Abstract by Chenyang Wu

Considering the growing interest in developing cancer nanomedicines capable of improving the delivery of conventional chemotherapeutic agents, particular attention has been directed toward pH-sensitive nanomedicines. These nanomedicines are attractive to exploit the mildly acidic pH of the tumor microenvironment, typically around pH 5.5–6.5, with the aim of reducing off-target effects while enhancing therapeutic efficacy. Here, non-lamellar liquid crystalline (LLC) nanoparticles are especially promising owing to their unique internal structural architectures, the biocompatibility of their major lipid constituents, and their ability to accommodate therapeutic agents with diverse physicochemical properties. The most widely investigated LLC nanoparticles include cubosomes (nanoparticles with an internal inverse bicontinuous cubic (Q₂) phase; hexosomes (nanoparticles with an internal inverse hexagonal (H₂) phase); and micellar cubosomes (nanoparticles with an internal inverse discontinuous (micellar) cubic of the space group Fd3m). The overall aim of this project was therefore to investigate in depth how diolein positional isomerism, monounsaturated fatty acid headgroup chemistry, pH, and PEGylation modulate the internal structure, morphology, and size characteristics of LLC nanodispersions, as well as their biological performance. By linking molecular lipid structure to pH-triggered self-assembly and therapeutic function, the project seeks to advance the rational design of pH-responsive LLC nanoparticles as injectable nanoplatforms for improved cancer therapy.

The potent anticancer agent 2-hydroxyoleic acid (2OHOA, known as Minerval^®) is an ionizable amphiphile that can be dispersed in excess water to form vesicular structures at pH values above its apparent pKa (pK_a^app), which varies from 4.3 to 6.1 depending on lipid type and composition. However, the colloidal stability of such nano-self-assemblies generally requires the presence of a stabilizer. Therefore, these nanodispersions are typically stabilized with Pluronic F127, which, based on previous studies in the literature, has been associated with complement activation and cytotoxicity. These limitations motivated the development of stabilizer-free pH-responsive lamellar and LLC nanocarriers as structurally tunable platforms for the delivery of 2OHOA. To address this, the first study introduced a stabilizer-free family of lamellar and LLC nanoparticles produced from binary combinations of 2OHOA with diolein and its positional isomers, 1,2-diolein and 1,3-diolein. This study demonstrated that subtle geometric differences arising from diolein positional isomerism strongly influence intermolecular interactions and molecular packing at the lipid–water interface, thereby governing the internal self-assembled architectures of the produced nanodispersions. At pH 9.0, increasing the content of either 1,2-diolein or 1,3-diolein in 2OHOA-containing nanodispersions induced a lipid composition-dependent phase transition from a lamellar (L_α) phase to an inverse hexagonal (H₂) phase. This transition was consistent with the classical vesicle-fusion pathway, as supported by cryo-transmission electron microscopy (cryo-TEM) observations, which indicated coexistence of vesicles with hexosomes and intermediate colloidal nanoobjects most likely representing hexosome precursors. Furthermore, 1,2-diolein/2OHOA and 1,3-diolein/2OHOA nanodispersions exhibited pronounced pH-sensitive structural behavior driven by the tunable protonation state of the 2OHOA carboxylic group. For instance, 1,2-diolein- and 1,3-diolein-based nanodispersions produced at a diolein/2OHOA weight ratio of 3:2 displayed hexosomes under basic conditions. Increasing the pH from 8.0 to 10.0 led to a reversible increase in the lattice parameter of the internal H₂ phase. Lowering the pH to neutral and mildly acidic conditions induced significant structural alterations, including an H₂-to-cubic Fd3m phase transition. Overall, this study established diolein positional isomerism and pH as key molecular and environmental determinants of the structural, morphological, and size characteristics of 2OHOA nanocarriers.

Building on the first study, the second study focused on clarifying how monounsaturated fatty acid headgroup chemistry influences the structural and morphological features and size characteristics of pH-responsive LLC nanoparticles by replacing 2OHOA with its nonhydroxylated analogue, oleic acid (OA). The role of diolein positional isomerism was then further examined in the produced OA-containing nanodispersions. By combining OA with either 1,2-diolein or 1,3-diolein, another family of stabilizer-free LLC nanoparticles was produced. Under basic conditions, replacing 1,2-diolein with 1,3-diolein in OA-containing nanodispersions induced an internal H₂-to-cubic Fd3m phase transition, indicating that the combination of 1,3-diolein with OA favors internal nanostructures with more negative spontaneous interfacial curvature at pH values above 8.0. Furthermore, both 1,2-diolein/OA and 1,3-diolein/OA nanodispersions exhibited distinct pH-sensitive behavior in the absence and presence of Pluronic F127. For instance, in stabilizer-free nanodispersions, 1,2-diolein/OA nanodispersions underwent an H₂-to-cubic Fd3m phase transition upon decreasing the pH from 9.0 to 8.0, whereas the corresponding 1,3-diolein/OA nanodispersions retained an internal cubic Fd3m phase. In the presence of Pluronic F127, the OA-containing nanodispersions exhibited cubic Fd3m-to-L₂ phase transitions upon decreasing the pH from 8.0 to 7.6 for 1,2-diolein/OA nanodispersions and from 9.0 to 8.0 for 1,3-diolein/OA nanodispersions. These findings demonstrate that fatty acid headgroup chemistry, diolein positional isomerism, pH, and polymeric stabilization collectively govern the internal nanostructures of OA-containing LLC nanodispersions, likely through modulation of the pK_a^app of embedded OA molecules at the lipid–water interface. Thus, the second study extended the mechanistic framework established for 2OHOA-containing nanodispersions and showed that both headgroup chemistry and positional isomerism can be used as molecular design parameters to tune pH-responsive LLC nanostructures.

Despite the growing attractiveness of LLC nanoparticles in drug delivery, their systemic administration remains challenged by the need to control their surface characteristics. Among various surface-engineering strategies, PEGylation is widely used to prolong systemic circulation time and reduce nonspecific interactions with blood components, thereby improving in vivo performance. Motivated by this strategy, the third study translated the biophysical insights obtained from the stabilizer-free 2OHOA- and OA-containing nano-self-assemblies into surface-PEGylated LLC nanoparticles, namely micellar cubosomes, produced from a binary combination of diolein and OA for the intravenous delivery of the conventional chemotherapeutic agent methotrexate (MTX). The PEGylated diolein/OA nanoparticles were produced at a diolein/OA weight ratio of 3:2, incorporating 0.5 wt% D-α-tocopheryl succinate poly(ethylene glycol)₂₀₀₀ (TPGS-mPEG₂₀₀₀), followed by MTX encapsulation at a final concentration of 0.75 mg/mL. The MTX-loaded PEGylated micellar cubosomes exhibited pH-sensitive structural features, displaying a pH-induced cubic Fd3m-to-L₂ phase transition upon lowering the pH from 8.0 to 7.4 and 6.5. Hemolysis studies demonstrated the high hemocompatibility of the drug-free PEGylated diolein/OA nanoparticles at pH 8.0 and at concentrations up to 100 µg/mL. Furthermore, fluorescently labeled PEGylated diolein/OA nanoparticles exhibited time-dependent cellular uptake by CT26 cells. Compared with MTX controls, defined as aqueous MTX solutions prepared at pH 8.0 and at different MTX concentrations, the MTX-loaded PEGylated micellar cubosomes demonstrated enhanced in vitro cytotoxicity and improved in vivo antitumor efficacy after intravenous (I.V.) administration in a CT26 tumor model, without inducing body weight loss.

Taken together, the first and second studies clarify how subtle structural variations in amphiphilic lipids, especially diolein positional isomerism and monounsaturated fatty acid headgroup chemistry, play a decisive role in modulating the structural and morphological features and size characteristics of pH-responsive lamellar and LLC nanoparticles. These findings provide molecular-level design principles for developing stabilizer-free pH-responsive lipid nanocarriers with tunable internal architectures and potential drug delivery applications. The third study extends these biophysical insights into a biologically relevant setting by demonstrating how PEGylated micellar cubosomes can combine pH-sensitive structural behavior with hemocompatibility, cellular uptake, and improved antitumor efficacy. More broadly, the investigated PEGylated diolein/OA micellar cubosomal nanocarrier represents a promising injectable drug delivery platform for cancer therapy, including colorectal cancer, and highlights the broader potential of LLC nanoparticles for the rational design of structurally tunable and biologically active nanomedicines.