Bis-Boronic Acid Liposomes for Carbohydrate Recognition and Cellular Delivery.

Liposomes are effective therapeutic nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of drugs and diagnostic agents. A primary area in which improvement is needed for liposomal drug delivery is to maximize the delivery of these nanocarriers to cells. Cell membrane glycans provide exciting targets for liposomal delivery since they are often densely clustered on cell membranes and glycan overabundance and aberrant glycosylation patterns are a common feature of diseased cells. Herein, we report a liposome platform incorporating bis-boronic acid lipids (BBALs) to increase valency in order to achieve selective saccharide sensing and enhance cell surface recognition based on carbohydrate binding interactions. In order to vary properties, multiple BBALs (1 a-d) with variable linkers in between the binding units were designed and synthesized. Fluorescence-based microplate screening of carbohydrate binding showed that these compounds exhibit varying binding properties depending on their structures. Additionally, fluorescence microscopy experiments indicated enhancements in cellular association when BBALs were incorporated within liposomes. These results demonstrate that multivalent BBALs serve as an exciting glycan binding liposome system for targeted delivery.

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics.

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Cell mimetic liposomal nanocarriers for tailored delivery of vascular therapeutics.

Liposomal delivery systems (LDSs) have been at the forefront of medicinal nanotechnology for over three decades. Increasing LDS association to target cells and cargo delivery is crucial to bolstering overall nanodrug efficacy. Our laboratory aims to develop LDSs for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNA interference in vascular cell types by using polyethylene glycol (PEG) decorated liposomes bearing an octa-arginine (R8) cell penetrating peptide (CPP). Further tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increasing cargo delivery. Here we aimed to develop liposomal formulations that could make use of diacylglycerol (DAG) and phosphatidylserine (PS), naturally occurring lipid species that are known to influence vascular cell function, as a facile and efficient means to increase nanodrug efficacy without compromising clinical viability. We investigated the ability of DAG and PS to amplify the cellular uptake of our previously established LDS platform loaded with small interfering ribonucleic acid (siRNA) cargo. Cellular fluorescence microscopy experiments were performed in conjunction with quantitative cell association assays and cytotoxicity assays to analyze the effect of DAG/PS on the differential delivery of fluorescently-tagged liposomes to vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) and on liposomal-mediated toxicity. In these studies, significant, dose-dependent increases in association to target cells were observed, as well as cell-type specific effects on cell viability. The stability and encapsulation-efficiency of the DAG/PS-modified LDSs were analyzed by standard nanoparticle characterization methods, and siRNA transfection efficacy was quantified to gauge delivery potential as a function of DAG/PS modification. Our results suggest that the signaling lipids tested here imbue our LDS architectures with increased therapeutic potential, without compromising stability, encapsulation efficiency, or biocompatibility, thus presenting a natural strategy to increase nanodrug efficacy and specificity.

Calcium-Responsive Liposomes via a Synthetic Lipid Switch.

Liposomal drug delivery would benefit from enhanced control over content release. Here, we report a novel avenue for triggering release driven by chemical composition using liposomes sensitized to calcium-a target chosen due to its key roles in biology and disease. To demonstrate this principle, we synthesized calcium-responsive lipid switch 1, designed to undergo conformational changes upon calcium binding. The conformational change perturbs membrane integrity, thereby promoting cargo release. This was shown through fluorescence-based release assays via dose-dependent response depending on the percentage of 1 in liposomes, with minimal background leakage in controls. DLS experiments indicated dramatic changes in particle size upon treatment of liposomes containing 1 with calcium. In a comparison of ten naturally occurring metal cations, calcium provided the greatest release. Finally, STEM images showed significant changes in liposome morphology upon treatment of liposomes containing 1 with calcium. These results showcase lipid switches driven by molecular recognition principles as an exciting avenue for controlling membrane properties.

Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications.

BACKGROUND: We have previously defined mechanisms of intimal hyperplasia that could be targets for molecular therapeutics aimed at vascular pathology. However, biocompatible nanocarriers are needed for effective delivery. Cationic liposomes (CLPs) have been demonstrated as effective nanocarriers in vitro. However, in vivo success has been hampered by cytotoxicity. Recently, neutral PEGylated liposomes (PLPs) have been modified with cell-penetrating peptides (CPPs) to enhance cellular uptake. We aim to establish CPP-modified neutral liposomes as viable molecular nanocarriers in vascular smooth muscle cells. METHODS: CLPs, PLPs, and CPP-modified PLPs (R8-PLPs) were assembled with short interfering RNA (siRNA) via ethanol injection. Characterization studies determined liposomal morphology, size, and charge. siRNA encapsulation efficiency was measured via RiboGreen assay. Vascular smooth muscle cells were exposed to equal lipid/siRNA across all groups. Rhodamine-labeled liposomes were used to quantify cell association via fluorometry, live/dead dual stain was used to measure cytotoxicity, and gene silencing was measured by quantitative polymerase chain reaction. RESULTS: R8-PLPs exhibited increased encapsulation efficiency equivalent to CLPs. PLPs and R8-PLP-5 mol% and R8-PLP-10 mol% had no cytotoxic effect. CLPs demonstrated significant cytotoxicity. R8-PLP-5 mol% and R8-PLP-10 mol% exhibited increased cell association versus PLPs. R8-PLP-10 mol% resulted in significant gene silencing, in a manner dependent on lipid-to-siRNA load capacity. CONCLUSIONS: The negligible cytotoxicity and enhanced cellular association and gene silencing capacity exhibited by R8-PLPs reveal this class of liposomes as a candidate for future applications. Further modifications for optimizing R8-PLPs are still warranted to improve efficacy, and in vivo studies are needed for translational development. However, this could prove to be an optimal nanocarrier for vascular gene therapeutics.

Artificial Membrane Fusion Triggered by Strain-Promoted Alkyne-Azide Cycloaddition.

Artificial systems for controlled membrane fusion applicable for drug delivery would ideally use triggers that are orthogonal to biology. To apply the strain-promoted alkyne-azide cycloaddition (SPAAC) to drive membrane fusion, oxo-dibenzocyclooctyne (ODIBO)-lipid 1 was designed, synthesized, and studied alongside azadibenzocyclooctyne (ADIBO)-lipids 2-4 to assess fusion with liposomes containing azido-lipid 5. Lipids 1-2 were first shown to be effective for liposome derivatization. Next, fusion was evaluated using liposomes containing 1 and varying ratios of PC and PE via a FRET dilution fusion assay, and a 1:1 PC-to-PE ratio yielded the greatest signal change attributed to fusion. Finally, lipids 1-4 were compared, and 1 yielded the greatest triggering of fusion, while 2-4 yielded varying efficacies depending on the structural features of each lipid. Fusion was further validated through STEM studies showing larger multilamellar assemblies after liposome mixing, and FRET assay results supporting the mixing of liposome aqueous contents. This work provides a platform for triggered fusion toward drug delivery applications and an understanding of the effects of lipid structure and membrane composition on fusion.

Triggered liposomal release through a synthetic phosphatidylcholine analogue bearing a photocleavable moiety embedded within the sn-2 acyl chain.

Liposomes represent promising carriers for drug delivery applications. To maximize this potential, there has been significant interest in developing liposomal systems encapsulating molecular cargo that are highly stable until their contents are released remotely in a controlled manner. Herein, we describe the design, synthesis, and analysis of a photocleavable analogue of the ubiquitous lipid phosphoatidylcholine (PC) for the development of highly stable and controllable photodisruptable membranes. Our strategy was to develop a lipid that closely mimics the structure of PC to optimize favorable properties including biocompatibility and stability of subsequent liposomes when mixed with lipids possessing a broad range of physicochemical properties. Thus, NB-PC was designed, which contains a photocleavable 2-nitrobenzyl group embedded within the acyl chain at the sn-2 position. Following the synthesis of NB-PC, liposome disruption efficacy was evaluated through photolysis studies involving the detection of nile red release. Studies performed using a range of liposomes with different percentages of NB-PC, PC, phosphatidylethanolamine (PE), cholesterol, and polyethylene glycol-PE (PEG-PE) demonstrated minimal background release in controls, release efficacies that correlate directly with the amount of NB-PC incorporation, and that release is only minimally impacted by the inclusion of the lipids PE and cholesterol that possess disparate properties. These results demonstrate that the NB-PC system is a highly stable, flexible, and tunable system for photoinitiated release from liposomal systems.