Cryo-TEM Reveals the Influence of Multivalent Charge and PEGylation on Shape Transitions in Fluid Lipid Assemblies: From Vesicles to Discs, Rods, and Spheres

Lipids, and cationic lipids in particular are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-assembly structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic transmission electron microscopy (cryo-TEM) allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol % MVL5. At a higher (75 mol %) content of MVL5, short- and intermediate-length worm-like micellar rods were observed, and in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles, and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on the assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications

The Influence of Multivalent Charge and PEGylation on Shape Transitions in Fluid Lipid Assemblies: From Vesicles to Discs, Rods, and Spheres

Lipids, and cationic lipids in particular, are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5 e ) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl- sn -glycero-phosphocholine (DOPC). This allowed us to elucidate lipid–liposome structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1 e ). Cryogenic TEM allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol% MVL5. At higher (75 mol%) content of MVL5, short and intermediate-length worm-like micellar rods were observed and, in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications

Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapes

Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs

Enhanced Loading of Paclitaxel in Cationic Liposomes by Replacement of Oleoyl with Linoleoyl Lipid Tails with Benefits in Cancer Therapeutics from In Vitro Studies

Lipid-based carriers of the hydrophobic drug paclitaxel (PTX) are used in clinical trials as next-generation agents for cancer chemotherapy. Improving the loading capacity of these carriers requires enhanced PTX solubilization. We compared the solubility of PTX in cationic liposomes (CLs) with lipid tails containing one (oleoyl; C18:1 Δ 9 ; DOTAP/DOPC) or two (linoleoyl; C18:2 Δ 9 ; DLinTAP/DLinPC) cis double bonds with newly synthesized cationic DLinTAP (2,3-dilinoleoyloxypropyltrimethylammonium methylsufate). We used differential-interference-contrast microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams representing the time-dependence of PTX solubility as a function of PTX content in the membrane. Replacing tails bearing one cis double bond (DO lipids) with those bearing two (DLin lipids) significantly increased PTX membrane solubility in CLs. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the membrane solubility limit which has been the focus of most previous fundamental studies and clinical trials) in DOTAP/DOPC CLs. The large increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX upon replacement of oleoyl by linoleoyl tails, rather than by the transition in membrane structure from lipid bilayers to inverse cylindrical micelles observed in small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against human prostate cancer (PC3) cells from measurements of the IC50 of PTX cytotoxicity was unaffected by changing the lipid tails, and toxicity of the CL carrier alone was negligible. Moreover, efficacy was approximately doubled against human melanoma (M21) cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. The findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading well over the typically used 3 mol% while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs

Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics

Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure–activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL–NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL–NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases

PEGylation of Paclitaxel-Loaded Cationic Liposomes Drives Steric Stabilization of Bicelles and Vesicles thereby Enhancing Delivery and Cytotoxicity to Human Cancer Cells

Poly(ethylene glycol) (PEG) is a polymer used widely in drug delivery to create "stealth" nanoparticles (NPs); PEG coatings suppress NP detection and clearance by the immune system and beneficially increase NP circulation time in vivo. However, NP PEGylation typically obstructs cell attachment and uptake in vitro compared to the uncoated equivalent. Here, we report on a cationic liposome (CL) NP system loaded with the hydrophobic cancer drug paclitaxel (PTX) in which PEGylation (i.e., PEG-CLPTX NPs) unexpectedly enhances, rather than diminishes, delivery efficacy and cytotoxicity to human cancer cells. This highly unexpected enhancement occurs even when the PEG-chains coating the NP are in the transition regime between the mushroom and brush conformations. Cryogenic transmission electron microscopy (TEM) of PEG-CLPTX NPs shows that PEG causes the proliferation of a mixture of sterically stabilized nanometer-scale vesicles and anisotropic micelles (e.g., bicelles). Remarkably, the onset of bicelles at sub-monolayer concentrations of the PEG coat has to our knowledge not been previously reported; it was previously thought that PEG-lipid in this composition regime was incorporated into vesicles but did not alter their shape. Confocal microscopy and flow cytometry reveal significantly greater PTX cell uptake from stabilized PEG-CLPTX NPs (vesicles and bicelles) in contrast to bare CLPTX NPs, which can aggregate in cell medium. This underscores the ability of steric stabilization to facilitate NP entry into cells via distinct size-dependent endocytic pathways, some of which cannot transport large NP aggregates into cells. This study highlights the value of understanding how PEGylation alters NP shape and structure, and thus NP efficacy, to design next-generation stealth drug carriers that integrate active cell-targeting strategies into NPs for in vivo delivery

Paclitaxel-loaded Cationic Fluid Lipid Nanodiscs and Liposomes with Brush-Conformation PEG Chains Penetrate Breast Tumors and Trigger Caspase-3 Activation

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1®) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol% PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol% of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol% PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol% PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol% (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.This research study was supported by the National Institutes of Health under award R01GM130769 (CRS, KKE, WF; mechanistic studies on developing lipid nanoparticles for drug delivery), the European Regional Development Fund (TT, Project No. 2014-2020.4.01.15-0012), the Estonian Research Council (TT, grants PRG230 and EAG79; PS, grant PSG38; LSG, grant MOBJD11), EuronanomedII projects ECM-CART and iNanoGun (TT), H2020 MSCA-RISE project Oxigenated (TT), and the Spanish Ministry of Science and Innovation grants RYC2020-028754-I and PID2021-122364OA-I00 (PS). Partial support was provided by the US National Science Foundation (NSF) under Award DMR-1807327 (CRS; kinetic phase behavior of cationic vesicles with incorporated hydrophobic molecules).N

Monoliths of Semiconducting Block Copolymers by Magnetic Alignment

Achieving highly ordered and aligned assemblies of organic semiconductors is a persistent challenge for improving the performance of organic electronics. This is an acute problem in macromolecular systems where slow kinetics and long-range disorder prevail, thus making the fabrication of high-performance large-area semiconducting polymer films a nontrivial venture. Here, we demonstrate that the anisotropic nature of semiconducting chromophores can be effectively leveraged to yield hierarchically ordered materials that can be readily macroscopically aligned. An n-type mesogen was synthesized based on a perylene diimide (PDI) rigid core coupled to an imidazole headgroup <i>via</i> an alkyl spacer. Supramolecular assembly between the imidazole and acrylic acid units on a poly(styrene-<i>b</i>-acrylic acid) block copolymer yielded self-assembled hexagonally ordered polystyrene cylinders within a smectic A mesophase of the PDI mesogen and poly(acrylic acid). We show that magnetic fields can be used to control the alignment of the PDI species and the block copolymer superstructure concurrently in a facile manner during cooling from a high-temperature disordered state. The resulting materials are monoliths, with a single well-defined orientation of the semiconducting chromophore and block copolymer microdomains throughout the sample. This synergistic introduction of both functional properties and the means of controlling alignment by supramolecular attachment of mesogenic species to polymer backbones offer new possibilities for the modular design of functional nanostructured materials

Claimed Detection of PH3 in the Clouds of Venus Is Consistent with Mesospheric SO2

Abstract The observation of a 266.94 GHz feature in the Venus spectrum has been attributed to phosphine (PH3) in the Venus clouds, suggesting unexpected geological, chemical, or even biological processes. Since both PH3 and sulfur dioxide (SO2) are spectrally active near 266.94 GHz, the contribution to this line from SO2 must be determined before it can be attributed, in whole or part, to PH3. An undetected SO2 reference line, interpreted as an unexpectedly low SO2 abundance, suggested that the 266.94 GHz feature could be attributed primarily to PH3. However, the low SO2 and the inference that PH3 was in the cloud deck posed an apparent contradiction. Here we use a radiative transfer model to analyze the PH3 discovery, and explore the detectability of different vertical distributions of PH3 and SO2. We find that the 266.94 GHz line does not originate in the clouds, but above 80 km in the Venus mesosphere. This level of line formation is inconsistent with chemical modeling that assumes generation of PH3 in the Venus clouds. Given the extremely short chemical lifetime of PH3 in the Venus mesosphere, an implausibly high source flux would be needed to maintain the observed value of 20 ± 10 ppb. We find that typical Venus SO2 vertical distributions and abundances fit the JCMT 266.94 GHz feature, and the resulting SO2 reference line at 267.54 GHz would have remained undetectable in the ALMA data due to line dilution. We conclude that nominal mesospheric SO2 is a more plausible explanation for the JCMT and ALMA data than PH3

Assembly of Building Blocks by Double-End-Anchored Polymers in the Dilute Regime Mediated by Hydrophobic Interactions at Controlled Distances

Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes