Strategies for Optimizing Liposomal Doxorubicin

Abstract

Liposome encapsulation of doxorubicin can dramatically alter its biological activity, resulting in decreased toxicity and equivalent or increased antitumor potency. Since the physical characteristics of the liposome carrier system (size, lipid composition, and lipid dose) can have profound effects on the pharmacologic properties of vesicles administered intravenously, it may be expected that the therapeutic activity of liposomal doxorubicin will be sensitive to these properties. To determine the influence of these variables on the toxicity and efficacy properties of liposomal doxorubicin, transmembrane pH gradient-dependent active encapsulation techniques have been utilized to generate liposomal doxorubicin preparations in which the vesicle size, lipid composition, and drug to lipid ratio can be independently varied. these studies indicate that the toxicity of liposomal doxorubicin is related to the stability of the preparation in the circulation. This property is dictated primarily by vesicle lipid composition, although the drug to lipid ratio can also exert an influence. In contrast, the antitumor activity of liposomal doxorubicin appears most sensitive to the size of the vesicle system. Specifically, antitumor drug potency increases as the vesicle size is decreased. these studies demonstrate that manipulating the physical characteristics of liposomal anticancer pharmaceuticals can lead to preparations with optimized therapeutic activity.     

Drug release rate influences the pharmacokinetics, biodistribution, therapeutic activity, and toxicity of pegylated liposomal doxorubicin formulations in murine breast cancer

The pharmacokinetics (PK), biodistribution (BD), and therapeutic activity of pegylated liposomal doxorubicin formulations with different drug release rates were studied in an orthotopic 4T1 murine mammary carcinoma model. The focus of these experiments was to study the effects of different release rates on the accumulation of liposomal lipid and doxorubicin (DXR) into the tumor and cutaneous tissues of mice (skin and paws). These tissues were chosen because the clinical formulation of pegylated liposomal doxorubicin (Caelyx)/Doxi) causes mucocutaneous reactions such as palmar-plantar erythrodysesthesia (PPE). Liposomes with different doxorubicin (DXR) leakage rates were prepared by altering liposome fluidity through changing the fatty acyl chain length and/or degree of saturation of the phosphatidylcholine component of the liposome. Liposomes with fast, intermediate, and slow rates of drug release were studied. The plasma PK of the liposomal lipid was similar for all formulations, while the plasma PK of the DXR component was dependent on the liposome formulation. Liposomal lipid accumulated to similar levels in tumor and cutaneous tissues for all three formulations tested, while the liposomes with the slowest rates of DXR release produced the highest DXR concentrations in both cutaneous tissues and in tumor. Liposomes with the fastest drug release rates resulted in low DXR concentrations in cutaneous tissues and tumor. The formulation with intermediate release rates produced unexpected toxicity that was not related to the lipid content of the formulation. The liposomes with the slowest rate of drug leakage had the best therapeutic activity of the formulations tested.     

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.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Proteins and peptides bound to long-circulating liposomes.

Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system. Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes. Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes. Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity. This strategy has been successfully applied to two proteins with known fast clearance rate, i.e. asialofetuin and ricin A-chain. The biological activities of both proteins are preserved when they are formulated in liposomes. Incorporation of a peptide, i.e. a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide.     

Characterization of sterically stabilized cisplatin liposomes by nuclear magnetic resonance

Extensive scientific efforts are directed towards finding new and improved platinum anticancer agents. A promising approach is the encapsulation of cisplatin in sterically stabilized, long circulating, PEGylated 100 nm liposomes. This liposomal cisplatin (STEALTH cisplatin, formerly known as SPI-77) shows excellent stability in plasma and has a longer circulation time, greater efficacy and lower toxicity than much free cisplatin. However, so far, the physicochemical characterization of STEALTH cisplatin has been limited to size distribution, drug-to-lipid ratio and stability. Information on the physical state of the drug in the liposome aqueous phases and the drug's interaction with the liposome membrane has been lacking. This study was aimed at filling this gap. We report a multinuclear NMR study in which several techniques have been used to assess the physical nature of cisplatin in liposomal formulations and if and to what extent the drug affects the liposome phospholipids. Since NMR detects only the soluble cisplatin in the liposomes and not the insoluble drug, combining NMR and atomic absorption data enables one to determine how much of the encapsulated drug is soluble in the intraliposomal aqueous phase. Our results indicate that almost all of the cisplatin remains intact during the loading process, and that the entire liposomal drug is present in a soluble form in the internal aqueous phase of the liposomes.     

Encapsulation of Vincristine in Liposomes Reduces its Toxicity and Improves its Anti-Tumor Efficacy

Abstract

Vincristine is a potent therapeutic agent with activity against a variety of tumor types. It is a cell-cycle specific agent which has exhibited enhanced anti-tumor activity when delivered in liposomal form. Vincristine can be encapsulated into large unilamellar vesicles in response to a transmembrane pH gradient with trapping efficiencies approaching 100%. The extent of vincristine encapsulation, and the subsequent retention of the drug within the liposomes, both in vitro and in vivo, are strongly dependent on the lipid composition of the liposome and on the magnitude of the transmembrane pH gradient. Liposomal formulations of vincristine have been optimized for both liposome circulation longevity, drug retention characteristics and in vivo antitumor activity. When compared to free vincristine, these formulations significantly increase the levels of vincristine remaining in the plasma after i.v. administration. These formulations also significantly increase the delivery of vincristine to tumor sites. As a consequence of the improved accumulation of vincristine at tumor sites, liposomal formulations of vincristine exhibit dramatically improved efficacy against a variety of ascitic and solid murine and human tumors than does free vincristine. Liposomal vincristine is expected to be of wide utility in a variety of human malignancies.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential

Among several promising new drug-delivery systems, liposomes represent an advanced technology to deliver active molecules to the site of action, and at present several formulations are in clinical use. Research on liposome technology has progressed from conventional vesicles ("first-generation liposomes") to "second-generation liposomes", in which long-circulating liposomes are obtained by modulating the lipid composition, size, and charge of the vesicle. Liposomes with modified surfaces have also been developed using several molecules, such as glycolipids or sialic acid. A significant step in the development of long-circulating liposomes came with inclusion of the synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition. The presence of PEG on the surface of the liposomal carrier has been shown to extend blood-circulation time while reducing mononuclear phagocyte system uptake (stealth liposomes). This technology has resulted in a large number of liposome formulations encapsulating active molecules, with high target efficiency and activity. Further, by synthetic modification of the terminal PEG molecule, stealth liposomes can be actively targeted with monoclonal antibodies or ligands. This review focuses on stealth technology and summarizes pre-clinical and clinical data relating to the principal liposome formulations; it also discusses emerging trends of this promising technology.     

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.     

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.     

Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.     

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     

Liposomes with entrapped doxorubicin exhibit extended blood residence times

The blood residence time of liposomes with entrapped doxorubicin is shown to be significantly longer than for identically prepared empty liposomes. Liposomal doxorubicin systems with a drug-to-lipid ratio of 0.2 (w/w) were administered at a dose of 100 mg lipid/kg. Both doxorubicin and liposomal lipid were quantified in order to assess in vivo stability and blood residence times. For empty vesicles composed of phosphatidylcholine (PC)/cholesterol (55:45, mole ratio) and sized through filters of 100 nm pore size, 15-25% of the administered lipid dose was recovered in the blood 24 h after i.v. injection. The percentage of the dose retained in the circulation at 24 h increased 2-3-fold when the liposomes contain entrapped doxorubicin. For 100 nm distearoyl PC/chol liposomal doxorubicin systems, as much as 80% of the injected dose of lipid and drug remain within the blood compartment 24 h after i.v. administration.     

Visualization of bioavailable liposomal doxorubicin using a non-perturbing confocal imaging technique

Commonly employed tissue processing techniques can significantly alter tissue drug distribution patterns for liposomal encapsulated drugs by virtue of drug leakage via loss of membrane integrity. We report here a method that has been developed to determine the fluorescence of bioavailable doxorubicin (DOX) in tissues after administration of liposomal DOX formulations. A non-perturbing confocal fluorescence microscopy (CFM) technique with image processing analysis was used with unprocessed fresh tissues. This method takes advantage of the fact that considerable quenching occurs when DOX is within liposomes, leading to the selective visualization of the fluorescence due to DOX released from liposomes. We demonstrate that fresh tissue confocal imaging can be applied to provide detailed drug distribution information with improved accuracy and is a superior method for analyzing tissue distribution of liposome entrapped fluorescent agents.     

Efficacy of Gel Formulations Containing free and Liposomal Foscarnet in a Murine Model of Cutaneous HSV-1 Infection

Abstract

The efficacy of gel formulations containing free and liposomal foscarnet has been evaluated in a murine model of cutaneous Herpes simplex virus type-1 infection. Both formulations were applied topically 3 times daily for 4 days and initiated 24 h post-infection. The penetration of liposomes incorporated into the gel in infected skin tissues was better than that of liposomes dispersed in buffer. Therein, their localization mostly matched that of viral antigen detected by immunoperoxydase staining. Despite these facts, the efficacy of gel formulations of both free and liposomal foscarnet in preventing the development of a zosteriform rash in mice was similar. Electron microscopic examination revealed that liposomes incorporated into the gel formed aggregates together with the micelles of gel. Diffusion studies showed that liposomes were trapped within these aggregates and were hardly able to diffuse across a polycarbonate membrane. In addition, although the liposomes were shown to be highly stable in vitro, the formation of these aggregates destabilized their membrane resulting in a premature release of foscarnet from liposomes. The efficacy of both gel formulations was higher than that of solutions of free or liposomal foscarnet suggesting that the gel formulation is a suitable matrix for the delivery of drugs. Thus, strategies aimed at reducing the interaction of liposomes with the gel could be a convenient approach to improve the efficacy of liposome-encapsulated drug over the free drug.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Rate of biodistribution of STEALTH® liposomes to tumor and skin: influence of liposome diameter and implications for toxicity and therapeutic activity

The influence of diameter on the pharmacokinetic and biodistribution of STEALTH® liposomes into the tumor (4T1 murine mammary carcinoma) and cutaneous tissues (skin and paws) of mice was studied to ascertain the time course of liposome accumulation and to determine if a preferential accumulation of liposomes into tumor over skin or paws could be achieved by altering liposome size. These tissues were chosen as the dose-limiting toxicity for Caelyx™/Doxil® in humans is palmar-plantar erythrodysesthesia, a cutaneous toxicity. We examined liposomes of four diameters: 82, 101, 154, or 241 nm. Liposomes with the three smallest diameters showed similar accumulation profiles that were significantly higher than the largest liposomes in all three tissues of interest. We were unable to achieve a preferential accumulation of liposomes into tumor over skin or paws based on size alone, as evidenced by the tumor to skin and tumor to paw ratios. However, there were differences in the time courses of liposome accumulation in these three tissues. Liposome levels plateaued in tumors and paws within 24 h, whereas skin levels plateaued between 24 and 48 h. The therapeutic activity of liposomal doxorubicin of three diameters (100, 157, and 255 nm) was tested in the same model. All formulations delayed tumor growth, with liposomes of 100 or 157 nm being equally efficacious and superior to liposomes of 255 nm.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

Doxorubicin entrapped within liposome-associated antigens results in a selective inhibition of the antibody response to the linked antigen

The generation of an immune response can dramatically alter the circulation lifetime of a targeted liposome, particularly when the response is generated against the surface-coupled ligand. Following repeated administrations, rapid elimination of the carrier system is observed, thereby limiting potential applications for targeted liposomes in a therapeutic setting. In this study, we have investigated whether the encapsulation of a toxic drug within the carrier could prevent an immune response against a surface-bound protein. Liposome clearance and humoral immune response were monitored throughout multiple administrations of liposomes containing doxorubicin with surface-conjugated ovalbumin. The results show that low doses of encapsulated doxorubicin can prevent humoral immunity against repeated administration of liposomes conjugated with ovalbumin. The immunosuppressive effect was specific for the ovalbumin coupled to the liposome surface. This selective suppression of immunity against a surface conjugated protein could prove advantageous for safe repeated administration of protein containing liposomal systems.