Stealth Liposomes: Five Years On

Abstract

Incorporation of polymers, such as polyethylene glycol (PEG-lipid derivatives, or glycolipids, such as monosialoganglioside GM_1; into liposomes results in sterically stabilized liposomes which have several advantages over liposome formulations traditionally used in the past, including reduced recognition and uptake by macrophages, extended circulation half-lives, dose-independent pharmacokinetics, and increased uptake in vivo by solid tumours. PEG-lipid derivatives such as PEG-distearoylphosphatidylethanolamine (PEG-DSPE) are particularily useful because of their ease of preparation and relative lack of expense. Optimum molecular weight of the PEG headgroup is approximately 2000 daltons and optimum concentration in the bilayer is 5 to 7 mol% of phospholipids. Pegylated liposomes have the additional advantage of allowing.     

Liposomes loaded with a dirhenium compound and cisplatin: preparation,properties and improved in vivo anticancer activity

Abstract

Liposomes loaded with the rhenium compound (bis-dimethylsulfoxido-cis-tetrachlorodi-μ-pivalatodirhenium(III) (cis–Re₂((CH₃)₃CCOO)₂Cl₄?2DMSO, I) and cisplatin in the molar ratio of 4:1 as well as those loaded only with I were synthesized and characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering and electronic absorption spectroscopy. The relative stability of liposomes loaded with I is reflected by a minimal change in the electronic absorption spectra over a period of 8 days whereas the stability of those loaded with both drugs is lower, which we ascribe to the formation of new Re-Pt species inside the liposomes. Furthermore, the investigations of the co-encapsulation effects on the anticancer activity of the Re-Pt system were undertaken. Importantly, the co-encapsulated liposomes exhibit synergistic or additive anticancer activities in vivo, e.g. introduction of these liposomes into tumor-bearing rats demonstrated their antianemic, nephro- and hepato-protecting effects. These liposomes, which are active in cancer treatment, protect the dirhenium compounds from hydrolysis and preserve the biological properties of the Re-Pt hybrid. This study reveals the importance of combined therapy in nanotechnology and medicine.     

Targeting of Liposomes Within Cardiovascular System

Abstract

Our studies on the targeting of liposomes and liposome-associated pharmaceuticals within the cardiovascular system are reviewed. The delivery of diagnostic and therapeutic agents in plain liposomes, immunoliposomes, long-circulating liposomes and long-circulating immunoliposomes into the sites of vascular injuries and myocardial infarction is discussed. In vitro, ex vivo, and in vivo experiments present a general view on the advantages and limitations of using liposome-mediated targeting. Liposomes capable of targeting pathological areas of the blood vessel wall both, in vitro and ex vivo are described, as well as liposome able to be internalized by normal endothelial cells. Liposome-mediated drug targeting to compromised myocardium is reviewed with a primary impact on liposomes with anti-cardiac myosin antibodies. Targeted visualization of myocardial infarction with diagnostic liposomes is discussed. Efficient accumulation of long-circulating immunoliposomes in the infarct zone is demonstrated, and a relative importance of different variables, such as liposome size, targetability, and prolonged circulation time, for target accumulation is analyzed. The use of immunoliposomes for targeted sealing of hypoxia-caused damages in plasmic membranes of cardiocytes is considered as a new approach in the therapeutic use of liposomes.     

Phospholipases. II. Enzymatic hydrolysis of lecithin: Effects of structure,cholesterol content,and sonication

Summary Hydrolysis of unsonicated liposomes of egg lecithin catalyzed by several phospholipases is markedly activated by addition ofn-alkanols [Jain & Cordes,J. Membrane Biol. 14:101 (1973)]. Further pursuit of these systems has established that several factors, including higher temperatures, increasing unsaturation of fatty acyl chains of the substrate, incorporation of cholesterol into the liposomes, and sonication, reduce the concentration ofn-hexanol required to elicit maximal activation for enzymatic hydrolysis. Moreover, sonication or incorporation of cholesterol into lecithin liposomes reduces from C₈ to C₇ and C₆, respectively, the chain length of that alcohol eliciting maximal activation. These results are consistent with the hypothesis that sonication and increasing cholesterol content lead to liposomes which have a diminished thickness of the hydrocarbon region compared to that for unmodified liposomes derived from the same lecithin.     

Targeted Delivery of Drugs and DNA with Liposomes

Abstract

This presentation is divided into three parts: long-circulating liposomes, immunoliposomes and gene transfer with liposomes. The mechanism of action for the poly(ethylene glycol)-phospholipid conjugates to prolong the circulation time of liposomes can be understood on the basis of steric barrier activity imposed by the flexible PEG chains on the liposome surface. The action of ganglioside GM₁, on the other hand, probably involves specific interactions with serum protein(s). Immunoliposomes can efficiently bind with the target only if the target is readily accessible and the liposomes stay in the circulation for a relatively long period of time. Coating the liposome surface with PEG chains or GM₁ enhances the target binding of immunoliposomes, except when PEG of greater than 5000 dalton is used. In this case, immunoliposome binding to the target is sterically hindered by the long PEG chains. To overcome the problem, antibody molecule is conjugated to the distal end of the PEG chain. This approach works well except that the liver uptake of immunoliposomes is somewhat enhanced. For the delivery of DNA into cells, a novel cationic amphiphile (DC-chol) is synthesized and is now used in clinical trials of gene therapy for melanoma. Current effort is concentrated on the means to enhance the level and duration of transgene expression.     

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.     

Synthesis of closo-Dodecaboryl Lipids and their Liposomal Formation for Boron Neutron Capture Therapy

High accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve efficient neutron capture therapy of cancers. We focused on liposomal boron delivery system to achieve a large amount of boron delivery to tumor. We succeeded in the synthesis of the double-tailed boron cluster lipids 4a–c and 5a–c, which has a B₁₂H₁₁S-moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols. Size distribution of liposomes prepared from the boron cluster lipid 4b, dimyristoylphosphatidylcholine, polyethyleneglycol-conjugated distearoylphosphatidylethanolamine, and cholesterol was determined as 100 nm in diameter by an electrophoretic light scattering spectrophotometer. Calcein-encapsulation experiments revealed that these boronated liposomes are stable at 37 °C in fetal bovine serum solution for 24 h.     

Doxorubicin Encapsulated in Long-Circulating Thermosensitive Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (TSL, 180-200 nm in mean diameter), prepared from dipalmitoyl phosphatidyl choline (DPPC)/distearoyl phosphatidyl choline (DSPC) (9:1, m/m) and either 3 mol% of amphipathic polyethylene glycol (PEG) with 1000 in average molecular weight or 6 mol% of ganglioside GMI (GMI), with 95-98% entrapping efficiency by the pH gradient method. 57% or 45% of the entrapped DXR was released from PEG/DPPC/DSPC or G_M1/DPPC/DSPC liposomes, respectively, by incubation with 20% serum at 42°C for 5 min. Inclusion of PEG or G_M1 endowed TSL with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system (RES) uptake over 6 hours after injection. Concomitantly, high DXR level in blood was kept for long time.

Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR-long-circulating TSL was 2 times or 7 times higher than that after treatment with DXR-TSL liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the systemic treatment with DXR-long-circulating TSL and hyperthermia resulted in effective tumor growth retardation and increased survival time. These results indicate that the combination of long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

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.     

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

Abstract

We review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macrophages more than the free antibiotic. Liposome-encapsulated paromomycin and sparfloxacin were effective against M. tuberculosis inside macrophages, including multi-drug-resistant strains. Streptomycin encapsulated in liposomes and delivered intravenously or subcutaneously reduced the number of viable M. tuberculosis in infected mice and prevented mortality.     

Systemic Activation of Macrophages by Liposomes Containing Muramyltripeptide Phosphatidylethanolamine for therapy of Cancer Metastasis

Abstract

The heterogeneous response of metastases to conventional therapy is a major cause of failure in cancer treatment. Evidence that activated macrophages can recognize and destroy neoplastic cells in vitro without regard to their phenotypic diversity has stimulated efforts to develop effective approaches to the activation of macrophages in situ. Systemic administration of liposomes containing immunomodulators activates macrophages in situ and augments host destruction of spontaneous metastases.

Liposomes are a useful carrier system for the transport of agents to phagocytic cells in vivo. Once in the circulation, liposomes are cleared by phagocytic cells, and this passive localization provides an effective mechanism for targeting liposome-entrapped materials, such as muramyltripeptide phosphatidylethanolamine (MTP-PE), to macrophages.

Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at start of treatment is minimal. For this reason, we advocate using chemotherapy or radiotherapy first to reduce the tumor burden in patients with metastases. Tumoricidal macrophages that can differentiate neoplastic from bystander nonneoplastic cells are then used to destroy the few tumor cells that escape destruction by conventional therapeutic methods.     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Toxicity and Biodistribution of Liposomes of the Main Phospholipid from the Archaebacterium Thermoplasma Acidophilum in Mice

Abstract

Toxicity and biodistribution of negatively charged liposomes of the main phospholipid (MPL) from the archaebacterium Thermoplasma acidophilum were tested in mice. MPL liposomes with a diameter of 160–220 nm were prepared by extrusion through polycarbonate filters, or by means of a French pressure cell and screened for central nervous system effects after intraperitoneal (i.p.) injection of 4–324 mg of liposomes per kg body weight in NMRI-mice. Besides increased behavioural activity no pharmacological or toxic effects were detected. No alterations were seen in the morphology of the tissues analyzed. Longterm toxicity after life-long oral application of 30 mg MPL per kg body weight per day starting at the age of 10 weeks was tested in immunosuppressed NMRI-mice. Again, there were no toxic effects on survival. Biodistribution of MPL liposomes labeled with ¹¹¹In-diethylenetriaminepentaacetic acid stearylamide was examined 15 min and 2.5 h after intravenous injection into ICR-mice. The liposomes were rapidly cleared from the circulation and the majority accumulated in the liver, followed by the spleen.     

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     

Complement-Dependent Phagocytosis of Liposomes: Suppression by “Stealth” Lipids

Abstract

Liposomes containing either ganglioside G_M1, phosphatidylinositol, sulfogalactosyl ceramide, certain other anionic phospholipids, prostaglandin E₂, or thromboxane B₂ have a reduced ability to undergo complement-dependent phagocytosis by cultured macrophages. We propose that this phenomenon is partially responsible for the prolonged circulation time that is observed after intravenous injection of certain liposomes that are said to have “stealth” properties.     

Liposomes in the Treatment of Parasitic,Viral, Fungal and Bacterial Infections

Abstract

The applicability of liposomes as carriers of immunomodulatory agents or antibiotics for improvement of treatment of severe infections is under investigation. The use of “classical'’ liposomes for targeting of macrophage modulators to enhance non-specific host resistance to infections caused by a variety of micro-organisms shows good results. The therapeutic prospects of “classical'’ liposomes as carriers of antibiotics are good, however are limited to the treatment of intracellular infections in mononuclear phagocyte system (MPS) tissues. The recent development of liposome formulations with reduced affinity to the MPS and long circulation half-lives creates new possibilities for obtaining improved delivery of antibiotics to infected tissues in general including infections in non-MPS tissues.     

Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

Summary The rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C₄ to C₈. For longer alcohols (C₉ to C₁₂), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C₄ to C₈ n-alkanols obey the relationshipp C=a logP _octanol+constant [cf. Hansch & Dunn,J. Pharm. Sci. 61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.     

Tissue distribution of 99mic-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues

Abstract

The tissue distribution of ^99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues was investigated for application to radiopharmaceuticals. The amphiphiles used were N,N-didodecyl-N α-[6-(trimethylammoniohexanoyl]-L-ala-ninamide bromide (N+C₅Ala2C₁₂), N,N-didodecyl-N_α-{6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl}-L-alaninamide bromide (CAC₂N⁺C₅Ala2C₁₂) and S-{l-carboxy-2-([2,3-bis (he xadecyloxy)propoxy]carbony1)ethyl}homocy ste ine. These liposomes were stable in saline and 50% serum at 37° for at least 24h in comparison with the liposomes prepared from phosphatidylcholine and cholesterol (1:1). Most of the radioactivity of N+C₅Ala2C₁₂ and CAC₂N+C₅Ala2C₁₂ liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. But the accumulation of three liposomes into the tumor of Ehrlich solid tumor-bearing mice after intravenous injection was low and most of the liposomes was taken up highly in liver and spleen which belong to the reticuloendothelial system (RES). Some approaches were made to reduce the RES uptake of N+C5Ala2C12 liposomes as follows: (1) the pretreatment of dextran sulfate depressed the uptake of the liposomes in the liver accompanied by increasing uptake in tumor and other tissues except stomach, (2) the modification of the liposomes with n-dodecyl glucoside or n-dodecyl sucrose depressed the uptake in liver and spleen, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, (3) the modification of the liposomes with ganglioside GM3 or GM1 reduced the uptake in liver and spleen, but increased scarcely the uptake in blood and tumor because of the rapid excretion into urine, (4) the intraperitoneal injection reduced the uptake of the liposomes in liver and increased significantly the accumulation in pancreas.     

Polymer-Grafted Liposomes: Physical Basis for the “Stealth” Property

Abstract

Polymer-bearing lipids have recently been incorporated into liposomes that are used in in vivo drug delivery. This strategy has improved the liposome's ability to avoid the reticuloendothelial system and has thereby increased its circulation time in the bloodstream. In order to understand the physical basis for this, so called, Stealth® effect, we have begun a series of studies that characterize the surface structure, interactive properties and in vivo performance of the polymer-bearing, Stealth lipids. For a 1900 g/mol polyethylene glycol (PEG) moiety, we have used x-ray diffraction and micropipet manipulation methods to show that, (i) the polymer chains extend ～50Å out from the lipid bilayer surface; (ii) this surface polymer exerts a significant long range mutual repulsion between adjacent bilayers that prevents bilayer-bilayer adhesion. Furthermore, the measured polymer extension and repulsive pressure are well modelled by polymer scaling laws. These results imply that the interaction of macromolecules and cellular surfaces with the Stealth liposome is probably limited to a distance of ～50Å from the liposome surface. We conclude that the origin of the Stealth effect lies in a steric stabilization mechanism. By using fluorescence video microscopy to observe implanted tumor tissue, we have also shown that fluorescent Stealth liposomes extravasate through the leaky vessel walls of tumors. This method allows us to characterize, in real time, the accumulation of liposomes and release of drug at an implanted tumor site.