How do Hydrophilic Surfaces Determine Liposome Fate in Vivo?

Abstract

Changing liposome physical, properties by designing vesicles with a hydrophilic/ steric barrier at the liposome surface has resulted in altered pharmacokinetics of these liposomes leading to increased blood levels of drug-carrying liposomes and reduced uptake by the RES. This discovery opens up new therapeutic opportunities for liposome-based drug delivery using hydrophilic coatings. Unravelling the mechanism of action of such coatings is an exciting challenge that will facilitate optimization of liposome surfaces for specific drug delivery applications. This article puts forward a series of assumptions and hypotheses to characterize the way hydrophilic coatings extend the plasma half-life of sterically - coated liposomes, to begin to explain how a steric barrier at the surface of liposomes may act. These speculations are examined in the light of current experimental evidence including that from non-liposome systems, and a model for particle removal from the circulation is proposed.

Introduction

Since the days when liposomes were first conceived for drug delivery, ways have been sought to increase the length of time injected vesicles circulate in the body (1). In the mid-eighties, manipulation of the liposomal lipid composition increased the amount of time liposomes remained in the circulation for a well-defined but relatively limited design of     

Solid Supported Vesicles for Bactericide Delivery

Abstract

Cationic and anionic liposomes have been prepared by extrusion from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with cholesterol and dimethyldioctadecyltrimethylammonium bromide (DDAB) and with phosphatidylinositol (PI) respectively covering a range of composition from 0 to 19 mole % DDAB and PI. The adsorption of liposomal lipid from the liposome dispersion onto particles of silica and titanium dioxide in suspension has been studied as a function of liposome composition and concentration. The adsorption isotherms have been fitted using a Langmuir equation from which the binding constants and maximum surface coverage were obtained. The Gibbs energies of adsorption for the cationic liposomes were on average -61.0 ± 2.1 kJ mol^?1 (on silica) and -50.6 ± 2.9 kJ mol^?1 (on titanium dioxide). On average saturation adsorption is equivalent to 3 to 10 lipid monolayers on silica and 3 to 7 on titanium dioxide. Using liposomes encapsulating D-glucose it is demonstrated that there is almost no release of glucose on adsorption of the lipid, indicating that the liposomes are adsorbed intact to form a liposome monolayer on the particle surfaces. Adsorption of intact liposomes to form a close-packed liposome monolayer of solid supported vesicles (SSV) is shown to be equivalent to on average 7.0 ± 0.2 phospholipid monolayers. The SSVs are shown to have increased stability to disruption by surfactants and when carrying the oil-soluble bactericide, Triclosan?, to be capable of inhibiting the growth of oral bacteria from immobilised biofilms.     

Enzyme-Controlled Release with Liposomes

Abstract

Achievements of avoiding the RES uptake and seccess in tumor targeting of liposomes in vivo have brought a great challenge to improve drug delivery efficiency of liposomes to cytoplasmic targets of a cell. Enzymes could be used as a built-in mechanism to regulate the drug release from liposomes. It involves disruption of the permeability barrier of liposome bilayer and has advantages of high specificity and high efficiency. Comparing to other methods, this system has increased the controllability of drug release from liposomes.     

Long-Circulating Liposomes: Development and Perspectives

Abstract

Long-circulating liposomes can be prepared by coating liposome surface with a hydrophilic layer of oligosaccharides, glycoproteins, polysaccharides and synthetic polymers in order to make liposomes “invisible” for scavenger cells of the mononuclear phagocyte system. Incorporation of lipid-anchored poly(ethylene glycol) in liposome bilayer allows to prolong its circulation at least tenfold. Various designs of glycolipid- and polymer-based liposomes are presented, possible mechanisms of action are discussed; potential of these liposomes for drug targeting is presented.     

ASSESSMENT OF LIPOSOME DELIVERY USING SCINTIGRAPHIC IMAGING

ABSTRACT

Scintigraphic imaging is a valuable tool for the development of liposome-based therapeutic agents. It provides the ability to non-invasively track and quantitate the distribution of liposomes in the body. Liposomes labeled with technetium-99 m (^99mTc) are particularly advantageous for imaging studies because of their favorable physical characteristics. Examples of how scintigraphic imaging studies have contributed to the evaluation and development of a variety of liposome formulations will be presented. These include liposomes for targeting processes with inflammation associated increased vascular permeability such as healing bone fractures and viral infections; liposomes for intraarticular delivery; and liposomes for delivery of agents to lymph nodes located in the extremities, the mediastinum and the peritoneum. Scintigraphic studies of liposome distribution are very informational and often suggest new drug delivery applications for liposomes.     

Evaluation of a New Extrusion Device for the Production of Stable Oligolamellar Liposomes in a Liter Scale

Abstract

A procedure for large-scale production of extruded oligolamellar liposomes was developed. the extrusion technique described is rapid, simple, and reproducible. Vesicles with a diameter in the range of 200-500 nm are obtained. the liposomal preparations were characterized by their specific turbidity (determined spectrophotometrically) and contamination with large particles (> 1 µm). Liposome physical stability was determined using a parameter (P) that relates the ratio of the specific turbidity before and after standardized centrifugation.

The extruded oligolamellar liposomes obtained were stable, and after 4 weeks storage at 4°C no irreversible aggregation or fusion of the vesicles occurs.

The extrusion process ensures sterilization of the final liposomal product, making it acceptable as a pharmaceutical preparation for parenteral use.     

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.     

Preclinical and Clinical Experience with a Doxorubicin-Liposome Preparation

Abstract

Entrapment of doxorubicin in liposomes results in increased drug concentrations in liver and spleen and decreased uptake by the heart muscle. these pharmacologic changes can be exploited to reduce the drug's toxicity and increase its therapeutic index in selected neoplastic conditions. We review here our preclinical and phase I clinical data with liposome-associated doxorubicin. these studies, together with preliminary observations on the pharmacokinetics of the liposome-associated drug and on the imaging of radiolabeled vesicles in patients, suggest that the maximal tolerated dosage is significantly increased over that of the free drug and that the reticuloendothelial system is responsible for the rapid and dominant pathway of liposome clearance. the implications of various pharmacologic aspects of liposome behavior in the circulation are also discussed.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions³, monitor drug delivery^4,5, and encapsulation⁴. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.⁶ Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)⁷. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking⁸. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions³. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest⁹. Drug encapsulation can be achieved as reported during liposome formation⁹. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication¹⁰, freeze-and-thaw¹⁰, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency^10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes^10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)¹⁰, electron microscopy¹¹ and nanoparticle tracking analysis (NTA)¹² were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.     

Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

The Use of Liposomes as Carriers of Lipophilic Anthracycline Antibiotics

Abstract

Tumor drug resistance and lack of tumor selectivity are the two main limitations of current systemic anticancer therapy. Liposomes have been shown to decrease certain doxorubicin (Dox)-related toxicities. By modifying liposome size and composition, the tumor localization of liposome entrapped drugs can be greatly enhanced. Through extensive structure-activity studies aimed at identifying anthracycline antibiotics which combine an enhanced affinity for lipid membranes and an ability to overcome multidrug resistance (MDR), we have identified Annamycin (Ann), which is ideally suited for entrapment in liposomes of different size and composition and has shown remarkable in vivo antitumor activity in different tumor models that display natural or acquired resistance to Dox. The unprecedented liposome formulation flexibility offered by Ann is expected to facilitate current efforts aimed at developing pharmaceutically acceptable liposomal-Ann formulations with optimal tumor targeting properties.     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These preclinical observations, suggesting that encapsulation of DOX in PEG-coated liposomes may lead to a significant improvement of the therapeutic index of DOX, have led to the initiation of clinical trials in cancer patients.     

Liposomes: From the Bench to the Bed

Abstract

Our recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process.     

Commentary: The Rationale for an International Liposome Society

Abstract

There is a considerable interest in establishing a liposome society for enhancing the scholarly exchange within our field, and with scientists in other relevant disciplines. In this respect we consider liposomes as a field of interest that includes a variety of lipid assemblies including stabilized emulsions, DNA-cationic lipid complexes, lipid micelles, as well as the lipid bilayer bounded particles referred to as liposomes. In this article I will develop the rationale for forming such a society.     

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.     

Introductory Remarks

Abstract

Sequestration of solutes within liposomes was the basis of the liposome drug-carrier concept, proposed and demonstrated in the early 70′s (1). Subsequent work established the feasibility of using liposomes in a multitude of therapies where site-directed delivery of drugs, coupled with avoidance of premature drug loss, inactivation and toxicity are required for optimal pharmacological action. Instrumental to the remarkable transition of the liposome from a laboratory curiosity 30 years ago to life-saving products today (2), were on the one hand substantial progress in the understanding of its behaviour in vivo (which led to strategies for behaviour control) and, on the other, sophisticated advances in the technology of the system.     

A Clinical Trial: In Vivo Studies on Dexamethasone Sodium Phosphate Liposomes in the Treatment of Human Aphthous Stomatitis

Abstract

Recurrent Aphthous Stomatitis (RAS) is the most common form of nontraumatic oral mucosal ulceration. Numerous studies have suggested a wide variety of the probable etiologic mechanisms (1). However, various therapeutic approaches that have been made to date are controversial since the etiopathology has not been evidently elucidated yet. Corticosteroids are the most commonly used drugs in the treatment of Aphthous Stomatitis. However, they cause side effects when they are used topically in conventional dosage forms (2-4). In earlier study, we tested the potential usage of liposomes as drug carriers to the ulcerated rat oral mucosa by determining whether liposomes could increase local and decrease systemic concentrations of a corticosteroid, dexamethasone sodium phosphate (DSP). We showed that liposomal encapsulation increased drug concentrations at the desired site of action and decreased concentrations at the internal organs (5).In this study by using the combination of two types of dosage forms i.e. liposome dispersion and adhesive gel (liposome dispersion in gel; Gelosome), the therapeutic effect of DSP liposomes was investigated comparing with control groups. The results were evaluated from clinical point of view and histopathologically. In addition, in vitro release and its fitting to different release models was investigated.     

Which Liposomes to Bypass Multidrug Resistance? A Study of Doxorubicin-Loaded Vesicles

Abstract

Liposomes used as antitumoral drug carriers have recently been designated as potential tools to overcome multidrug resistance. In order to understand better the mechanism of such an effect, we have investigated the capacity of liposomes exhibiting a pH gradient to trap efficiently the antitumoral drug doxorubicin in conditions of high dilution such as those used for cell cultures treatment. A simple calculation described the transmembrane pH gradient and the thermodynamic equilibrium of the neutral form, on both sides of the membrane. It showed that liposomes, even efficiently loaded with doxorubicin in response to the pH gradient will lose most of their content upon dilution because of the neutral form physicochemical gradient. Using fluorescence properties of the drug, we found that liposomes made of egg yolk phosphatidylcholine (EPC), phosphatidyl serine (PS) and cholesterol in the ratio 10:1:4 rather closely fitted the situation predicted by the calculation and that the equilibrium state after dilution was reached within one hour. We also showed using liposomes made of dipalmitoyl phosphatidyl choline (DPPC) and cholesterol in the ratio 11:4 that the drastic leakage could be overcome by changing the physical state of the liposome membrane at 37°C.

Referring to the drug release characteristics of other colloidal systems having demonstrated significant capacitiy to oververcome doxorubicin resistance, we concluded that liposomes made of lipids in gel (Lβ or Lβ') state at 37°C could be interesting tools in MDR bypass because they very efficiently retain their content under high dilution conditions.     

Liposomes and Skin Tolerance

Abstract

The authors evaluate different situations where there is an increase or a decrease of complexed liposome tolerance on the skin and in particular of the substances they convey. Since liposomes are made of physiological membrane components, namely phospholipids, they are considered as atoxic microspherules, that are non immunogenic and histocompatible when applied to the skin. When produced in the laboratory (synthetic liposomes) the purity and stability of the lipids making up the liposomes must be kept under strict control. Any undesired side effect should not be attributed to the phospholipids as liposomes, but to the substances (both medical and non) that they convey; indeed side effects result from the interaction and activity of these active principles. On the other hand, the drugs or active principles delivered to the skin by the liposomes have a longer and more concentrated effect and they enter into the systemic circulation in very small amounts, and consequently local and general tolerance is very much enhanced.