Stealth Liposomes

Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their interior. The size of a liposome ranges from some 20 nm up to several micrometers and they may be composed of one or several concentric membranes, each with a thickness of about 4 nm. Liposomes possess unique properties owing to the amphiphilic character of the lipids, which make them suitable for drug delivery. . Liposomal drug delivery systems not only enable the delivery of higher drug concentrations , but also a possible targeting of specific cells or organs . Harmful side effects can therefore be reduced owing to minimised distribution of the drug to non-targeted tissues.

Clinical medicine possesses an extremely broad range of drug molecules currently in use, and new drugs are added to the list every year. One of the main goals of any treatment employing xenobiotics is to increase the therapeutic index of the drug while minimizing its side-effects. The clinical utility of most conventional chemotherapeutics is limited either by the inability to deliver therapeutic drug concentrations to the target tissues or by severe and harmful toxic effects on normal organs and tissues. Different approaches have been attempted to overcome these problems by providing “selective” delivery to the affected area; the ideal solution would be to target the drug only to those organs, tissues, or cells affected by the disease. Selected carriers, such as molecular conjugates and colloidal particulates, can be suitable for this purpose. Colloidal particulates result from physical incorporation of the drug into a particulate colloidal system such as liposomes, niosomes, micro- and nano-spheres, erythrocytes, and polymeric and reverse micelles. Among these carriers, liposomes have been most studied.Liposomal formulations of several active molecules are currently in pre-clinical and clinical trials in different fields, with promising results. Two of the key problems in drug therapy (biodistribution throughout the body and targeting to specific receptors) can be overcome by using liposomal formulations: liposomes protect encapsulated molecules from degradation and can passively target tissues or organs that have a discontinuous endothelium, such as the liver, spleen, and bone marrow. Their attraction lies in their composition, which makes them biocompatible and biodegradable. They consist of an aqueous core entrapped by one or more bilayers composed of natural or synthetic lipids. Liposomes composed of natural phospholipids are biologically inert and weakly immunogenic, and they possess low intrinsic toxicity. Further, drugs with different lipophilicities can be encapsulated into liposomes: strongly lipophilic drugs are entrapped almost completely in the lipid bilayer, strongly hydrophilic drugs are located exclusively in the aqueous compartment, and drugs with intermediate logP easily partition between the lipid and aqueous phases, both in the bilayer and in the aqueous core. On intravenous administration, liposomes are rapidly captured by the mononuclear phagocyte system (MPS) and removed from the blood circulation. This behavior has been exploited for efficient delivery of antiparasitic and antimicrobial drugs to treat infections localized in the mononuclear phagocytic system (e.g. antimonial drugs against leishmaniasis), or in order to encapsulate immunomodulators in activated macrophages in cancer models, to produce tumoricidal agents.

Liposomes can be classified according to their lamellarity (uni-, oligo-, and multi-lamellar vesicles), size (small, intermediate, or large) and preparation method (such as reverse phase evaporation vesicles, VETs). Unilamellar vesicles comprise one lipid bilayer and generally have diameters of 50–250 nm. They contain a large aqueous core and are preferentially used to encapsulate water-soluble drugs. Multilamellar vesicles comprise several concentric lipid bilayers in an onion-skin arrangement and have diameters of 1–5 μm. The high lipid content allows these multilamellar vesicles to passively entrap lipid-soluble drugs.

Since liposomes were first developed (around 1980) the related technology has made considerable progress, and several important formulations for the treatment of different diseases are now available commercially or in advanced clinical trials. As shown in Figure 1, the interest in liposome technology and clinical applications remains high, and almost 800 papers and 50 reviews were published in 2005 alone.  

However, when the target site is beyond the MPS, efficient liposome uptake by the macrophages, and their consequent removal from circulation, is one of the main disadvantages for possible use of liposomes as drug delivery systems.

Binding of selected serum proteins (opsonins) is the first signal for removal of liposomes: the MPS does not recognize the liposomes themselves but, rather, recognizes opsonins, which are bound to the surface of the liposomes. A limited number of possible opsonizing proteins that affect the fate of liposomes have been identified e.g. immunoglobulins, fibronectin, beta 2-glycoprotein, C-reactive protein (CRP), and beta 2-macroglobulin. Complement components comprise another important system able to recognize liposomes, which evolved as an immediate host defense against invading pathogens. This system acts through initiating membrane lysis and enhancing uptake by the MPS cells (neutrophils, monocytes, macrophages). In particular, the assembly of C5b-9 complexes (membrane attack complex: MAC) of the complement system is able to produces lytic pores, which induce cell lysis or, in the case of liposomes, the release of their contents. The complement-dependent release of liposomal contents appears to be one of the dominant factors in determining the biological fate of liposomes. However, serum components that inhibit the phagocytosis of pathogens or particles, referred to as dysopsonins, have also been identified. Human serum albumin and Ig A possess dysopsonic properties and their presence on particle surfaces has been shown to reduce recognition and phagocytosis. A balance between blood opsonic proteins and suppressive proteins has been found to regulate the rate of liposome clearance. The instability of liposomes in plasma due to their interaction with high (HDL) and low density (LDL) lipoproteins is another limitation, since this interaction results in the rapid release of the encapsulated drug into the plasma. 

The physicochemical properties of liposomes, such as net surface charge, hydrophobicity, size, fluidity, and packing of the lipid bilayers, influence their stability and the type of proteins that bind to them. One of the first attempts to overcome these problems was focused on manipulation of lipid membrane components in order to modify bilayer fluidity. Damen et al (2005) demonstrated that incorporation of cholesterol (CHOL), by causing increased packing of phospholipids in the lipid bilayer, reduces transfer of phospholipids to HDL; Senior (1982) demonstrated that liposomes obtained from phosphatidylcholine (PC) with saturated fatty acyl chains (with a high liquid crystalline transition temperature) or from sphingomyelin (SM) are more stable in the blood than liposomes prepared from PC with unsaturated fatty acyl chains.

Several approaches have also involved modulating liposome size and charge, so as to reduce MPS uptake. In general, larger liposomes are eliminated from the blood circulation more rapidly than smaller ones. Small unilamellar vesicles (SUVs) have a half-life longer than that of multilamellar liposomes (MLVs) (500–5000 nm). This suggests that phagocytes can distinguish between the sizes of foreign particles.

Based on these observations, it is evident that the binding of opsonins to liposomes depends on the size of the liposomes, and that in consequence the enhanced MPS uptake of liposomes by the liver is likewise size-dependent. Negatively charged liposomes have a shorter half-life in the blood than do neutral liposomes, although the contrary has also been found; positively charged liposomes are toxic and thus quickly removed from circulation.

The complement system has been reported to be activated by both negatively charged and positively charged liposomes in man. Chonn et al (1991) report that, for both human serum and guinea pig serum, surface charge is a key determinant in complement-system activation by liposomes: negatively charged liposomes activate the complement system via the classical pathway, while positively charged liposomes activate it via the alternative pathway.

Clinical applications of conventional liposomes

Based on these studies, “conventional” liposomes composed of neutral and/or negatively charged lipids plus CHOL have been prepared; some of these formulations have reached the market or are now entering clinical trials. Ambisome® (Gilead Sciences, Foster City, CA, USA) in which the encapsulated drug is the antifungal amphotericin B, Myocet® (Elan Pharmaceuticals Inc., Princeton, NJ, USA) encapsulating the anticancer agent doxorubicin, and Daunoxome® (Gilead Sciences), in which the drug incorporated is daunorubicin, are the principal examples of such formulations. To obtain stable formulations incorporating a constant amount of drug, various mechanisms are exploited. Ambisome is lyophilized; Myocet is supplied in three separate vials, one containing doxorubicin as dry powder, one a solution of empty liposomes in citric buffer, and the third a solution of sodium carbonate. In this case drug entrapment must be achieved immediately prior to administration. Daunoxome is at present the only pure-lipid MPS-avoiding liposomal formulation; it is available as a stable ready-to-inject liposomal formulation.

Active agent (product name) Composition Company, year of product marketing Application Trial phase

Daunoxome® (daunorubicin) DSPC/CHOL Nexstar Pharmaceuticals, 1995 Kaposi’s sarcoma Approved

DOXIL®/Caelyx® (doxorubicin) SoyHPC/CHOL/DSPE-PEG Sequus Pharmaceuticals, 1997 Kaposi’s sarcoma Approved

Myocet®/Evacet® (doxorubicin) EPC/CHOL Elan Pharma, 2000 Metastatic breast cancer Approved

SPI-077 (cisplatin) SoyHPC/CHOL/DSPE-PEG Sequus Pharmaceuticals Head and neck cancer, Lung cancer Phase I/II

Lipoplatin™ (cisplatin) SoyPC/DPPG/CHOL Regulon Inc. Several cancer type Phase II/III

S-CKD602 (camptothecin analog) — Alza Co. Several cancer type Phase I

Aroplatin (oxaliplatin analog) DMPC/DMPG Antigenics Inc Colorectal cancer Phase II

Depocyt DOPC/DPPG/CHOL/triolein SkyePharma 1999 Lymphomatous meningitis Approved

LEP-ETU (paclitaxel) DOPE/CHOL/cardiolipin NeoPharm Inc ovarian, breast, and lung cancer Phase I

LEM-ETU (mitoxantrone) DOPE/CHOL/cardiolipin NeoPharm Inc leukemia, breast, stomach, liver, ovarian cancers Phase I

LE-SN38 (irinotecan) DOPE/CHOL/cardiolipin NeoPharm Inc advanced cancer Phase I

MBT-0206 (paclitaxel) DOPE/DO-  trimethylammonium

propane MediGene AG Anti-angiogenic proprieties Breast cancer Phase I

OSI-211 (lurtotecan) SoyHPC/CHOL Enzon Co. Ovarian cancer Head and neck cancer Phase II

Marqibo® (vincristine) DSPPC/CHOL/

sphingosine Inex Pharm Non-Hodgkin’s lymphoma Phase II/III

Atragen® (t-retinoic acid) DMPC, and soybean oil Aronex Pharm advanced renal cell ca, acute pro-myelocytic leukemia Phase I/II

INX-0125 (vinorelbine) DSPPC/CHOL/

sphingosine Inex Pharm breast, colon and lung cancer Preclinical Phase I

INX-0076 (topotecan) DSPPC/CHOL/

sphingosine Inex Pharm advanced cancer Preclinical

Liposomal-Annamycin® DSPC/DSPG/Tween MD Anderson CC breast cancer Phase II

Ambisome® (amphotericin) SoyHPC/DSPPC/CHOL Fujisawa USA Inc. and Nexstar Pharm 1997 Fungal infections in immuno-compromised patients Approved

Nyotran® (nistatin) DMPC/DMPG/CHOL Aronex Pharm Fungal infections in immuno-compromised patients Phase II/III

By addition of sphingomyelin and saturated fatty acid chain lipids to the lipidic formulation, two commercial liposome formulations have been produced. A novel liposomal formulation of vincristine (Marqibo®, formerly Onco TCS; Inex Pharmaceuticals Co., Vancouver, BC, Canada and Enzon Pharmaceuticals Inc., Bridgewater, NJ, USA), based on sphingomyelin-cholesterol uni-lamellar vesicles, has recently been shown to be efficacious in the treatment of relapsed non-Hodgkin’s lymphoma. Inex Pharmaceuticals Co. has in development two further candidate formulations: INX-0125™ (liposomal vinorelbine) and INX-0076™ (liposomal topotecan), also based on the use of sphingomyelin–CHOL mixture.

Another liposomal formulation composed of hydrogenated soy phosphatidylcholine (HSPC) and CHOL and containing lurtotecan has been developed and named OSI-211™ (OSI Pharmaceuticals, Inc., Melville, NY, USA). Clinical results have shown that incorporation of OSI-211 in the acid aqueous core of the vesicle, in addition to providing the known therapeutic advantages of a liposomal carrier, can also favor maintenance of lactone ring closure of lurtotecan (the active form), which increases stability of the active compound, consequently improving its tumor toxicity.

Two other liposome formulations employing saturated phospholipids have been launched for clinical development: Nyotran® (Aronex Pharmaceuticals, The Woodlands, TX, and USA) and Aroplatin® (Antigenics Inc., Lexington, MA, USA). These are multilamellar liposomal formulations consisting of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). Nyotran contains nystatin A1, a membrane-active polyene antifungal antibiotic that is structurally related to amphotericin B. Aroplatin is a multilamellar liposomal formulation of cis-bis-neodecanoato-Trans-R, R-1, 2-diaminocyclohexane platinum (II), a hydrophobic structural analog of oxaliplatin. Aroplatin is in clinical trials for a wide range of tumors and more recently for advanced solid tumors and B-cell lymphoma.

Aronex Pharmaceuticals has also developed a liposomal-all/trans/retinoic acid formulation (Atragen®) containing tretinoin, dimyristoyl phosphatidylcholine (DMPC), and soybean oil. The formulation has shown efficacy in the treatment of acute promyelocytic leukemia and other retinoid-responsive cancers.

Several interesting liposome preparations have been developed by NeoPharm (NeoPharm Inc., Waukegan, IL, USA including LEM-ETU™, LEP-ETU™, and LE-SN38™). In LEM-ETU, mitoxantrone is encapsulated in multilamellar liposomes (composed of dioleoyphosphatidyl choline [DOPC] and CHOL) after charge interaction with cardiolipin. LEP-ETU, a liposome-entrapped “easy to use” paclitaxel formulation, recently demonstrated bio-equivalence with Taxol® (Bristol-Myers Squibb, New York, NY, USA) and interesting activity in Phase I trials. This formulation, composed of DOPC, CHOL, and cardiolipin, is capable of carrying paclitaxel in the liposome bilayer at a maximum mole percent of about 3.5%. This is a higher loading capacity than those obtained in our previous studies with paclitaxel and docetaxel. In LE-SN38™, the active metabolite of irinotecan (7-ethyl-10-hydroxycamptothecin) is encapsulated in PC, CHOL, and cardiolipin liposomes. LE-SN38 was in Phase I trials in 2004.

Paclitaxel has also been encapsulated in other modern formulations of cationic lipid complexes (MBT-0206) that have been shown to be bounded and internalized selectively by angiogenic tumoral endothelial cells after intravenous injection. A Phase II clinical trial is in progress for candidate drug EndoTAG™-1 (MediGene A.G., Martinsried, Germany) in the treatment of advanced pancreatic cancer. Cytarabine is an anti-metabolite, anti-neoplastic agent used in clinical applications for acute lymphoid leukemia, myeloid leukemia and meningeal leukemia. DepoCyt® (SkyePharma PLC, London, UK) is a slow-release formulation of cytarabine designed for intrathecal administration in the treatment of neoplastic meningitis due to breast cancer. In DepoCyt, cytarabine is encapsulated in the aqueous compartment of a spherical 20-μm matrix comprised of lipids biochemically similar to normal human cell membranes (phospholipids, triglycerides and CHOL) (also called DepoFoam™).

Liposomes include controlled retention of entrapped drugs in the presence of biological fluids, prolonged vesicle residence in circulation and enhanced vesicle uptake by target cells. Accumulated (in vivo) evidences suggest that some liposome-entrapped drugs and vaccines exhibit superior pharmacological properties to those observed with conventional formulations especially in cancer chemotherapy, antimicrobial therapy, vaccines, diagnostic imaging and the in treatment of ophthalmic disorders. It is an established fact that, like other colloidal particles, drugs and conventional liposomes are usually rapidly cleared from the circulation by reticuloendothelial system (RES); primarily of kupfer-cells of liver and fixed macrophages of spleen. The rate of liposome uptake by RES is believed to be related to the process of opsonization or dysopsonization of liposomes. There are two different ways with which liposomes may interact with macrophages. Liposomes may directly interact with the surface receptor(s) of macrophages or indirectly via certain serum proteins. Liposomes can be assumed to be effective “Therapeutic System” only when we can keep them out of RES, allowing them to be directed to other sites in vivo. Except for the cases where the drugs have to be directed to RE system, RES uptake presents some major problems with regard to therapeutic applications of conventional liposomes. The problems associated with the conventional liposomes are, 

1. The pronounced tendency of liposomes to localize in RES raises concerns about RES impairment and its consequences particularly during continued liposomes administration.

2. Short circulation times severely limit the use of liposomes as micro-reservoir systems for the slow release of biologically active molecules which are normally degraded rapidly within the vasculature.

3. Rapid uptake of liposomes into liver and spleen greatly reduces the possibility of extra vascularization of liposomes and substantially prevents targeting of liposomes to the cells within the vasculature or targeting to non-RE tissues,

Liposomes that are formulated to “escape” from being recognized by RES can remain in circulation for prolonged periods and may serve as micro-reservoir system and may minimize the problems associated with conventional liposomes. The term “Stealth” liposomes was coined to describe this evasive property (stealth is a registered Trademark of Liposome Technology Inc, Menlo park, CA (USA) and polyethylene glycol lipids (PEG-lipids) are commonly referred to as Stealth Lipids. Among the different polymers investigated in the attempt to improve the blood circulation time of liposomes, poly-(ethylene glycol) (PEG) has been widely used as polymeric steric stabilizer. It can be incorporated on the liposomal surface in different ways, but the most widely used method at present is to anchor the polymer in the liposomal membrane via a cross-linked lipid (i.e. PEG-distearoylphosphatidylethanolamine [DSPE] as schematized in Figure 2).

Table. Composition of few stealth liposomal systems and their performance in vivo.

Sr.

No. Composition of the system Performance in vivo

Polyethylene glycols

1. Hydrogenated SOY Phosphotidyl choline/cholesterol/polyethylene glycol-distearoyl phosphotidyl ethenolamine. Stealth liposome formulation was significantly more effective than conventional liposome formulation (Egg phosphotidyl glycerol/Egg phosphotidyl choline/cholesterol/dI-alpha tocopherol) in reducing the incidence of metastases from intramammory implants of tumor MC2A, tumor MC2B and tumor MC65.

2. Phosphotidyl choline liposomes casted with upto 10 mol% PE with a covalently attached PEG 5000 head group. Circulation time of stealth liposomes within first 24 hrs exceeded 8000% over pure phosphotidyl choline liposomes. 

3. DSPE/cholesterol (2:1) liposomes were

grafted with PEG 1900 mg/ml. Membrane bound PEGs can exert a significant interbilayer

repulsion thereby polymer chain extends to 50°A from lipid bilayer surface, which may reduce interactions with plasma proteins and phagocytic cells.

1. Liposomes were prepared with saturated phospholipids and sphingomyelin. These liposomes attracted serum dysopsonins. which inhibit their uptake by liver cells. Inclusion of cholesterol in these liposome preparations enhanced this uptake in splenic cells but not liver cells.

2. Egg PC/sphingomyelin/cholesterol/ganglioside GM1 in molar ratio 1:1:1 :0.14 The ability of GM1 to reduce leakage of aqueous contents from liposomes there by reducing opsonization was due to

(i) Molecular confirmation.

(ii) Location of negative charge relative to phospholipid bilayer and

carbohydrate back bone

(iii) Packing characteristics of GM1 in phospholipid bilayers.

1. Sphingomyelin/cholesteroI/egg PC (1 :l :l) liposomes were prepared and a carramate

derivative of PEG 1900 with distearyl phosphotidyl ethanolamine (DSPE) was incorporated into these liposomes. (PEG-DSPE) liposomes had the greatest ability to decrease the uptake by mononuclear phagocyte system.

1. PEG is a linear polyether diol 

2. Biocompatibility, 

3. Solubility in aqueous and organic media, 

4. Lack of toxicity, 

5. Very low immunogenicity and antigenicity, and 

6. Good excretion kinetics. 

These properties allow its use in a variety of applications, including the biomedical field.

Moreover, unlike GM1, molecular weight and structure of PEG molecules can be freely modulated for specific purposes, and it is easier and cheaper to conjugate the polymer with the lipid. Poly-ethylene glycols have been used to derivatize therapeutic proteins and peptides, increasing drug stability and solubility, lowering toxicity, increasing half-life, decreasing clearance and immunogenicity. These benefits have been particularly observed using branched PEG in the derivatization. For the most part, reaction with PEG derivatives does not alter the mechanism of action of a therapeutic protein; rather it enhances its therapeutic effect by altering its pharmacokinetics. PEG-ademase (utilized to treat immunodeficiency), PEG-visomant (human growth hormone), PEG-aspargase (for leukemias), PEG-interferon-alpha (for chronic hepatitis C), PEG-aldesleukin (PEG-IL-2) (an anticancer agent), and PEG-filgrastim (for chemotherapy-induced trans ferase neutropenia) are the principal PEGylated proteins in clinical use.Surface modification of liposomes with PEG can be achieved in several ways: by physically adsorbing the polymer onto the surface of the vesicles, by incorporating the PEG-lipid conjugate during liposome preparation, or by covalently attaching reactive groups onto the surface of preformed liposomes. Grafting PEG onto liposomes has demonstrated several biological and technological advantages. The most significant properties of PEGylated vesicles are their strongly reduced MPS uptake and their prolonged blood circulation and thus improved distribution in perfused tissues. Moreover, the PEG chains on the liposome surface avoid the vesicle aggregation, improving stability of formulations. The presence of PEG on the liposome surface provides a strong interbilayer repulsion that can overcome the attractive Van der Waals forces, thus stabilizing liposome preparations by avoiding aggregation. In particular, from X-ray analysis of bilayers incorporating PEG1900-lipid, their research showed that the grafted polymer moiety extends about 50Å from the lipid surface and gives rise to strong inter-membrane repulsive forces. Regarding MPS uptake, Blume et al have found that the increased blood circulation time is due to a reduced interaction with plasma proteins and cell-surface proteins, although other studies have found no direct evidence of this reduced interaction with plasma components . One possible explanation for the reduced interaction is the steric hindrance effect, which is generated by the surface-grafted methoxy-PEG molecules. Complement fixation on PEG-bearing liposomes thus appears to occur in a cryptic location inaccessible to ligation to complement receptors. Another possible contributor to the stealth behavior of such vesicles is competition for CR3 between surface-bound and free-complement proteins iC3b. Furthermore, degradation of surface-bound C3b to fragments inhibiting recognition by phagocytic complement receptors might also explain the anti-phagocytic effect. Studies with freshly isolated macrophages have also indicated the presence of unidentified serum factors (called dysopsonins) that act synergistically with the steric barrier of long circulating particles, thereby further suppressing particle recognition by phagocytic cells. A number of reports have indicated that PEG does not completely avoid cumulative uptake by cells of the MPS, and an interesting review updates progress in this area.  Moghimi and Szebeni (2003) critically examine the supposed mechanisms that contribute to prolonged circulation times of sterically protected liposomes. They point out that PEGylated liposomes are not completely biologically inert and that there is some evidence the polymer can still induce activation of complement systems: a PEGylated liposomal doxorubicin (PLD) (DOXIL® in the US, Caelyx® in Europe, Schering-Plough, Kenilworth, NJ, USA), is a strong activator of the human complement system, with activation taking place within minutes. The behavior of PEGylated liposomes depends on the characteristics and properties of the specific PEG linked to the surface. Figure 3 represents the regimens proposed by deGennes, when polymers are attached to the liposome surface, depending on the graft density of the polymer . The molecular mass of the polymer, as well as the graft density, determine the degree of surface coverage and the distance between graft sites.

The most evident characteristic of PEG-grafted liposomes (PEGylated-liposomes) is their circulation longevity, regardless of surface charge or the inclusion of stabilizing agent such as cholesterol. Figure 4 represents the degree of longevity as determined by a pharmacokinetic evaluation of PEGylated liposomes containing docetaxel.

The ability of the hydrophilic shell of PEG to avoid aggregation between liposomal particles and to decrease the extent of particle-protein interaction in biological fluids is due not only to the molecular mass of the bound polymer and its uniformity (“molecular cloud”) but also to its considerable conformational flexibility . A more rigid polymer, like dextran, grafted to liposomes and used in comparable quantities, does not cause an analogous decrease in liposome-protein interactions.

Liposome membranes containing bilayer-compatible species such as poly (ethylene glycol)-linked lipids (PEG-lipid) or the gangliosides are being used to prepare stealth liposomes. These, so called “Stealth” liposomes have a relatively longer half-life approximately 1 day (whereas the conventional liposomes have only -minutes) in blood circulation and show an altered biodistribution in vivo. Vaage et al prepared stealth liposomes of doxorubicin and used to treat recently implanted and well established, growing primary mouse carcinomas, and to inhibit the development of spontaneous metastases from intra-mammary tumor implants and concluded that long circulation time of the stealth liposomes of doxorubicin formulation accounts for its superior therapeutic effectiveness. A brief review of different approaches undertaken for the preparation and characterization of stealth liposomes are discussed.

Several workers have recently tried to prolong the circulation times of the liposomes by grafting the polymers like PEGs and gangliosides into the lipid vesicles. In addition, the attachments of similar polymers are being investigated in order to minimize or prevent the adsorption of proteins and cells from the blood stream and as a model polymer covered surfaces. A brief account of the composition and performance of few stealth liposomal systems is given in Table 2.

Hershfield et al prepared stealth liposomes of doxorubicin composed of hydrogenated

SOY phosphotidyl choline/ cholesterol/ polyethylene glycol-distearoyl phosphotidyl ethanolamine and the therapeutic efficacy was compared in mice with conventional liposomes composed of egg phosphotidyl glycerol or egg phosphotidyl choline or cholesterol or dl-alpha tocopherol. Stealth Liposome formulation was significantly more effective than the conventional liposome formulation in reducing the incidence of metastases from intra-mammary implants of tumor MC19 and tumor MC65 in curing mice with recent implants of tumor MC2A, tumor MC2B and tumor MC65 and in increasing the 8-week survival of mice with well established implants of tumor MC2B.

Senior et al prepared liposomes which quantitatively retain aqueous markers, were covalently coupled via dipalmitoyl phosphotidyl ethanolamine (DPPE), to the hydrophilic polymer, monoethoxy poly (ethylene glycol) (MPEG 5000). These liposomes retained the coating in the presence of plasma, and appeared to adsorb plasma components more slowly than liposomes without the polymer. MPEG- coupled liposomes were cleared from the blood circulation up to 30% more slowly than liposomes without MPEG after i.v. administration in mice. New lipid carriers consisting of phosphotidyl choline (PC) liposomes casted up to 10 mol% phosphotidyl ethanolamine (PE) with a covalently attached PEG 5000 head group PE-PEG were prepared by Blume et al and in vivo studies were performed in mice. Vesicles exhibited very long circulation time after an i.v. administration in mice. The improvement over pure PC liposomes within the first 24 hours exceeded 8000%, at this point nearly 25% of the applied PE-PEG liposome being still in the circulation. Needham et al have performed X-ray diffraction studies to characterize the surface structures that promote steric stability of PEG (1900 mg/ml, degree of polymerization - 43 mers) grafted lipid vesicles [DSPE/cholesterol (2:1)] and concluded that membrane-bound PEGs can exert a significant inter bilayer repulsion thereby polymer chain extends a distance of 50OAfrom the lipid bilayer surface, which inhibits mutual aggregation and likely reduces interactions with plasma proteins and phagocytic cells that normally lead to conventional liposome disintegration and uptake. The stealth property was not related to any augmentation of mechanical stability due to incorporation of PEG lipid.

Moghimi et al prepared liposomes from sphingomyelin and saturated phospholipids and their affinity to different serum opsonins (liver and spleen) was characterized. Neither liver nor spleen specific opsonins have affinity for sphingomyelin saturated phospholipid liposomes since serum fails to enhance their uptake.

On the contrary, these liposomes attract serum dysopsonins which inhibit their uptake by liver cells. Inclusion of cholesterol in these liposome preparations enhanced their uptake in spleenic cells but not liver cells. It was concluded that the fluidity and hydrophobicity of liposomal membranes play an important role in attracting the right opsonins which determine their phagocytic fate. Allen et al and Gabizons used gangliosides like GMI, GMz, GMs, G&s, GDla and glucolipids like sulfatides, globosides, glycosyl ceramide and several others for the preparation of stealth liposomes. But GMI, only has shown the ability to prolong circulation half-life and other negatively charged glycolipids with bulky head groups i.e., sulfatides and phosphotidyl inositol, had some effect in prolonging circulation half-life. Bilayer rigidity, imparted by sphingomyelin or other high phase transition lipids acted synergistically with negatively charged components especially GM1 in extending circulation time. This exclusive ability of GM1 may be accounted for different reasons; 

(a) Presence of screened negative charge may contribute to RE avoidance, perhaps by decreasing or preventing opsonization of the bilayers. The negative charge of GM1 is shielded from the surface by the presence of two neutral sugars, while with GD, GT, GM2, GM3 this is not the case; 

(b) It has been predicted that the surface hydrophobicity may be a key factor in the phagocytosis of particulate matter. At the critical concentration of GM1 (7mol %) used, it imparts minimum charge density and sufficient surface hydrophilicity to the liposomes to prevent opsonization.

Park et al worked on dioleoyl phosphotidyl ethanolamine (DOPE) derivatives (negatively charged phospholipids). A series of negatively charged phospholipid derivatives has been synthesized by coupling aliphatic dicarboxylic acids to DOPE. The individual derivatives were incorporated into egg PC/cholesterol liposomes and injected into mice to test its effect on liposome circulation in vivo.

i. Liposomes containing negatively charged phospholipids are more rapidly removed from circulation and localized in the RES cells of liver, spleen and bone marrow than the neutral or positively charged liposomes.

ii. DOPE derivatives with n= 1 or 2 accelerated the clearance of liposomes from circulation while those with n=3 or 4 delayed the clearance. Derivatives with a longer hydrocarbon chain than n= 1 appeared not to effect the liposome clearance in either way. Allen et al incorporated a carbamate derivative of PEG 1900 with distearoyl phosphotidyl ethanolamine (DSPE) (PEG-DSPE) into liposomes (sphingomyelin/ cholesterol/egg PC 1:1:1) in concentrations of 5-7mol% and compared for circulation half life with liposomes bearing 10% mono siaganglioside GM~, (PEG-DSPE) liposomes had the greatest ability to decrease the mononuclear phagocyte system uptake of liposomes. Altering vesicle size for liposomes containing PEG-DSPE resulted in only minor changes in blood levels of liposomes. 

Gabizons and papahadjopoulos have divided various negatively charged lipids into two categories.

(i) A diacetyl phosphate type of lipid has negatively charged groups which are exposed to the aqueous environment. The exposed negative charge promotes opsonization of liposomes via charge- mediated interaction with certain proteins in serum.

(ii) The other type of lipid is some glycolipids such as GMT, phosphotidyl inositol or sulfatides, which have a negative charge shielded by surrounding bulky, neutral, hydrophilic groups. 

It is suggested that this “Shielded negative charge” was responsible for prolonged circulation of liposomes. But Park et al reported that, negatively charged phospholipids with the exposed and unshielded carboxylic group such as N-glutaryl DOPE (NGPE) and Nadipyl DOPE (NAPE) show considerable activity to prolong the circulation time of liposomes.

For the derivatives with a short hydrocarbon chain i.e., N-malonyl DOPE (NMPE) and N-succinyl  DOPE (NSPE) the position of the carboxyl groups is close to the interfacial surface of liposome. Nonspecific adsorption of opsonins responding to the surface negative charges may be responsible for the increased RES uptake of liposomes containing negatively charged phospholipids such as phosphotidyl serine (PS) and phosphotidyl glucine (PG). Chann et al have reported that liposome adsorption of the activated complement component Cs, a liposome opsonin, is significantly enhanced with the presence of negatively charged phospholipid. Such Non-specific adsorption of the opsonin(s) would be completely inhibited when the length of the hydrocarbon chain increases. In addition, the adsorption of dysopsonin to the liposome surface, which might require the terminal carboxyl group, should be located at a certain distance from the liposome surface. The activity of the dysopsonin is to decrease the uptake of liposomes by RES.

Sr. no. Composition of the system Performance in vivo

1. Hydrogenated SOY Phosphotidyl choline/ cholesterol/polyethylene glycol-distearoyl phosphotidyl ethanolamine. Stealth liposome formulation was significantly more effective than conventional liposome formulation (Egg phosphotidyl glycerol/Egg phosphotidyl choline/cholesterol/ Di-alpha tocopherol) in reducing the incidence of metastases from intramammory implants of tumor MC2A, tumor MC2B and tumor MC65.

2. Phosphotidyl choline liposomes casted with up to 10 mol% PE with a covalently attached PEG 5000 head group. Circulation time of stealth liposomes within first 24 hrs exceeded 8000% over pure phosphotidyl choline liposomes.

3. DSPE/cholesterol (2:1) liposomes were

grafted with PEG 1900 mg/ml. Membrane bound PEGs can exert a significant interbilayer repulsion thereby polymer chain extends to 50°A from lipid bilayer surface, which may reduce interactions with plasma proteins and phagocytic cells.

1. Liposomes were prepared with saturated phospholipids and sphingomyelin. These liposomes attracted serum dysopsonins which inhibit their uptake by liver cells. Inclusion of cholesterol in these liposome preparations enhanced this uptake in splenic cells but not liver cells.

2. 

Egg PC/sphingomyelin/cholesterol/

ganglioside GM1 in molar ratio 1:1:1 :0.14 

The ability of GM1 to reduce leakage of aqueous contents from liposomes there by reducing opsonization was due to,

(i) Molecular confirmation.

(ii) Location of negative charge relative to phospholipid bilayer and carbohydrate backbone

(iii) Packing characteristics of GM1 in phospholipid bilayers.

1.  Liposomes were prepared and a carbamate 1. Sphingomyelin/cholesterol/egg PC 

(1: l: l) derivative of PEG 1900 with distearylphosphotidyl ethanolamine (DSPE) was incorporated into these liposomes. (PEG-DSPE) liposomes had the greatest ability to decrease the uptake by mononuclear phagocyte system.

A detailed characterization of structure of stealth liposomes including particle size distribution, lamellarity, and bilayer repeat distance and encapsulated volume has to be performed, since it gives information about differences in structure caused by changes in method of preparation and lipid composition. These differences in structure affect the behavior of the vesicles in vitro (stability) as well as in vivo (disposition).

1. Morphology: 

Small-angle X-ray scattering (SAXS) is a small-angle scattering (SAS) technique where the elastic scattering of X-rays (wavelength 0.1 to 0.2 nm) by a sample which has in homogeneities in the nm-range, is recorded at very low angles (typically 0.1 - 10°). This angular range contains information about the shape and size of macromolecules, characteristic distances of partially ordered materials, pore sizes, and other data. SAXS is capable of delivering structural information of macromolecules between 5 and 25 nm, of repeat distances in partially ordered systems of up to 150 nm.

In a SAXS instrument a monochromatic beam of X-rays is brought to a sample from which some of the X-rays scatter, while most simply go through the sample without interacting with it. The scattered X-rays form a scattering pattern which is then detected at a detector which is typically a 2-dimensional flat X-ray detector situated behind the sample perpendicular to the direction of the primary beam that initially hit the sample. 

31P-NMR has been one of the most accurate and straightforward technique that determines lamellarity of the liposomes. The technique exploits 31P-NMR to monitor to phospholipid phosphorous signal intensity. In particular, adding an impermeable paramagnetic shift or non-permeable broadening agent to the external medium will decrease the intensity of the external 31P-NMR signal by an amount proportional to the fraction of lipid exposed to the external medium. Mn2+ ions interact with the outer leaflet of the outermost bilayer. Thus, 50% reduction in the signal indicates unilamellar vesicles where as subsequent reduction indicates multilamellar vesicles.

C. Freeze fracture electron microscopy (FFEM):

Freeze fracture electron microscopy can be used not only to assess the shape of the liposomes but also the topology of liposomes. In this technique the fracture plan passes through the vesicles, which are randomly positioned in the freezed position. The Fracture plane may not necessarily pass through the mid-plane thus non-mid-plane may result in erroneous readings. The observed distribution profile thus depends on the distances of the vesicles center from the plane of fracture. Furthermore, heterogeneous population requires a careful monitoring before analyzing the final results. However, quick freeze and deep etching techniques give much better lamellarity evaluation. It is reported that etching of freeze fractured specimen can provide information about fractures of vesicles that are unilamellar in a given population. After 5 mins of etching, cross-fractured vesicles are clearly seen and the numbers of the lamellae can be readily determined.

Allen and Clelanda developed a technique to study the leakage rates of the drugs or entrapped substances (fluorescent) from liposomes.

Fluorescence increase accompanying leakage was studied using Perkin Elmer MPF-A spectrofluorimeter at 37 0C in 25% human plasma. Exchange/Transfer with High density lipoproteins (HDL): The radiolabel led phospholipids are used to prepare the stealth liposomes. Liposomes are incubated for 2-16 hrs at 37OC with HDL followed by chromatography over a Sepharose CL-4B column and quantization of the radiolabel associated with the HDL peak.

Stealth liposomes prepared using radiolabelled phospholipids are injected (known quantity) into mice and after approximate 7 hrs, anaesthetized and radioactivity of each internal organ is counted in a gamma counter.

1. Targeting of anticancer drugs to tumor sites.

2. Targeting of drugs to non-RE tissues which has not been possible with conventional liposomes.

3. Stealth liposomes may be used in depot applications for slow release of contents for prolonged periods.

4. Stealth liposomes can be used for controlled release within the vasculature by manipulating the phospholipid composition of bilayers.

5. For the diseases of vasculature origin, stealth liposomes provide the best therapeutic effect over conventional drug delivery system

In addition to the common applications possible with conventional liposomes stealth liposomes broaden the area of liposome usage as a drug delivery system by their ability to retain in blood circulation relatively for longer periods.

Ahmed et al reported that PEG-Iiposomes, containing entrapped doxorubicin, targeted to KNL- 205 squamous cell carcinoma of the lung by means of specific antibodies attached at the liposome surface were capable of reducing tumor burden to a high degree and eradicating tumor in a significant percentage in mice. 

PEGylated liposomal doxorubicin (PLD) (DOXIL/ Caelyx) was the first and is still the only stealth liposome formulation to be approved in both the USA and Europe for treatment of Kaposi’s sarcoma and recurrent ovarian cancer. 

DOXIL®/Caelyx is now undergoing trials for treatment of other malignancies such as multiple myeloma, breast cancer, and recurrent high-grade glioma. Several studies are under way to investigate the anticancer activities of PLD in combination with other therapeutics, including the taxanes (paclitaxel or docetaxel), temozolomide (Temodal® Schering-Plough, Kenilworth, NJ, USA and vinorelbine. 

Nearly 100% of the drug detected in the plasma after PLD injection was in liposome-encapsulated form; plasma clearance is clearly slow (0.1 L/hour) and the distribution volume small (4 L). The rigid bilayer of PLD is composed of HSPC, CHOL, and mPEG-DSPE (molecular weight 2000) at a molar ratio of 55:40:5. Liposomes with a mean diameter of 85 nm are able to incorporate doxorubicin at a concentration of 2 mg/mL. The pharmacokinetics is very slow: plasma elimination follows a biexponential curve, with half-lives of 1.5 and 45 hours (median values); in comparison, plasma half-lives are 0.2 hours for free drug, 2–3 hours for Myocet and 5 hours for Daunoxome. 

Due to its pharmacokinetic behavior, cardiotoxicity, myelosuppression, alopecia and nausea are significantly decreased with PLD compared with an equal effective dose of conventional doxorubicin. These bio-distribution characteristics also make skin treatment of localized cancers such as Kaposi’s sarcoma possible; on the other hand, due to its reduced clearance, the palmar-plantar skin reaction and stomatitis / mucositis are the chief dose-related toxicities of PLD.

Gabizon and Papahadjopoulos have reported that when small liposomes of size 0.1 mm were administered by i.v. / i.p. routes elevated tumor levels were achieved compared to conventional liposomes. This suggests the use of liposomes for targeting the drugs to non-RE tissues which has not been feasible with conventional liposomes.

When stealth liposomes were administered by subcutaneous route, they were observed to remain at the site of injection in significant quantities for several days. It is therefore possible that these formulations may be useful in depot applications where slow release of contents is desired over a long period of time. Allen et al developed a new liposome system of 1-beta-D-arabino furanosyl cytosine with prolonged circulation half-life and dose-independent pharmacokinetics for the treatment L121 O/C2 leukemia in mice.

Another stealth liposome formulation is SPI-077™ (Alza Corporation, Mountain View, CA, USA), in which cisplatin is encapsulated in the aqueous core of sterically stabilized liposomes (fully hydrogenated soy HSPC, CHOL, and DSPE-PEG). The stealth behavior of these compounds is evident from their apparent half-life of approximately 60–100 hours. Phase I/II clinical trials have been run to treat head and neck cancer and lung cancer. Although the toxicity profile was promising, the therapeutic efficacy requires improvement. To obtain the desired balance between encapsulation and release of cisplatin from liposomes, another formulation was evaluated by Alza Corporation (SPI-077 B103); they chose B103, in which HSPC is replaced by unsaturated phospholipids, because of its greater theoretical propensity to release cisplatin (Alza Corporation, data on file). However, Zamboni et al (2004) were not able to detect released cisplatin in vitro systems, plasma, or tumor extracellular fluid after administration of either stealth formulation of liposomal cisplatin.

Recently, S-CKD602 (Alza Corporation), a PEGylated stealth liposomal formulation of CKD-602, which is a semi-synthetic analog of camptothecin, was submitted for a Phase I trial. After administration of S-CKD602 at doses of 0.5 mg/m2, the plasma AUC was 50-fold that of non-liposomal CKD-602; S-CKD602 showed minimal toxicity and interesting activity.

Lipoplatin™ (Regulon Inc. Mountain View, CA, USA) is another liposomal cisplatin formulation composed of dipalmitoyl phosphatidyl glycerol (DPPG), soy PC, CHOL, and mPEG2000-DSPE. Its reported half-life is 60–117 hours, depending on the dose. The study found that Lipoplatin™ has no nephrotoxicity up to a dose of 125 mg/m2 every 14 days without the serious side effects of cisplatin.

Stealth liposomes with a large range of contents leakage rates*4P8 can be formulated and can be used for controlled release within the vasculature. Especially for the diseases of vasculature origin Iike atherosclerosis, stealth liposomes may provide the best therapeutic effect over the conventional drug delivery systems.

The development of liposomes as carriers for therapeutic molecules is an ever-growing research area. With the recent developments achieved in the field of liposome technology especially stealth liposomes, a number of therapeutic applications are possible which have not previously been possible. However the mechanisms involved in reducing opsonization of liposomes by grafting the vesicle membranes with suitable polymers has not yet been clearly understood. Although much clinical and laboratory research is required to ascertain for practical utility of stealth liposomes, the likely directions appear to be established. The possibility of modulating the technological characteristics of the vesicles makes them highly versatile both as carriers of several types of drug (from conventional chemotherapeutics to proteins and peptides) and in therapeutic applications (from cancer therapy to vaccination). In recent years, several important formulations for the treatment of different diseases have been developed. Among these, PEG-coated liposomes are becoming increasingly important, giving technological and biological stability to liposomal systems. At present, few PEGylated liposomal formulations have been approved or are in advanced trials (DOXIL®, Lipoplatin™) but stealth technology for different applications is destined to continue developing. PEG-derivatized liposomes with increased stability can easily be modified using a wide array of targeting moieties (MAb, ligands) to deliver the drug specifically to the target tissues with increasing accuracy. Moreover, PEG grafted onto the liposome surface can guide the liposome to a specific intracellular target, using for example cell-penetrating proteins and peptides as targeting agents. The development of liposome delivery to particular subcellular compartments is a field of great interest in different fields, such as gene therapy and vaccination. The interaction of stealth liposomes with cell membranes, and release of the drug in the neighborhood of target tissues are still under investigation, but some recent studies indicate that the use of detachable PEG may facilitate cell penetration and/or intracellular delivery of vesicles. Taking into account these considerations and the great advantages of PEGylated liposomes in decreasing aspecific drug toxicity and in passively targeting the incorporated molecules to the site of action, new and “improved” stealth liposomal formulations designed for different therapeutic and diagnostic areas may soon be expected to arrive on the market.

1. Vyas S.P., Khar R.K., Targeted and controlled drug delivery system (Novel carrier system), CBS publishers and distribution. 

2. Brahmankar D.M., Jaiswal S.B., Biopharmaceutics and pharmacokinetics (A treatise), Vallabh publication.

3. Srinath P., Diwan P.V., Indian Journal of Pharmacology 1994; Vol.: 26, Page no. 179 – 184.

4. Patel HM, Moghimi SM. Optimization strategies. In: Gregoriadis G, ed. targeting drugs. New York: Plenum Press, 1990:87.

5. Papahadjopoulos D, Allen TM, Gabizon A et al. sterically stabilized liposomes: Improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natal Acad Sci. (USA) 1991; 88:11460-4. 

6. Allen TM, Hansen C, Martin F, Redeman C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly ethylene glycol show prolonged circulation half-lives in vivo. Biochem Biophysics Acta 1991; 1066:29-36.

7. Theresa M. Allen, Tarun Mehra, Christian Hansen, and Yeen Cheel Chin, Cancer research 52, 2431-2439. May 1. I992.

8. Allen, T. M. Toxicity of drug carriers to the mononuclear phagocyte system. Adv. Drug Del. Rev., 2: 55-67, 1988.

9. Blume G. and Cevc G. Liposomes for the sustained drug release in vivo. Biochem. Biophysics. Acta, I029: 91-97. 1990.

10. Ahmad I, longlenecker M, Samuel J, Allen TM. Antibody targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice Cancer Research 1993; 53: 1 484-8. 

11. Needham D, McIntosh JJ, Lasic D. Repulsive interactions and mechanical stability of polymer-grafted lipid membranes. Biochem Biophys Acta 1992; 1108:40-8.

12. Ganapathi. R.. Krishnan, A., Wodinsky I. Zubrod, C. G., and Lesko, L. J. Effect of cholesterol content on antitumor activity and toxicity of liposome encapsulated 1-d-o-arabinofuranosylcytosine in vivo. Cancer Research, 40: 630- 633, 1980.

13. Hershfield MS, Buckley K. Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. N Engl J Med 1987; 316:589-96. 

14. Senior J, Delcada C, Fischer D, Tilcock C, Gregoriadis G. Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly (EG)-coated vesicles. Biochem, Biophys Acta 1991; 1062:79-82.

15. Lasic D. D., Martin F. J., Gabizon A., Huang S. K., Papahadjopoulos D. Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times.. Biochim. Biophys. Acta, 1070: 187-192, 1991.

16. Oku N., Namba Y., Okada S. Tumor accumulation of novel RES-avoiding liposomes.. Biochim. Biophys. Acta, 1126: 255-260, 1992. 

17. Peterson, H. I. (ed.). Tumor Blood Circulation: Angiogenesis, Vascular Morphology and Blood Flow of Experimental and Human Tumors. Boca Raton, FL: CRC Press, 1979. 

18. Connolly D. T., Heuvelman D. M., Nelson R., Olander J. V., Eppley B. L., Delfino J. J., Siegel N. R., Leimgruber R. M., Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.. J. Clin. Investig., 84: 1470-1478, 1989. 

19. Maeda H. SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy.. Adv. Drug Delivery Rev., 6: 181-202, 1991. 

20. Duncan R. Drug-polymer conjugates: potential for improved chemotherapy.. Anticancer Drugs, 3: 175-210, 1992. 

21. Huang S. K., Lee K-D., Hong K., Friend D. S., Papahadjopoulos D. Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.. Cancer Res., 52: 5135-5143, 1992. 

22. Yuan F., Leunig M., Huang S. K., Berk D. A., Papahadjopoulos D., Jain R. K. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft.. Cancer Res., 54: 3352-3356, 1994. 

23. Haran G., Cohen R., Bar L. K., Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases.. Biochim. Biophys. Acta, 1151: 201-215, 1993. 

24. Harrington, K. J., Mohammadtaghi, S., Uster, P. S., Glass, D., Peters, A. M., Vile, R. G., and Stewart, J. S. Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled PEGylated liposomes. Clin Cancer Res., 7: in press, 2001. 

25. Harrington K. J., Rowlinson-Busza G., Syrigos K. N., Uster P. S., Abra R. M., Stewart J. S. Biodistribution and pharmacokinetics of 111In-DTPA-labelled PEGylated liposomes in a human tumour xenograft model: implications for novel targeting strategies.. Br. J. Cancer, 83: 232-238, 2000. 

26. Woodle M. C. 67Gallium-labeled liposomes with prolonged circulation: preparation and potential as nuclear imaging agents.. Nucl. Med. Biol., 20: 149-155, 1993. 

27. Hubert A., Lyass O., Pode D., Gabizon A. Doxil (Caelyx).. An exploratory study with pharmacokinetics in patients with hormone-refractory prostate cancer. Anti-Cancer Drugs, 11: 123-127, 2000. 

28. Lyass O., Uziely B., Ben-Yosef B., Tzemach D., Heshing N. I., Lotem M., Brufman G., Gabizon A. Correlation of toxicity with pharmacokinetics of PEGylated liposomal doxorubicin (Doxil) in metastatic breast cancer.. Cancer (Phila.), 89: 1037-1047, 2000. 

29. Koukourakis M. I., Koukouraki S., Giatromanolaki A., Archimandritis S. C., Skarlatos J., Beroukas K., Bizakis J. G., Retalis G., Karkavitsas N., Helidonis E. S. Liposomal doxorubicin and conventionally fractionated radiotherapy in the treatment of locally advanced non-small-cell lung cancer and head and neck cancer.. J. Clin. Oncol., 17: 3512-3521, 1999. 

30. Koukourakis M. I., Koukouraki S., Giatromanolaki A., Kakolyris S., Georgoulias V., Velidaki A., Archimandritis S., Karkavitsas N. N. High intratumoral accumulation of stealth liposomal doxorubicin in sarcomas–rationale for combination with radiotherapy.. Acta Oncol., 39: 207-211, 2000. 

31. Harrington K. J., Rowlinson-Busza G., Syrigos K. N., Abra R. M., Uster P. S., Peters A. M., Stewart J. S. Influence of tumour size on uptake of 111ln-DTPA-labelled PEGylated liposomes in a human tumour xenograft model.. Br. J. Cancer, 83: 684-688, 2000. 

32. Safra T., Groshen S., Jeffers S., Tsao-Wei D. D., Zhou L., Muderspach L., Roman L., Morrow C. P., Burnett A., Muggia F. Treatment of patients with ovarian carcinoma with pegylated liposomal doxorubicin.. Cancer (Phila.), 

33. Jain R. K. Delivery of molecular and cellular medicine to solid tumors.. Adv. Drug. Delivery Rev., 

34. Gabizon, A. PEGylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy. Cancer Invest., in press, 2001. 

35. Cummings J., McArdle C. S. Studies on the in vivo disposition of Adriamycin in human tumors which exhibit different responses to the drug.. Br. J. Cancer, 53: 835-838, 1986.