Receptor-Specific Delivery of Liposomes Via Folate-Peg-Chol

Abstract

A novel lipophilic conjugate of folate, folate-PEG-Chol, was synthesized and evaluated for receptor-mediated targeting of liposomes to tumor cells. Liposomes composed of DSPC/Chol/PEG-DSPE/folate-PEG-Chol (60/ 34/5/1, m/m) were taken up by cultured folate receptor-bearing KB cells via a saturable mechanism. Cellular binding of these liposomes could be competitively inhibited by free folic acid with an IC₅₀ of 0.39 mM, indicating an extraordinarily high binding affinity. Fluorescence micrographs of KB cells treated with targeted liposomes encapsulating calcein showed that they were distributed both on the cell surface and in intracellular vesicular compartments. Targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to KB cells than non-targeted control liposomes. A biodistribution study in receptor-positive tumor-bearing C57BL/6 mice showed no significant differences between the tumor uptake of folate-PEG-liposomes and non-targeted control liposomes. This study has demonstrated that cholesterol could be used as an alternative to phospholipids as an effective anchor for incorporation of a targeting ligand into liposomes.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Spectroscopic characterization of nanoErythrosomes in the absence and presence of conjugated polyethyleneglycols: an FTIR and P-NMR study

We have recently developed from red blood cells a new delivery system called nanoErythrosomes. These nanovesicles offer a high degree of versatility for the encapsulation of biological or nonbiological compounds and for the binding of targeting agents. In particular, polyethyleneglycols can be conjugated by a covalent link to the basic amino acid residues constitutive of the different proteins. The binding of polyethyleneglycols to the nanoErythrosome membrane could be interesting for the therapeutic use of this delivery system since it could overcome heterologous immunogenicity and reduce rapid clearance from circulation. In the present study, we have investigated the effect of temperature on the nanoErythrosome behavior in the absence and presence of conjugated polyethyleneglycols. More specifically, Fourier transform infrared (FTIR) spectroscopy has been used to evaluate the lipid order and dynamics, the hydration and the degree of protein aggregation of the nanoErythrosomes after covalent binding of polyethyleneglycols having molecular weights of 2000 and 5000 g mol⁻¹. The results indicate that the nanoErythrosome lipid chain order is not significantly affected by heating the nanoErythrosomes at temperatures up to 50 °C. They also indicate that the nanoErythrosome proteins aggregate irreversibly at temperatures above 37 °C, this effect being abolished in the presence of polyethyleneglycols. The presence of polyethyleneglycols decreases the accessibility of water to the lipid head groups. On the other hand, ³¹P-nuclear magnetic resonance (NMR) and electron microscopy results reveal that the presence of polyethyleneglycols prevents the aggregation of the nanoErythrosome structures.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pK_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of PEG can sufficiently encapsulate siRNA, improve cellular uptake and the efficiency of gene silencing.     

Water stress and root formation in pea cuttings

The stock plants of pea (Pistum sativium L. cv. Alaska) grown for 11 days at 16 W m^?2 38 W m^?2 were subjected to different degrees of moisture stress, simulated with polyethyleneglycol (PEG, 6000) for different periods. The cuttings were made at the end of stress treatments, planted in perlite and allowed to root in a mist propagation chamber. The number of adventitious roots formed on the cuttings from non-stressed plants was significantly higher under low (16 W m^?2) than under high (38 W ^m?2) irradiance. However, under the influence of short duration stress the number of roots increased significantly under high but not under low irradiance. There was significantly poor rooting after prolonged stress under both irradiances. The leaf osmotic potential ψ_π showed a greater reduction with increasing degree and duration of stress at 38 W m^?2 than at 16 W m^?2. The differential rooting behaviour as a result of stress levels and irradiances is discussed in the light of available literature on adventitious root formation.     

INTERFERENCE OF MACROPHAGES WITH IMMUNOTARGETING OF LIPOSOMES

ABSTRACT

We investigated the binding, uptake and intracellular degradation of immunoliposomes by isolated rat liver macrophages in vitro. Immunoliposomes were prepared either by coupling a randomly thiolated anti-CC531 rat colon adenocarcinoma monoclonal antibody to bilayer-incorporated MPB-PE by means of a thioether linkage or by attaching it through its F_c moiety to the distal terminus of hydrazide-modified PEG-DSPE. The two immunoliposome preparations clearly differ in their interaction with the tumor target cells, as well as with the macrophages. At comparable antibody densities both cell types show 1.5–2-fold higher levels of association for the Hz-PEG-immunoliposomes than for the MPB-PEG-immunoliposomes. We provide evidence that immunoliposome macrophage-interaction is both F_c-receptor and scavenger receptor mediated to about equal extents. At low antibody density the hydrazide immunoliposomes favor interaction with the tumor cells to that with macrophages. At higher antibody densities, on the other hand, interaction of these liposomes with the macrophages is increasingly favored, mostly due to enhanced scavenger receptor mediated uptake. The rate of intracellular degradation of (immuno)liposomes internalized by liver macrophages is barely influenced by the presence of either PEG or immunoglobulins on the liposomal surface.     

Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus α-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H⁺, Zn²⁺ and Ca²⁺ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with α-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[¹²⁵I]iodophenyl)diazirine(¹²⁵I-TID) labels monomeric and—predominantly—hexameric forms of liposome-associated α-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H⁺ and Zn²⁺ but not of Ca²⁺—all of which inhibit calcein leakage—the distribution of label between hexamer and monomer is perturbed in favor of the latter. In α-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; they differ only in being more sensitive to ``closure' by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increases with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the α toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of α-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells. Received: 2 October 1995/Revised: 3 November 1995     

Effect of Low Temperature lmbibition on Mito chondrium Respiration and Phosphorylation of PEG Primed Soybean Seed

Cotyledon mitochondrium respiration and oxidative phosphorylation activity of PEG primed and unprimed (control) soybean seeds which have been exposed to low temperature imbibition before germination are studied. The ADP stimulated respiration rates of control mitochondria are evidently higher than state Ⅲ respiration rates of mitochondria from primed seed when L-Mal, α-Kg and Succ are used as substrates respectively. The mitochondria from the unprimed do not possess respiratory control (RC.) On the contrary, mitochondria from the primed, even after seeds being exposed to 2–3 ℃ imbibition for 24 h, phosphorylate normally. The ADP/O and RC values are consistent with those of theoretical expectation. When NADH is used as substrate, unprimed seed mitochondria still possess oxidative phosphorylation activity, while ADP/O and RC values are obviously lower than those of mitochondria from the primed. The emerging sequence of the activity of the diverse phosphorylation sites during germination is also studied. When a different substrate is used, the emerging sequence of the primed is as follows: 1. NADH (12 h), 2.α-Kg (24 h), 3. L-Mal and Succ (48 h). This corresponds to occurrence sequence of ADP stimulated respiration in control mitochondria. The above results show that low temperature imbibition has an irreversible destructive effect on oxidative phosphorylation activity of control mitochondria, and PEG priming has a protective effect on structure and function of the mitochondria under low temperature imbibition stress. The mechanism of soybean imbibitional chilling injury and protective effect of PEG priming are discussed.     

Liposome fragment-mediated introduction of multiple plasmids into Bacillus subtilis

Transformation of microorganisms by plasmid introduction is one of the central techniques in modern biotechnology. However, applicable transformation methods for simultaneous introduction of multiple plasmids are still limiting. Here, we reported a liposome-mediated method that efficiently introduces multiple plasmids into B. subtilis. In this method, liposomes containing three kinds of plasmids were mixed with B. subtilis protoplasts in the presence of 36% polyethylene glycol (PEG), and the resultant protoplasts were grown in cell wall-regeneration media. We found that the rates of introduction of multiple plasmids were significantly increased in the presence of liposomes. We also found that an intact liposome structure was not required for introduction, and the presence of phosphatidylglycerol (PG) was important for efficient introduction of multiple plasmids. Therefore, the liposome- or liposome fragment-mediated transformation method reported here can advance studies utilizing multiple plasmids.