Essential oils encapsulated in liposomes: a review

Abstract

In the recent years there has been an increased interest toward the biological activities of essential oils. However, essential oils are unstable and susceptible to degradation in the presence of oxygen, light and temperature. So, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, microspheres, nanoemulsions and liposomes. This review focuses specifically on encapsulation of essential oils into liposomes. First, we present the techniques used to prepare liposomes encapsulating essential oils. The effects of essential oils and other factors on liposome characteristics such as size, encapsulation efficiency and thermal behavior of lipid bilayers are then discussed. The composition of lipid vesicles membrane, especially the type of phospholipids, cholesterol content, the molar ratio of essential oils to lipids, the preparation method and the kind of essential oil may affect the liposome size and the encapsulation efficiency. Several essential oils can decrease the size of liposomes, homogenize the liposomal dispersions, increase the fluidity and reduce the oxidation of the lipid bilayer. Moreover, liposomes can protect the fluidity of essential oils and are stable at 4–5?°C for 6 months at least. The applications of liposomes incorporating essential oils are also summarized in this review. Liposomes encapsulating essential oils are promising agents that can be used to increase the anti-microbial activity of the essential oils, to study the effect of essential oils on cell membranes, and to provide alternative therapeutic agents to treat several diseases.     

Beneficial Effects of Additional Adjuvants on the Immune Response to Haptenated Liposomes

Abstract

The humoral immune response to haptenated liposomes is well documented. This review summarizes our efforts in this field of research. The immunogenicity of haptens, small oligosaccharides and peptides linked to phosphatidyl ethanolamine are studied in mice. Special attention is given to the immunomodulating properties of lipid A, its derivatives and the synthetic adjuvants non-ionic block polymers and ditnethyldioctadecylammonium bromide on the outcome of the response to these haptenated liposomes.     

Macrophage Activation by Liposomal Lipid A: Implications for Vaccines

Abstract

Introduction

Vaccine technology has fostered many promising immunologic strategies for disease prevention, especially vaccination with synthetic peptides and recombinant proteins. Such products, even if considered good candidates for vaccines, are often incomplete in that they may require the 'help' of an adjuvant to become immunogenic (1). However, an immunologist considering peptide epitopes for immunization, must in tandem carefully reflect on how the adjuvant directs the immune response. In our experience, while the expected response to an antigen has often been extensively characterized, less attention is given to adjuvant-immune system interactions beyond the desire to enhance the immune response. This is in part due to the lag in studies focusing on adjuvant-immune system interactions when compared to the immunizing antigens themselves, although the study of adjuvants has begun to receive increasing attention. The adjuvant effect on the immune response can be the determining factor in terms of the effector arm of the immune system recruited (e.g., cytoxoxic T lymphocytes, particular IgG subclass antibodies, etc.) (2-4).

Our laboratory has had a long-standing interest in the immune response to liposomal antigens, beginning with phospholipid antibodies (5). This interest has grown and our laboratories are currently participating in several vaccine trials in humans (6-9). This is in large part due to the effectiveness of liposomes containing lipid A in inducing strong antibody and cytotoxic lymphocyte responses to antigens, coupled with minimal or no adverse reactions (6, 10). Our understanding of the mechanisms by which liposomes containing lipid A exert such adjuvant effects is incomplete but is rapidly growing. This review endeavors to highlight some of the known interactions between liposomes containing lipid A and the macrophage that might be of interest to the vaccinologisl, with a particular focus on macrophage activation and the mechanisms by which liposomes containing lipid A cause this to occur.     

Lipid reducing potential of liposomes loaded with ethanolic extract of purple pitanga (Eugenia uniflora) administered to Caenorhabditis elegans

Abstract

The ethanolic extract obtained from purple pitanga fruit (Eugenia uniflora – PPE) has been previously described by its potential to reduce lipid accumulation in vitro. In this study, we aimed to study this potential in vivo using Caenorhabditis elegans as animal model. Considering the low pH of the extract, its hydrophilic characteristic, its absorption by the medium where the worms are cultivated and the need of a chronic exposure in the worms solid medium, we have loaded liposomes with PPE and investigated its potential for oral administration. Following 48?h exposure to the PPE-loaded liposomes on worms nematode growth medium, we did not observe any toxic effects of the formulation. Under high cholesterol diet, which increased worms total lipid and also triacylglycerides levels, liposomes containing PPE were able to significantly attenuate these alterations, which could not be observed when worms were treated with free PPE. Furthermore, we could evidence that liposomes were ingested by worms through their labelling to uranin fluorescence dye. Through total phenolic compounds quantification, we estimated an entrapment efficacy of PPE into liposomes of 87.7%. The high levels of phenolic compounds present in PPE, as previously described by our group, indicate that these antioxidants may interfere in worms lipid metabolism, which may occur through many and intricated mechanisms. Although the use of conventional liposomes for human consumption may not be pragmatic, its application for oral delivery of a hydrophilic substance in C. elegans was absolutely critical for our experimental design and has proven to be efficient.     

Enhancement Of Immunogenicity of an Immunoprotective Glycophospholipid Antigen of Mycobacteria Using Liposomes Containing Lipid A

Abstract

Role of liposomes as the carriers of mannophosphoino-sitides (PIMs) antigen of mycobacteria have been investigated. PIMs when incorporated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH)/ in a molar ratio of 2: 1. 5 respectively/ elicited both humoral (anti-PIMs IgG) and cell-mediated (DTH) immune responses in mice. Further/ lipid A containing liposomes as carriers of PIMs were observed to induce almost two times more anti-PIMs immune responses as compared to those without lipid A. These findings clearly establish that the lipid A containing liposomes not only replace FIA, but are even better adjuvants for PIMs.     

Liposome Elimination by Non-Phagocytic Cells of the Liver

Abstract

In this contribution we summarize our observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. We demonstrate that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in accessibility is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to hypothesize at least four different mechanisms of interaction of liposomes with hepatocytes.     

Target-Sensitive Liposomes

Abstract

Target-sensitive liposomes are liposomes which spontaneously destablize when they come into contact with target membrane/surface. The principle lipid in the liposomes ingredient is dioleoyl phosphatidylethanolamine (DOPE) which readily forms inverted micelle at physiological conditions. Earlier design of the liposomes uses acylated antibody as both a bilayer stabilizer and a targeting ligand. Although the immunoliposomes specifically release then-contents upon binding with the target membrane, they are not stable enough for long-term storage. Recent improvement in the design uses a charged phospholipid as a bilayer stabilizer and uses acylated antibody or other ligands at a much lower concentration. The new liposomes are stable for long-term storage, yet still destablize when bound with a target membrane. The rate of destabilization is significantly enhanced at elevated temperatures. The physical and biological properties of these liposomes are reviewed in this paper.     

Archaeobacterial Ether Lipid Liposomes (Archaeosomes) as Novel Vaccine and Drug Delivery Systems

ABSTRACT:?

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeo-bacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

Lipid Dependent Cardio- Haemodynamic Tolerability of Liposomes in Rats

Abstract

Rationale and Objectives:

The use of contrast-carrying liposomes in diagnostic applications (1) or of haemoglobin liposomes in blood replacement therapy (2) requires infusion of large lipid doses. Saturated lipids like HSPC are often used in these formulations to render the liposomes more stable (3). Previous studies have indicated that intravenous injection of such liposome preparations can result in significant haemodynamic changes in rats (14). The purpose of this study was to systematically evaluate cardio- and haemodynamic effects of liposomes prepared from saturated and unsaturated phosphatidylcholine alone or in combination with other lipid components.

Methods;

Liposomes made from SPC, HSPC, DSPC, DSPC/CH, DSPC/DSPG, DSPC/CH/DSPG were infused in anaesthetized rats (total lipid dose: 300 mg lipid/kg BW) and cardio-heamodynamic parameters were measured.

Results:

DSPC-liposomes significantly reduced blood pressure (BP) and total peripheral resistance (TPR) by -53.7 % and -45.7 % of prevalue, respectively. Similar results were obtained for HSPC-liposomes. Marked ECG-changes were recorded for both formulations. SPC-liposomes caused a transient and moderate reduction of BP and TPR (-17.0 % and -22.3 %, respectively). Short-lasting ECG changes were also observed. The addition of cholesterol or DSPG to DSPC liposomes reduced cardiac and haemodynamic side effects in rats.

Conclusion;

The lipid composition of liposomes is of major importance for the incidence of cardiovascular side effects in rats. Liposomes composed of pure saturated phosphatidylcholine cause significant changes which can be diminished by the addition of other lipid components like cholesterol.     

Saturated phospholipids are required for nano- to micron-size transformation of cholesterol-containing liposomes upon QS21 addition

Abstract

Liposomes containing cholesterol and monophosphoryl lipid A (such as ALFQ and AS01B) are vaccine adjuvants. During construction of the formulations, addition of QS21 to nano-size (50–100?nm) liposomes resulted in extremely large (up to ～30 µm) liposomes in ALFQ, but AS01B liposomes remained small nano-vesicles. Here, we show that saturation of phospholipid chains is essential for production of large liposomes by QS21.     

Diffusion and Catalysis Processes in Unilamellar Liposomes

Abstract

This research concerns the study of enzyme loaded unilamellar vesicles as bioreactors. The plant enzyme Ascorbic Acid Oxidase (AAO) was entrapped in unilamellar liposomes.

Kinetic study shows that the enzyme activity is largely criptic and that the enzyme in liposomes is partially protected against proteolysis. In order to describe the reactivity of such a system, a kinetic model was developed which accounts both for the enzyme location and for the substrate diffusion across the lipid membrane. For the building of the kinetic model we have used information derived from previous studies of freeze-fracture and label fracture microscopy, which have shown that the protein is located both inside the aqueous core and across the lipid membrane of the vesicles.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Efficient Encapsulation of Plasmid DNA in Anionic Liposomes by a Freeze/Thaw-Extrusion Procedure

Abstract

In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity during extrusion. A solution containing supercoiled plasmid pCMVβ (7164 base pairs) was added to dry lipid films, and after freezing and thawing, the suspension was extruded through a filter with 0.2 μm pores. About 20% of the DNA became encapsulated, as evidenced by protection from degradation by endonuclease added externally. Plasmid isolated from the liposomes was structurally intact, and had essentially the same transfection activity as untreated DNA. These results show that plasmid DNA can be reliably and efficiently encapsulated in anionic liposomes by freeze/thaw and extrusion.     

Carbon Dioxide Hydration with Liposomes Entrapping Carbonic Anhydrase

Abstract

Liposomes entrapping enzymes behave as small membrane bioreactors which can be used in biotechnological and biomedical applications. Furthermore, liposomes loading enzymes are being used as model to mimic cellular and subcellular organel behaviour. This work deals with the study of the kinetic behaviour of carbonic anhydrase (CA) entrapped in lipid vesicle. CA is nearly ubiquitous, being present in many tissues of different species, in cells, subcellular particles and membranes. The carbonic anhydrase entrapment in liposomes, as a function of the lipid cocktail used, was studied preliminarily. Then kinetics of carbon dioxide hydration reaction, catalysed by CA entrapped in liposomes, has been studied.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.     

Are Anti-Phosphoupid Antibodies to be Expected after Proteoliposomal Hepatitis A Vaccination?

Abstract

Introduction

The so-called IRIV- (Immunopotentiating Reconstituted Influenza Virosomes) system (1) is a new type of vaccine. During the past five years extensive experience has been accumulated concerning the immunological and side effects in organisms, both animals and humans, in order to draw up an assessment of the new vaccine Although IRIV could be placed in the category of liposomal vaccines this classification is, however, not quite correct. Such a classification could raise certain questions regarding its use in human medicine. These questions are derived from toxicological studies in animals to which liposomes combined with lipid A were administered It would be valuable to clear up the difference between an IRIV and a liposomal vaccine, with and without lipid A The IRIV is made up of a combination of liposomes (without lipid A) and influenza vims envelopes. Above all, the inclusion of liposomes imposes itself since they already receive much consideration as a new immunological adjuvant and will probably, in the near future, find a large place both as vaccines and as therapeutics.     

Cytotoxicity to a Tumor Cell Line of Epoxy-Phosphatidylinositol Liposomes

Abstract

Selective cytotoxicity of tumor cells induced by liposomal plant phosphatidylinositol (Ptdlns) has been studied. We could not always obtain cytotoxic plant Ptdlns liposomes in a series of experiments. Moreover, animal Ptdlns occasionally showed cytotoxicity towards tumor cells. By ¹H nuclear magnetic resonance analysis of non-and cytotoxic Ptdlns, it has been suggested that oxidized acyl residues, such as hydroperoxide or dioxetan, may have been present in the cytotoxic Ptdlns. We have prepared epoxy-Ptdlns, as an analogous compound of the oxidized lipid, from noncytotoxic Ptdlns by chemical synthesis. the epoxy-PtdIns liposomes showed cytotoxicity towards tumor cells. In the presence of 100 µM epoxy-Ptdlns liposomes, normal human peripheral lymphocytes survived for 3 days, but Raji human lymphoblastoid-like cells were almost all killed. However, at higher concentrations, epoxy-PtdIns liposomes were also cytotoxic to normal cells.     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.     

Morphology of Cationic Liposome/DNA Complexes in Relation to Their Chemical Composition

Abstract

Electron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE¹), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-strand and short oligonucleotides seem not to form such structures during interaction with monovalent cationic liposomes. Neither fusion nor spaghetti formation is observed during interaction of DNA with liposomes made of polyvalent cationic amphiphiles. In general, small complexes consisting of some few semifused liposomes bearing the self-encapsulated nucleic acid and additionally the spaghetti-like structure, free or connected with these complexes, seem to be candidates for the transfectionactive structure rather than large extended H_II¹-lipid arrangements.     

Optimization of drug loading to improve physical stability of paclitaxel-loaded long-circulating liposomes

Abstract

The effect of formulation and process parameters on drug loading and physical stability of paclitaxel-loaded long-circulating liposomes was evaluated. The liposomes were prepared by hydration–extrusion method. The formulation parameters such as total lipid content, cholesterol content, saturated–unsaturated lipid ratio, drug–lipid ratio and process parameters such as extrusion pressure and number of extrusion cycles were studied and their impact on drug loading and physical stability was evaluated. A proportionate increase in drug loading was observed with increase in the total phospholipid content. Cholesterol content and saturated lipid content in the bilayer showed a negative influence on drug loading. The short-term stability evaluation of liposomes prepared with different drug–lipid ratios demonstrated that 1:60 as the optimum drug–lipid ratio to achieve a loading of 1–1.3?mg/mL without the risk of physical instability. The vesicle size decreased with an increase in the extrusion pressure and number of extrusion cycles, but no significant trends were observed for drug loading with changes in process pressure or number of cycles. The optimization of formulation and process parameters led to a physically stable formulation of paclitaxel-loaded long-circulating liposomes that maintain size, charge and integrity during storage.