Interaction of unilamellar liposomes with serum lipoproteins and apolipoproteins

The effect of rat whole blood plasma, serum, serum lipoproteins, and apolipoproteins on the stability of unilamellar liposomes prepared with French pressure cell was evaluated by measuring the release of entrapped carboxyfluorescein and by electron microscopy. In the absence of serum components, dye escaped very slowly (hours) from egg phosphatidylcholine and phosphatidylcholine-cholesterol (43 mol % cholesterol) vesicles without apparent change in liposomal structure. This slow release was both temperature- and size-dependent. serum and some of its constituents induced a far more rapid (seconds) loss of entrapped dye from phosphatidylcholine liposomes, associated with structural changes. For equal masses of protein the order of potency of this induced activity was: free apolipoproteins (apo A-I, apo E) > isolated lipoproteins (HDL and VLDL) > whole serum or whole plasma. Substantial activity was found in three preparations of bovine serum albumin. This activity could be attributed to small and variable amounts of contaminating lipoprotein-like particles and apolipoprotein A-I. Induced release of dye from liposomes by apolipoproteins was usually associated with rapid formation of discs although other structures were sometimes formed. Purified rat apolipoproteins A-I and E appeared to interact identically with liposomes to induce dye release. This effect was progressively impaired for both apoproteins by increasing amounts of cholesterol and was completely inhibited when liposomes contained 37 mol % cholesterol.     

Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems

SUMMARY

The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn²⁺ containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.     

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.     

Liposomes in immunology: Evidence that their adjuvant effect results from surface exposition of the antigens

The immune responses against human serum albumin (HSA) and bovine gamma globulin (BGG) were studied in rabbits after intravenous injections of various preparations of these antigens. Antigens were injected free in saline, coated on “empty” liposomes or both coated on liposomes, and entrapped in their inner compartments. The earlier established adjuvant effect of the liposomes was confirmed for both antigens. Although the amount of antigen entrapped in the liposomes was much higher than the amount coated on their outer surfaces, liposomes containing the antigen both in their inner compartments and on their outer surface showed no stronger adjuvant effect than “empty” liposomes coated with the antigen only. The results support the hypothesis that the adjuvant effect of liposomes is mediated by antigens exposed on the outer surfaces of the liposomes. Suggestions are made for the use of liposomes as a practical immunoadjuvant with definite advantages over many other adjuvants.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Immunoassay for human serum albumin using capillary electrophoresis–semiconductor laser-induced fluorometry

Capillary electrophoresis combined with semiconductor laser-induced fluorometry was applied to an immunoassay of human serum albumin. Human serum albumin was labeled with a fluorescent molecule (Cy5), which has an absorption maximum at 649 nm. The labeled albumin was purified by ultrafiltration in order to reduce signals, which are unreacted labeling reagent, product, and fragment products derived therefrom. After the purification, no signal for unreacted labeling reagent and fragment products was detectable in the electropherogram of the labeled albumin. The labeled albumin was then reacted with anti-albumin to form an immunocomplex, which was separated from the excess free albumin. The competitive immunoassay was used in the determination of human serum albumin in a controlled serum sample, using the labeled albumin. The obtained value was found to be 0.21±0.02 mg/ml, which is in good agreement with other known values.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.     

Effect of diglucosamine on the entrapment of protein into liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Effect of Diglucosamine on the Entrapment of Protein into Liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Association of an albumin antigen with phosphatidylcholine liposomes alters the nature of immunoglobulins produced during the immune response against the antigen

Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG responses against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.     

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.     

Red laser-induced fluorescence energy transfer in an immunosystem

We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

Bilirubin diffusion through lipid membranes

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.     

Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Use of liposomes to aid intestinal absorption of entrapped insulin in normal and diabetic dogs

The effectiveness of liposomes in aiding intestinal absorption of entrapped insulin was studied in normal and diabetic dogs. Intraduodenal administration of free insulin (490 and 1630 U) or free insulin (88 U) plus empty liposomes to normal conscious dogs produced no change in plasma immunoreactive insulin or glucose Administration of 40–80 U insulin entrapped in liposomes composed of either phosphatidylcholine, distearoylphosphatidylcholine, or dipalmitoylphosphatidylcholine with cholesterol and dicetylphosophate ( in the ratio 10:2:1 by weight) to normal dogs produced substantial rises in peripheral plasma immunoreactive insulin after 45–60 min. However, the magnitude of these rises was neither reproducible nor dose-dependent. No fall in plasma glucose was observed. Intraduodenal administration of 50–100 U insulin entrapped in liposomes to diabetic dogs also produced rises in plasma immunoreactive insulin levels after 45–60 min but again these rises were not dose-related. However, unlike the results in normal dogs, a small fall in plasma glucose followed the plasma immunoreactive insulin rise in diabetic dogs. This glucose fall was not dose-dependent nor was it related to the magnitude of the rise in plasma immunoreactive insulin. In conclusion, it seems that administration of insulin in liposomes may allow absorption of partially degraded insulin into the circulation but the rise in plasma immunoreactive insulin observed in normal and diabetic dogs and the fall in plasma glucose in diabetic dogs are not influenced by the dose of insulin entrapped nor the lipid composition of the liposomes.     

Kinetic measurements of protein conformation in a microchip

This paper presents a microchip-based system for collecting kinetic time-based information on protein refolding and unfolding. Dynamic protein conformational change pathways were studied in microchannel flow using a microfluidic device. We present a protein-conserving approach for quantifying refolding by dynamically varying the concentration of the chemical denaturants, guanidine hydrochloride and urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. To validate this protein assay method, folding transition experiments were performed using two well-characterized proteins, human serum albumin (HSA) and bovine carbonic anhydrase (BCA). Transition events were monitored through fluorescence intensity shifts of the protein dye 8-anilino-1-naphthalenesulfonic acid (ANS) during dilutions of protein from urea or guanidine hydrochloride solutions. The enzymatic activity of refolded BCA was measured by UV absorption through the conversion of p-nitrophenyl acetate (p-NPA). The microchip protein refolding transitions using ANS were well-correlated with conventional plate-based experiments. The microfluidic platform enables refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material.     

Cross-reactivity of a commercial chemiluminescence immunoassay for human chorionic gonadotropin with the free β-subunit

An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens from oncology patients has been assessed with respect to its cross-reactivity with the free HCG ββ-subunit (HCG-β). The assay, standardized against the First International Reference Preparation 75/537, had a crossreactivity with the free β-subunit of 625% (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-β in serum of patients with seminomatous or nonseminomatous testicular cancers. Results of both assays, the in-house immunoradiometric assay (+ HCG-β) and the Amerlite HCG-60 assay, showed a close correlation (R =0.854?0.960) when serum samples from tumour patients were analyzed. Moreover, the content of free β-subunit determined in a specific HCG-β assay, could be quantitatively measured in the enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, but also highlights the limitations of measuring HCG in serum samples.     

Liposome-based immunoaffinity chromatographic assay for the quantitation of immunoglobulin E in human serum

Immunoglobulin E (IgE)-mediated type I allergies affect over 25% of the world's population; they are among the most common diseases in developed countries. Therefore, simple and rapid in vivo and in vitro methods for diagnosing allergies are becoming increasingly important. In this paper, we demonstrate the feasibility of using sulforhodamine B, a fluorescent dye, entrapped inside immunoliposomes, the outer surfaces of which were sensitized with IgE, as a signal amplifier for the development of a simple, rapid, and inexpensive colorimetric affinity chromatographic immunoassay for the detection of total IgE in serum. This assay operates based on competition between standards (or human serum samples) containing IgE and IgE-sensitized immunoliposomes for the limited number of antigen binding sites of immobilized anti-IgE antibodies at the antigen capture (AC) zone on the nitrocellulose membranes. The color density of the AC zone is indirectly proportional to the number of IgE units present in the test sample. The detection limit of this liposome-based immunoaffinity chromatographic assay was 0.37 ng in IgE-free serum solution (equivalent to 20 μL of a 18.5 ng mL⁻¹ solution). A commercially available ELISA kit was used as a reference method to validate the proposed assay through the analysis of three human serum samples.     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.