Zexi Xu

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Delivery of chemotherapy drugs specifically to cancer cells raises local drug doses in tumors and therefore kills more cancer cells while reducing side effects in other tissues, thereby improving oncological and quality of life outcomes. Cubosomes, liquid crystalline lipid nanoparticles, are potential vehicles for delivery of chemotherapy drugs, presenting the advantages of biocompatibility, stable encapsulation, and high drug loading of hydrophobic or hydrophilic drugs. However, active targeting of drug-loaded cubosomes to cancer cells, as opposed to passive accumulation, remains relatively underexplored. We formulated and characterized cubosomes loaded with potential cancer drug copper acetylacetonate and functionalized their surfaces using click chemistry coupling with hyaluronic acid (HA), the ligand for the cell surface receptor CD44. CD44 is overexpressed in many cancer types including breast and colorectal. HA-tagged, copper-acetylacetonate-loaded cubosomes have an average hydrodynamic diameter of 152 nm, with an internal nanostructure based on the space group Im3m. These cubosomes were efficiently taken up by two CD44-expressing cancer cell lines (MDA-MB-231 and HT29, representing breast and colon cancer) but not by two CD44-negative cell lines (MCF-7 breast cancer and HEK-293 kidney cells). HA-tagged cubosomes caused significantly more cell death than untargeted cubosomes in the CD44-positive cells, demonstrating the value of the targeting. CD44-negative cells were equally relatively resistant to both, demonstrating the specificity of the targeting. Cell death was characterized as apoptotic. Specific targeting and cell death were evident in both 2D culture and 3D spheroids. We conclude that HA-tagged, copper-acetylacetonate-loaded cubosomes show great potential as an effective therapeutic for selective targeting of CD44-expressing tumors.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5–7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

There is a growing demand to develop smart nanomaterials that are structure-responsive as they have the potential to offer enhanced dose, temporal and spatial control of compounds and chemical processes. The naturally occurring pH gradients found throughout the body make pH an attractive stimulus for guiding the response of a nanocarrier to specific locations or (sub)cellular compartments in the body. Here we have engineered highly sensitive lyotropic liquid crystalline nanoparticles that reversibly respond to changes in pH by altering the connectivity within their structure at physiological temperatures. At pH 7.4, the nanoparticles have an internal structure consisting of discontinuous inverse micellar “aqueous pockets” based on space group Fd3m. When the pH is ≤6, the nanoparticles change from a compartmentalized to an accessible porous internal structure based on a 2D inverse hexagonal phase (plane group p6mm). We validate the internal symmetry of the nanoparticles using small-angle X-ray scattering and cryogenic transmission electron microscopy. The high-resolution electron microscopy images obtained have allowed us for the first time to directly visualize the internal structure of the Fd3m nanoparticles and resolve the two different-sized inverse micelles that make up the structural motif within the Fd3m unit cell, which upon structural analysis reveal excellent agreement with theoretical geometrical models.