some cases, more complex morphologies have been observed; for instance, LLC nanoparticles coexist with a relatively large fraction of distinct vesicular-like structures as well as with elongated and cylindrically shaped vesicles, including a fraction adhered to LLC nanoparticles (Figure a,b and Figure a,b). ,, In another example, combinations of binary lipid mixtures (e.g., glycerol monooleate and vitamin E) and the stabilizer d -α-tocopheryl succinate mPEG 2000 (TPGS-mPEG 2000 ) have generated more complex architectures such as micellar cubosomes but are covered by lamellar petals (i.e., flower-like tubular vesicular structures as shown in Figure a,b) …”

“…Thus, such approaches and through further refinements in microfluidic manufacturing technologies, enabling high precision liquid handling, an efficiency in microscale mixing and a control of timing and flow conditions might resolve separation of typically coexisting small vesicles and micelles of different sizes (in the range 10–50 nm) and shapes from LLC nanodispersions. This would be particularly useful, given the potential tendency of nonionic mPEG-lipids, to display normal micelles and stabilize vesicles in excess buffer , and the expected relatively high percentage of vesicles in LLC nanodispersions produced from phospholipids. ,, Finally, coupling X-ray compatible microfluidic platforms to synchrotron SAXS/wide-angle X-ray scattering (WAXS) might allow multivariate analysis on nanoparticle formation kinetics and mapping structural transition pathways . Currently, limited work has been devoted toward designing X-ray compatible microfluidic platforms for real-time monitoring of dynamic structural events during microfluidic synthesis of LPNs. , Also, 2D X-ray compatible HFF microfluidic platform has been employed for online SAXS characterization studies of liposomes during their microfluidic synthesis reporting on fast development of multilamellar vesicles within fractions of seconds .…”

“…For example, using citrem as stabilizer minimizes, and depending on the lipid composition of the LLC nanodispersion totally overcomes, nanoparticle-mediated complement activation in the blood. , Finally, a wide range of biophysical modalities is available for the physiochemical characterization of LLC nanoparticles including size, morphology, and inner architectural arrangements (Table ). − , …”

“…In the absence of any LLC nanoparticles, the ULVs (black arrowheads) and BLVs (blue arrowheads) in the PEGylated 3:2 DOPG/DHA nanodispersion were still observed after 30 min incubation in mouse plasma (d) and their sizes were in the range 10–200 nm. Reprinted with permission from ref . Copyright 2022, American Chemical Society.…”

“…To further realize the pharmaceutical potential of LLC nanodispersions, numerous efforts are being directed to broaden characteristics of LLC nanoparticles and improving their production. 3−5 Thus, most recent notable developments include (i) introduction of simpleby-design and "green" approaches for LLC nanoparticle preparation in absence of any stabilizer or organic solvent, 7,10,19,20 (ii) microfluidic methodologies for LLC nanoparticle assembly, 21 and (iii) engineering of pH-responsive LLC nanodispersions. 7,20,22,23 While these advances are essential in thriving the development of LLC nanopharmaceuticals, evidence from integrated synchrotron small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and nanoparticle tracking analysis (NTA) has revealed considerable size, morphology, and inner structural heterogeneity in LLC nanodispersions.…”