Shape transformations of lipid vesicles by insertion of bulky-head lipids

Lipid vesicles, in particular Giant Unilamellar Vesicles (GUVs), have been increasingly important as compartments of artificial cells to reconstruct living cell-like systems in a bottom-up fashion. Here, we report shape transformations of lipid vesicles induced by polyethylene glycol-lipid conjugate (PEG lipids). Statistical analysis of deformed vesicle shapes revealed that shapes vesicles tend to deform into depended on the concentration of the PEG lipids. When compared with theoretically simulated vesicle shapes, those shapes were found to be more energetically favorable, with lower membrane bending energies than other shapes. This result suggests that the vesicle shape transformations can be controlled by externally added membrane molecules, which can serve as a potential method to control the replications of artificial cells

Osmotic properties of polyethyleneglycols: quantitative features of brush and bulk scaling laws

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander - de Gennes theory of polymer brushes successfully describes the intermembrane distance vs. applied osmotic stress data of Kenworthy et al. for PEG-grafted multilamellar liposomes [Biophys. J. (1995) 68:1921]. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semi-dilute regime of bulk polymer solutions). The condition that attached polymers behave as semi-dilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander - de Gennes brush scaling laws yield effective monomer lengths of 3.3 to 3.5 AA, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal content of PEG-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.Comment: 26 pages with figure

Development of a novel exosome-based platform for anticancer drug delivery

In this project, exosomes surface has been decorated with folate end-tipped PEG-lipids to actively redirect the homing capacity of such vehicles towards folate receptor expressing cells. Paclitaxel was loaded on exosomes colloidal systems to exert drug delivery to prostate cancer

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

AbstractIn order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG2000-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000). Transfer of DSPE-PEG2000 to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG2000 was transferred efficiently even at 4°C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG2000 to become evenly distributed between the two liposome populations (TEQ) at 4°C and 37°C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue

Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids

AbstractLiposomes containing the acidic phospholipid phosphatidylserine (PS) have been shown to avidly interact with proteins involved in blood coagulation and complement activation. Membranes with PS were therefore used to assess the shielding properties of poly(ethylene glycol 2000)-derivatized phosphatidylethanolamine (PE-PEG2000) with various acyl chain lengths on membranes containing reactive lipids. The desorption of PE-PEG2000 from PS containing liposomes was studied using an in vitro assay which involved the transfer of PE-PEG2000 into multilamellar vesicles, and the reactivity of PS containing liposomes was monitored by quantifying interactions with blood coagulation proteins. The percent inhibition of clotting activity of PS liposomes was dependent on the PE-PEG2000 content. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 which transferred out slowly from PS liposomes was able to abolish >80% of clotting activity of PS liposomes at 15 mol%. This level of DSPE-PEG2000 was also able to extend the mean residence time of PS liposomes from 0.2 h to 14 h. However, PE-PEG2000 with shorter acyl chains such as 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-PEG2000 were rapidly transferred out from PS liposomes, which resulted in a 73% decrease in clotting activity inhibition and 45% of administered intravenously liposomes were removed from the blood within 15 min after injection. Thus, PS facilitates the desorption of PE-PEG2000 from PS containing liposomes, thereby providing additional control of PEG release rates from membrane surfaces. These results suggest that membrane reactivity can be selectively regulated by surface grafted PEGs coupled to phosphatidylethanolamine of an appropriate acyl chain length

Neutron reflectivity of supported membranes incorporating terminally anchored polymers: Protrusions vs. blisters

The effect of terminally anchored chains on the structure of lipid bilayers adsorbed at the solid/water interface was characterized by neutron reflectivity. In the studied system, the inner leaflet, closer to the substrate, consisted of head-deuterated 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and the outer leaflet comprised a mixture of DSPC and polyethylene glycol (PEG) functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. The DSPC headgroups were deuterated to enhance sensitivity and demarcate the bilayer/water interface. The effect on the inner and outer headgroup layers was characterized by w 1/2, the width at half-height of the scattering length density profile. The inner headgroup layer was essentially unperturbed while w 1/2 of the outer layer increased significantly. This suggests that the anchored PEG chains give rise to headgroup protrusions rather than to blister-like membrane deformations. © 2013 EDP Sciences, SIF, Springer-Verlag Berlin Heidelberg.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

SURFACE MODIFICATIONS OF CAPILLARY-CHANNELED POLYMER (C-CP) FIBER STATIONARY PHASES: IMPROVING THE EFFICIENCY OF HIGHLY SELECTIVE ANALYTE SEPARATIONS

High performance liquid chromatography (HPLC) is a fundamental methodology for the characterization and purification of macromolecules. Since its development in the 1970\u27s, HPLC has significantly advanced its instrumentation and column technology to become a powerful analytical technique. A considerable amount of research focuses on advancements to the stationary phase, or solid support, on which molecules interact and separate. To overcome limitations of other phases that lead to poor mass transfer, slow speeds, and low efficiency, nonporous and superficially porous phases have been developed. Specifically, fiber based polymer stationary phases show great promise as stationary phases for protein separations. The Marcus laboratory has investigated capillary-channeled polymer (C-CP) fibers as an HPLC stationary phase for the past decade. These novel shaped fibers have advantageous attributes including increased surface area over cylindrical cross section fibers, low cost, rapid mass transfer, and the ability to operate at high linear velocities without high pressure. The fibers are available in several base which allow for a wide variety of chemical interactions. What\u27s more, surface modifications can allow for a broader range of chemical specificity. Therefore, the focus of this work is on adosprtion-based surface modification of C-CP fibers. First, modification with recombinant protein A ligand allowed for the capture and recovery of immunoglobulin G (IgG) antibody. This small scale study evaluated maximum protein A ligand density, the stability of the modification, and demonstrated the selective capture of IgG from a mixture with myoglobin (as a surrogate host cell protein) with minimal non-specific binding through the use of a sodium citrate (pH 4) wash buffer. IgG was recovered with high yield with a 0.1 M acetic acid elution buffer. The second modification involves the adsorption of head-group functionalized poly(ethylene glycol) lipids (PEG-lipids), where the lipid tail strongly adsorbs to the polypropylene surface and the hydrophilic PEG group extends away from the surface allowing for the functional ligand to interact with the analyte of interest. This modification was first evaluated as an initial proof-of-concept study, where biotin-PEG-lipid modified PP C-CP fibers were able to selectively capture streptavidin from a complex mixture that also contained a green fluorescent protein. Non-specific binding was minimized through the use of a 0.1 % PBS-Tween buffer. Next, a more in-depth study of surface loading characteristics determined maximum binding capacity when using FITC-PEG-lipid to modify the fibers. This modified surface was also exposed to several test solvents to reveal a highly robust surface modification. Finally, the mechanism of interaction between the lipid and the polypropylene was determined through modification with an environmentally sensitive probe, NBD. Fibers were modified with lipids containing an NBD group attached to either the head group or the fatty acid tail. Fluorescence imaging revealed that the lipid tail intercalates into the PP structure to yield an efficient, robust surface modification. Overall, modifications of the polypropylene fiber surface show promise in increasing the efficiency of affinity separations

Development of Targeted Lipid Nanoparticles for Delivery of siRNA to Neuroblastoma

High-risk neuroblastoma is an aggressive childhood cancer, for which 5-year survival remains less than 50%. Current therapies are non-specific and can lead to acute and long-term side effects. Improved therapies that directly target tumour associated genes are required. Polo-like kinase 1 (PLK1) regulates cell cycle progression, and its over expression in tumours encourages growth. Gene silencing using short interfering RNA (siRNA), is a selective method to target PLK1, with reduced off-target effects. However, efficient delivery of siRNA to tumours requires improved delivery vehicles. Whilst lipid nanoparticles (LNPs) are the most clinically advanced RNA delivery vehicle, clinical siRNA-LNPs exhibit rapid accumulation in liver tissue. I hypothesised that changes in LNP formulation, and active targeting of LNPs to antigens such as epidermal growth factor receptor (EGFR) and disialoganglioside GD2 would improve siRNA delivery to neuroblastoma. siRNA-LNPs were formulated with PEG-lipids that have different detachment kinetics from the LNP surface; DMG-PEG (fast detachment), DSG-PEG and DSPE-PEG (slow detachment). Bispecific antibodies (BsAbs) that recognise either EGFR or GD2 and PEG were used to create targeted LNPs. EGFR targeting improved uptake of siRNA-LNPs in high EGFR-expressing SH-SY5Y neuroblastoma cells in vitro. Similarly, GD2 targeting improved uptake of siRNA-LNPs in GD2-expressing CHP-134 cells. BsAb targeting of LNPs containing PLK1 siRNA (siPLK1-LNPs) resulted in improved PLK1 silencing efficiency compared to untargeted siPLK1-LNPs. Additionally, GD2 targeting reduced the IC50 of siPLK1-LNPs up to 13-fold in CHP-134 cells. Next, the biodistribution of siPLK1-LNPS with and without BsAbs was compared in a neuroblastoma xenograft model. We found that DSG-PEG or DSPE-PEG had higher tumour accumulation compared to DMG-PEG siPLK1-LNPs. However, the presence of BsAb did not significantly impact tumour accumulation. Finally, efficacy studies with untargeted and GD2-targeted siPLK1-LNPs demonstrated that siPLK1 has anti-tumour effects in vivo, although further work is required to optimise therapeutic delivery. In conclusion, this study demonstrates the potential of BsAb targeting and increased PEG-lipid stabilities in improving cell specific uptake and PLK1 gene silencing in neuroblastoma

Tresylated PEG-sterols for coupling of proteins to preformed plain or PEGylated liposomes

AbstractA simple and inexpensive method for functionalization of preformed liposomes is presented. Soy sterol–PEG1300 ethers are activated by tresylation at the end of the PEG chain. Coupling of bovine serum albumin as an amino group containing model ligand to the activated lipids can be performed at pH 8.4 with high efficiency. At room temperature, the mixture of sterol–PEG and sterol–PEG–protein inserts rapidly into the outer liposome monolayer with high efficiency (>100 μg protein/μmol total lipid). This method of post-functionalization is shown to be effective with fluid or rigid and plain or pre-PEGylated liposomes (EPC/Chol, 7:3; HSPC/Chol 2:1, and EPC/Chol/MPEG2000–DSPE 2:1:0.16 molar ratios). The release of entrapped calcein upon the insertion of 7.5 mol% of the functionalized sterols is lower than 4%. Incubation of post-functionalized liposomes with serum for 20 h at 37 °C shows stable protein attachment at the liposome surface