Biodistribution and clearance of intra-articular liposomes in a large animal model using a radiographic marker

The intra-articular (IA) route of administration in treating arthritis has potential for targeting drug delivery to affected tissues, thereby minimising the attendant side-effects of systemically administered drugs. The ultra-structure of the synovium however facilitates rapid drug efflux from the joint; effectively the IA route is equivalent to other non-IV parenteral routes with regards absorption and redistribution into the systemic circulation. The aim of this study was to extend the drug residence time within the knee joint by using a liposome formulation. DPPC-based liposomes were prepared with the radio contrast agent iohexol as a drug marker. 8 sheep had their right knees injected IA with iohexol liposomes and the contralateral joints with either free iohexol or empty liposomes. Joints were radiographed at multiple time points up to 16 days post-injection. Iohexol-mediated radiopacity was quantified by densitometer. Sheep were sacrificed at the end of the study for microscopy of synovial tissues. Good visualization of iohexol-mediated radiopacity with fine anatomical definition was possible throughout the experiment. Also evident on the films was extra-articular radiopacity with liposomes tracking along muscle facial planes. Cellular and tissue localization with light microscopy was possible through use of frozen sections and because of the large liposome size. Residence of encapsulated iohexol within the knee joint was greatly prolonged. Liposomal iohexol declined bi-exponentially with a terminal elimination half-life of 134 hours. In contrast, free iohexol was undetectable at 3 hours post-injection.     

Long-Circulating Liposomes: Development and Perspectives

Abstract

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

Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics

Abstract

The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is – at least to some extent – influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.     

Encapsulation of 8-azaguanine in single multiple compartment liposomes

Incorporation of [2-¹⁴C]-8-azaguanine into positively charged single and multiple component dipalmitoyl-DL-α-phosphatidylcholine and egg yolk phosphatidylcholine liposomes has been established. The extent of encapsulation in single compartment liposomes is 0.70–1.80% and it depends on the concentration of the added 8-azaguanine and the sonication time. Utilizing dialysis, the leakage of the drug from the single compartment liposomes after 20 hours was determined to be 4–34%. At high concentration of the added drug it is possible to encapsulate 18×10^?9 pmole of 8-azaguanine per liposome. Percentages of uptake into multicompartment liposomes are 3.6–5.4%. A preliminary study has been carried out on the effects of free and single and multiple compartment encapsulated 8-azaguanine on the survival and weight gains of leukemia L-1210 bearing mice.     

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.     

Distribution of liposome-entrapped cations in tumor-bearing mice.

The effect of entrapment of ⁸⁶Rb⁺ and ²²Na⁺ in multilamellar, negatively charged phospholipid liposomes on their clearance from the bloodstream and uptake into a variety of tissues in tumor-bearing mice was studied. Although differences were seen between the distribution of free and entrapped ions, these were smaller than might be expected from the uptake of liposomes containing labelled phospholipid. One problem detected was that addition of mouse serum caused a large increase in the efflux of ²²Na⁺ from liposomes, suggesting that a large amount of the injected, trapped ions may have been free in the bloodstream within 1–2 hours. It is concluded that if liposomes are to be fully effective in increasing the uptake of entrapped substances into tissues, the type of liposome used as well as the nature of the entrapped substance are important variables.     

Liposomes and diagnostic imaging: the potential to visualize both structure and function

Abstract

Liposomes have been studied over the past several decades because of their usefulness as a model membranes. The knowledge gained in these studies has been applied in the design of liposomes for use as therapeutic drug carriers. Currently, several liposome-based therapeutics are in the final stages of clinical trials. The field of liposome-based diagnostics is less advanced. For liposome-based materials to reach the clinic, it wIII be necessary for them to show significant advantages over currently employed diagnostic agents. It is the thesis of this review that the major advantage of liposomes is their ability to deliver agents in a manner that makes it possible to image and/or quantitate physiological processes. This ability, along with the demonstrated ability of liposomes to target or constrain contrast agents to specific tissues or structures, constitute the chief advantages of liposome-based diagnostics. Several examples of how these characteristics are being exploited in animal studies are given. It is postulated that continued refinement of liposome carriers and further increases in the ability to control the interactions of liposomes with the in vivo environment wIII allow liposome-based diagnostics to assume a place in the clinic with liposome-based therapeutic agents.     

Oral ingestion of insulin liposomes: Effects of the administration route

We prepared an insulin liposome suspension by hot dispersion (50 °C) of a lipid mixture comprising dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (7:2 molar ratio) in an 80 UI/ml acid bovine insulin solution, followed by two minutes of cold sonification (4 °C). Free insulin was removed by ultracentrifugation and the washed insulin liposomes were resuspended in a 1% aqueous saline solution (pH 3). Administration of these liposomes in the buccal cavity of normal rats caused clear hypoglycemia (?37% of the initial glycemia after one hour and ?44% after $\text{2}\text{1}\text{2}$ hours), but the solution was inactive when introduced by a strictly intragastric route. Hypoglycemic effects were also obtained when a mixture containing a liposome suspension devoid of insulin and 10 UI/100 g b.w. of free insulin was given by the buccal route (?56% of initial glycemia one hour later and ?55% after $\text{2}\text{1}\text{2}$ hours). These results show that the route of liposomal insulin administration strongly influences its biological effects.     

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.     

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.     

Liposomes as carriers for paramagnetic gadolinium chelates as organ specific contrast agents for magnetic resonance imaging (mri)

Abstract

Small unilamellar liposomes were used as carriers for chelates of gadolinium as organ specific magnetic resonance imaging (MRI) contrast agents. The pharmacokinetic and imaging properties of the lipophilic liposome membrane associated chelate diethylenetriaminepentaacetate-stearylamide (DTPA-SA) were investigated. Gadolinium-DTPA-SA liposomes accumulated in the liver of rats at a peak concentration of 60% of the injected dose 4 hours after application. The elimination half-life from the liver was 61 h. Tl-weighted MR images of this liposomal Gd-chelate in rats and dogs gave a strong signal enhancement of the abdominal organs, liver and spleen. High blood concentrations of the Gd-DTPASA liposomes, reaching 60% of the injected dose after 30 min., decreasing to 40% after 2 hours, suggest their potential as a contrast agent for the blood pool. The gadolinium chelate benzoyloxypropionictetraacetate (Gd-BOPTA) was entrapped in liposomes of different lipid composition. Pharmacokinetic studies of liposome preparations containing a poly(ethylene)glycol (PEG) modified lipid showed that high levels of 80 - 60 % of the injected dose remained in the blood, 15 to 60 minutes after application. Peak blood concentrations of liposomes without PEG reached only 30%, with a correspondingly higher uptake in the liver and the spleen. Thus, both the lipophilic chelate Gd-DTPA-SA, as well as Gd-BOPTA entrapped within the aqueous volume of liposomes possess not only a potential as a liver and spleen specific contrast agent, but also for the imaging of the vascular system.     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

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     

New approaches in the chemical design of gd-containing liposomes for use in magnetic resonance imaging of lymph nodes

Abstract

Several approaches to Improve Gd-containing liposomes as magnetic resonance contrast medium for the visualization of lymph nodes are discussed. The modification of the liposome surface with a polymer was chosen as a chemical solution to control the contrast enhancement properties of the medium. It was found that liposome modification with Gd-diethylenetriaminepentaacetic acid (DTPA)-polylysine-based chelating polymer can increase several fold the metal load per vesicle, while surface modification with polyethylene glycol (PEG) might lead to the increased relaxivity of paramagnetic vesicles. Examples are given on how chemical modification of the liposome surface can improve the performance of Gd-containing liposomes in the visualization of lymph nodes.     

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e., gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.     

Commentary: Rantosomes and Ravosomes

Abstract

Those who cannot remember the past are condemned to repeat it.—George Santayana

I am fortunate to have entered the liposome research field in its infancy. In that “golden age” of liposome research, scientific advances related to lipid vesicles appeared in the literature on a regular basis and there was little danger of repeating a study because there was so little published. Many of the potential uses of liposomes in drug delivery that have come to be, were discussed in two creative and prescient articles published in 1976 in the New England Journal of Medicine by Gregory Gregoriadis (1). At liposome/ drug carrier meetings, discussions raged over materials, mechanisms, models, methods and structures. My colleagues and I published a few papers dealing with liposome preparation and extrusion through polycarbonate membranes to form defined diameter populations of liposomes (2-4). The extrusion method is widely used and like the original Bangham method (5) for making MLV, it has become so a part of making liposomes that it is no longer cited. For those entering the liposome field the failure to correctly attribute is understandable, given the vast number of liposome publications (Table 1) that have appeared over the ensuing years.     

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.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

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

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.