Antibacterial activity of liposome-entrapped ampicillin in vitro and in vivo in relation to the lipid composition

The antibacterial activity of liposome-entrapped ampicillin against Listeria monocytogenes was investigated in relation to the lipid composition of the liposomes. This was studied in vitro in mouse peritoneal macrophages infected with L. monocytogenes, as well as in vivo in experimental L. monocytogenes infection in mice. Two types of liposomes, a relatively fluid type, consisting of cholesterol-phosphatidylcholine-phosphatidylserine (5:4:1), and a less fluid type, consisting of cholesterol-distearoylphosphatidylcholine-dipalmitoylphosphatidylglyc erol (10:10:1), were used. The uptake of both types of liposomes by macrophages in vitro was similar. However, the rate of intracellular degradation appeared to be dependent on the lipid composition. A correlation was found between the relatively slow degradation of the less fluid liposomes and a delayed intracellular release of the encapsulated ampicillin, as reflected in absent or delayed intracellular killing of L. monocytogenes in vitro. The results obtained in vitro were confirmed by the observations in vivo. Slow degradation of the less fluid liposomes in vivo resulted in a decrease in the therapeutic effect of the antibiotic.     

An in vitro assay based on surface plasmon resonance to predict the in vivo circulation kinetics of liposomes

The adsorption of blood proteins onto liposomes and other colloidal particles is an important process influencing the circulation time. Proteins adsorbed to the surface of liposomes can mediate recognition of the liposomes by macrophages of the reticuloendothelial system (RES) facilitating their clearance from the circulation. Coating liposomes with poly(ethylene glycol) (PEG) decreases the blood clearance considerably, most likely due to reduced protein adsorption and/or liposome aggregation. By using the relation between clearance and protein binding, the present study introduces an in vitro assay measuring interactions of liposomes with proteins to predict their blood clearance in vivo. Such assay is valuable since it limits time and costs, and importantly reduces the number of animals required for pharmacokinetic investigations of new formulations. In the current study, Surface Plasmon Resonance (SPR) and fluorescence Single Particle Tracking (fSPT) were used to study liposome–protein interactions and blood induced liposome aggregation in vitro. By means of SPR the interactions between proteins and liposomes coated with PEG of different molecular weights and at different densities (PEG₂₀₀₀ in 2.5%, 5% and 7%; PEG₅₀₀₀ in 0.5%, 1.5% and 2.5%), were measured for several plasma proteins: human serum albumin (HSA), apolipoprotein E (ApoE), α2-macroglobulin (α2-M), β2-glycoprotein (β2-G) and fibronectin (Fn). Liposomes coated with PEG interacted less with all proteins, an effect which increased with the PEG surface density. In parallel, fSPT analysis showed that the exposure of liposomes to full blood did not change the liposome size, indicating that aggregation is not a strong attributive factor in the clearance of these liposomes. In addition, the SPR measurements of the interactions between liposomes and proteins were correlated with the blood clearance of the liposomes. For each protein, the degree of protein–liposome interaction as determined by SPR showed a moderate to strong positive correlation with the clearance of the liposome type.     

Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time.

Cationic liposomes have been widely sensed as good DNA compacting delivery agents. Although their use generally met with encouraging results in vitro, the results in vivo were rather disappointing, as they strongly interact with the blood components before they can reach the therapeutic target. Polyethyleneglycol (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex (zeta potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.     

Preclinical and clinical evidence for disappearance of long-circulating characteristics of polyethylene glycol liposomes at low lipid dose

This study describes the effect of the lipid dose of (99m)Tc-polyethylene glycol (PEG) liposomes in the low-dose range (0. 02-1.0 micromol/kg) on the pharmacokinetics and biodistribution in rats, rabbits, and humans. The biodistribution and pharmacokinetics of (99m)Tc-PEG liposomes at various dose levels were studied in rats and rabbits with a focal Escherichia coli infection. Scintigraphic images were recorded on a gamma camera. In addition, the role of macrophages in the biodistribution of a low-dose PEG liposome injection was studied. Finally, the pharmacokinetics of (99m)Tc-PEG liposomes at two lipid dose levels was studied in four patients. At a dose level of 0.03 micromol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 micromol/kg). The same effect was observed in rabbits where enhanced clearance was observed at a dose level of 0.02 micromol/kg. The circulatory half-life decreased from 10.4 to 3.5 h (at 1.0 and 0. 02 micromol/kg, respectively). At the lowest dose level, liposomes were mainly taken up by the liver and to a lesser extent by the spleen. Injection of a low dose of PEG liposomes in macrophage-depleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid elimination of the liposomes from the circulation. Most importantly, the rapid clearance of low-dose PEG liposomes was also observed in humans when relatively low lipid doses were administered. This study showed that at very low lipid doses the biodistribution of PEG liposomes is dramatically altered.     

Protection of oligonucleotides against nucleases by pegylated and non-pegylated liposomes as studied by fluorescence correlation spectroscopy.

Antisense phosphodiester oligonucleotides (ONs), complexed to carriers such as cationic liposomes, inhibit the production of proteins. The biochemical and biophysical phenomena that govern the extent of this inhibition are still not fully understood. Major biological barriers limiting a pronounced antisense effect are the cellular entry and endosomal escape of the ONs containing liposomes, the release of the ONs from the liposomes and the extra- and intracellular degradation of the ONs. In this paper we focus on the latter barrier and evaluate, by fluorescence correlation spectroscopy (FCS), to what extent phosphodiester ONs complexed to DOTAP/DOPE liposomes, are protected against degradation by nucleases. Liposomes studied were either with or without a polyethyleneglycol (PEG) moiety at the surface. Using non-pegylated liposomes the phosphodiester ONs were initially adequately protected when exposed to DNase I. Indeed, in the mechanism for lipoplex formation as suggested by others, the ONs become trapped between lipid bilayers and are therefore shielded from the environment. However, after a few hours the phosphodiester ONs no longer stayed intact. This was explained by a gradual fusion of the lipoplexes in time thereby spontaneously releasing phosphodiester ONs. Using pegylated liposomes, a substantial fraction of the phosphodiester ONs degraded immediately after exposing the complexes to DNase I. Based on experimental evidence we suggest that the presence of the PEG-chains influences lipoplex formation so that the ONs are not trapped between lipid bilayers and therefore remain accessible by the DNase I enzyme.     

Polymer coating of liposomes with a modified polyvinyl alcohol and their systemic circulation and RES uptake in rats.

The objective of this study was to evaluate the in vivo characteristics of liposomes coated with a polyvinyl alcohol having a long alkyl chain at the end of the molecule (PVA-R) as an injectable drug carrier for passive targeting of drugs. A fluorescence marker, cholesteryl-anthracene-9-carboxylate, was incorporated into the liposomes to detect their concentration in the plasma and organs. The small unilamellar liposomes (100 nm in diameter) with various lipid compositions, such as the different cholesterol contents or the different charges, were prepared by the hydration method followed by sonication and coated with PVA or PVA-R by just mixing the resultant liposomal suspensions with the polymer solutions. The circulation and distribution of the liposomes were tested with their intravenous administration in rats. The PVA-R-coated liposomes showed significantly higher circulation compared to that of non-coated ones in any liposomal formulation tested. The prolonged circulation of PVA-R-coated liposomes was attributed to their fewer uptake in liver and spleen. The extent in improvement in the in vivo characteristics were well interpreted by the hydrophobicity of liposomes and their coating amount of PVA-R. It was also demonstrated that the liposomes having a positive and a negative charge, which showed the completely different circulating profiles, showed almost the same profile by coating with PVA-R. When the liposomes were coated with PVA the improvement in blood circulation was much less in any cases, although coating layer of PVA was detected. These results confirm that not only the hydrophilic property but the sterically stabilizing effect of the coating layer is important to prolong the circulation of the particulate drug carriers with less RES uptake, and the PVA-R having a hydrophobic moiety at the end of the molecule of PVA is a suitable material for the coating of liposomes.     

Functional improvement of an IRQ-PEG-MEND for delivering genes to the lung

The targeted delivery of genes to endothelial cells is a potential strategy for curing certain types of disorders including cancer, inflammation and obesity. We previously reported that a liposome (IRQ-LP) modified with the IRQ peptide (IRQRRRR) was taken up by cells via a unique pathway, namely caveolar endocytosis, a cellular uptake pathway that is involved in the blood-to-tissue uptake of macromolecules in vascular endothelial cells. In the present study, we initally investigated the effect of IRQ peptide-modification on the biodistribution of poly(ethyleneglycol) (PEG)-coated liposomes (PEG-LP) after i.v. administration. The IRQ peptide-modified PEG-LP (IRQ-PEG-LP), as well as the PEG-LP were found to be mainly accumulated in the liver. Nevertheless, the fold increase in the lung accumulation of IRQ-PEG-LP, compared to the PEG-LP (approximately 20-folds) was substantially higher than other tissues (< 5-fold). Thus, IRQ could function as a target ligand for lungs. We then used the IRQ peptide as a model for a ligand for targeting normal tissue endothelial cells, and then applied it to a gene delivery system. We previously developed a multifunctional envelope-type nano device (MEND), in which plasmid DNA is condensed using a polycation to form a core particle that is encapsulated in a lipid envelope. We modified the IRQ-modified PEG to the MEND (IRQ-PEG-MEND) and marker gene expression was evaluated after i.v. administration. However the transgene expression of the IRQ-PEG-MEND in lungs was low. This is most likely due to the inhibitory effect of the PEG spacer on intracellular trafficking (especially endosomal escape) of the IRQ-PEG-MEND. To overcome the dilemma associated with PEGylation, we improved the MEND system from the point of view of PEG length, lipid chain of the PEG derivative, the polycation and cationic lipid. As a result, transgene expression in lungs was enhanced in stepwise manner, and was finally improved by 5 orders of magnitude compared with the original IRQ-PEG-MEND. Overcoming the dilemma of PEGylation is critical issue for in vivo applications of gene delivery targeting endothelial cells.     

Evaluation of circulation profiles of liposomes coated with hydrophilic polymers having different molecular weights in rats.

The purpose of this study was to evaluate the circulating properties of liposomes coated with modified polyvinyl alcohol (PVA-R) having different molecular weights (6000, 9000 and 20000). The size controlled liposomes (egg phosphatidylcholine (or distearoylphosphatidylcholine):cholesterol=7:3 in a molar ratio) were prepared by the hydration method followed by sonication. Polymer coated liposomes were prepared by just mixing the resultant liposomal suspension and a polymer solution. The effects of polymer coating were evaluated by measuring the circulation time of the injected liposomes after i.v. administration in rats and the dispersing property of the liposomes in a biological condition. The circulation of the PVA-R coated liposomes was prolonged with increasing the molecular weight of PVA-R. The aggregation and/or fusion of the liposomes in the presence of serum in vitro was also depressed more by coating the liposomes with PVA-R having higher molecular weight. There was a good correlation between the circulation time and the physical stability of non-coated and the various PVA-R coated liposomes. The prolonged circulation time of PVA-R (molecular weight: 20000) coated liposomes (ca. 1.3 mol% coating) was comparable to that of a stealth liposome prepared with 8 mol% of DSPE-PEG (molecular weight of PEG: 2000).     

Prevention of antibody-mediated elimination of ligand-targeted liposomes by using poly(ethylene glycol)-modified lipids

One of the major obstacles in the development of ligand-targeted liposomes is poor liposome circulation longevity as a result of antibody-mediated elimination of these highly immunogenic carriers. Because studies from our laboratory suggest that it is not possible to reduce the immunogenicity of ligand-conjugated liposomes by using surface-grafted poly(ethylene glycol) (PEG), we investigated the usefulness of PEG in protecting hapten-conjugated liposomes from elimination by an existing immune response that was previously established against the hapten. Using biotin as a model hapten, a strong biotin-specific antibody response was generated in mice by using bovine serum albumin-biotin. When these animals were challenged with liposomes containing biotin-conjugated lipid (1 or 0.1%), these liposomes were rapidly eliminated. Incorporation of PEG-lipids into these liposomes substantially reduced biotin-specific antibody binding as measured using an in vitro antibody consumption assay. However, depending on the hapten concentration, significant reductions in antibody binding through the use of PEG-lipids may not be sufficient to protect these liposomes from rapid elimination in vivo. Complete protection of liposomes was only achieved when the biotin concentration on liposome surface was low (0.1%) and with 5 mol% of either 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] or 1,2-dipalmatoyl-sn-glycero-3-phosphoethanolamine-n-methoxy(polyethylene glycol)-2000]. The use of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (up to 15 mol%) was not effective in protecting liposomes from rapid elimination in vivo, indicating the limited usefulness of this highly exchangeable PEG-lipid. In conclusion, our in vivo and in vitro data indicate that liposomes can be protected from antibody-mediated elimination by using the right type and concentration of PEG-lipids. This result has important implication in the development of ligand-targeted liposomes.     

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16 ± 0.2 and 0.32 ± 0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06 ± 0.02 and 0.14 ± 0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.     

Intracellular drug delivery by sulfatide-mediated liposomes to gliomas.

We described here a liposomal carrier system in which the targeting ligand was sulfatide, a glycosphingolipid known to bind several extracellular matrix (ECM) glycoproteins whose expression was highly up-regulated in many tumors. In vitro experiments with human glioma cell lines demonstrated that robust intracellular uptake of the liposomes depended specifically on the presence of sulfatide as the key liposomal component. Significant amount of the liposomes remained largely intact in the cytoplasm for hours following their internalization. When anticancer drug doxorubicin (DOX) was encapsulated in such liposomes, most of the drug was preferably delivered into the cell nuclei to exert its cytotoxicity. Use of this drug delivery system to deliver DOX for treatment of tumor-bearing nude mice displayed much improved therapeutic effects over the free drug or the drug carried by polyethylene glycol (PEG)-grafted liposomes. Our results demonstrate a close link between effective intracellular uptake of the drug delivery system and its therapeutic outcome. Moreover, the sulfatide-containing liposomes (SCL) may represent an interesting ligand-targeted drug carrier for a wide spectrum of cancers in which sulfatide-binding ECM glycoproteins are expressed.     

Liposomes and disaccharides as carriers in spray-dried powder formulations of superoxide dismutase.

In this study, we aim to investigate the effects of disaccharides and liposome carriers on the activity, solid state characteristics, structural preservation, and aerosol powder performance of spray-dried superoxide dismutase (SOD) formulations. Sucrose, trehalose, and lactose were selected as stabilizing adjuvants in the spray-drying process. Dipalmitoyl phosphatidylcholine (DPPC) was the major lipid component for preparing liposomes. These SOD formulations were characterized with SOD activity assay, particle size, residual moisture content, scanning electron microscopy (SEM), electron spectroscopy for chemical analysis (ESCA), differential scanning calorimeter (DSC), and Fourier transform infrared (FT-IR) spectroscopy. We found that the inlet/outlet temperature of spray drying can be performed up to 168/122 degrees C with maintaining the activity of SOD in the SOD\DPPC\sucrose formulation for 98%. The SEM image of this formulation showed wrinkled and raisin-like appearance. Aerosol powder performance test demonstrated that this formulation exhibited excellent emitted dose (ED, 71%), aerodynamic diameter (2 microm), and respirable fraction (RF, 72%). DSC study suggested an indication of initial electrostatic stabilization of SOD by DPPC and sucrose, the following lipid perturbation by SOD, and the formation of an inclusion complex, thus minimizing the individual transition peaks of SOD and DPPC. FT-IR study showed that the major secondary structure of SOD, beta-sheet, was maintained in this formulation. The surface ESCA analysis of this formulation suggested the absence of SOD on the surface region of the powders, indicating that SOD was well surrounded and protected by DPPC and sucrose. Spray drying has been demonstrated to be a feasible process to preserve the activity of SOD in the formulation of DPPC liposomes with sucrose.     

Preparation of in vivo cleavable agglomerated liposomes suitable for modulated pulmonary drug delivery.

In an attempt to achieve post-inhalation modulation of drug release rate, Bhavane et al. have recently proposed a microparticle agglomerate of nano-sized liposomal particles, with the agglomeration process consisting of chemical cross-linkages that are capable of cleavage [Bhavane et al. J. Cont. Rel 93 (2003) 15-28.]. There, the in vitro modulation of release from agglomerated liposomes encapsulating the antibiotic ciprofloxacin was demonstrated. However, the cleaving agents used in the previous studies are not acceptable for in vivo use. In the present work therefore, a new generation of in vivo compatible agglomerated liposomes has been developed. The release rate of encapsulated compounds from these carriers can be modulated by the addition of mild thiolytic cleaving agents such as cysteine. Specifically, an amino terminated PEG conjugate has been successfully synthesized, similar to the conjugate proposed by Zalipsky [Bioconjugate Chemistry, 10 (5) (1999) 703-707.]. This conjugate contains a dithiobenzyl urethane linkage between the lipid and the PEG, cleavable by the addition of cysteine. The amines at the distal ends of the PEG are used to cross-link the liposomes into agglomerates by the addition of a suitable cross-linking agent reactive towards amines. The cross-linkages were cleaved by cysteine at the DTB sites, resulting in changes in the size distribution of the agglomerates, as well as changes in the release rate of the encapsulated drug.     

Basic approach to application of liposomes for cancer chemotherapy.

The method for augmentation of systemic in vivo anticancer effect of liposomes (Lip) containing adriamycin (ADM) and endocytosis activity of cancer cells to liposomal preparations have been studied. Encapsulation of ADM in liposomes increases its maximal tolerated dose and pretreatment of animals bearing tumor with tumor necrosis factor alpha (TNF) resulted in effective targeting of ADM-Lip to tumor, leading to its augmented therapeutic effect, but only when TNF and ADM-Lip were administered with an appropriate interval. All human tumor cell lines tested showed endocytosis activity to liposomes but the activity was differed among different tumor cell lines.     

Folate-Targeted Gene Transfer in Vivo

Nonviral systemic delivery is one of the most attractive approaches for cancer gene therapy. To achieve this goal, various laboratories have developed cationic liposomes. However, when injected intravenously, cationic lipid–DNA complexes accumulate mostly into and transfect lung tissue. Here, we describe a method by which these complexes can be targeted to tumors using folic acid. Adding polyethylene glycol (PEG)–lipids to the complexes dramatically reduced both lung accumulation and gene transfer to lungs and tumors after intravenous administration. The presence of folic acid at the distal end of the PEG–lipid did not modify tumor accumulation of the complexes. However, with folate-targeted complexes, gene transfer activity was restored in tumors while the activity in lungs was reduced by 50- to 100-fold compared with nontargeted lipid–DNA complexes. This approach provides a first in vivo proof of concept to achieve targeted tumor gene delivery.     

Antioxidant status and hepatic lipid peroxidation in chloramphenicol-treated rats

The present study reports the enzymatic and non-enzymatic antioxidant status and hepatic microsomal lipid peroxidation in chloramphenicol treated rats. Chloramphenicol at a dose of 28 mg/kg body weight orally administered to rats increased the activity of cytosolic superoxide dismutase by 63% while the activities of glutathione peroxidase and catalase were decreased by 57% and 44%, respectively. In vitro, chloramphenicol altered the activities of these enzymes though not as pronounced as the effect of the drug on the enzymes in vivo. The levels of serum vitamins A, C and beta-carotene were significantly decreased following chloramphenicol treatment. Microsomal lipid peroxidation was markedly and significantly increased by chloramphenicol treatment. The drug elicited 69% and 71% increases in the levels of malondialdehyde and lipid hydroperoxide respectively. Glutathione level and glutathione S-transferase activity were decreased by 42% and 58%, respectively, compared to untreated controls. Overall, the results of the present investigation indicate alteration of enzymatic and nonenzymatic antioxidant status and induction of lipid peroxidation by chloramphenicol. The clinical implications in the detoxification of toxic metabolites of lipid peroxidation caused by chloramphenicol warrant co-administration with antioxidant vitamins in chloramphenicol treatment regimen.     

Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice.

Using mice as a model, we recently reported that the long-circulating properties of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG(2000)-liposomes) disappeared when they were intravenously injected at certain intervals [referred to as the "accelerated blood clearance (ABC) phenomenon"]. Herein, we report on a study of issue of whether physicochemical properties of a prior dose of liposomes such as degree of PEGylation, PEG chain length, lipid dose, surface charge, size, play a role in inducing this phenomenon. The injection of conventional liposomes (without a PEG-coating) significantly induced the phenomenon. The PEGylation of conventional liposomes attenuated the induction of the phenomenon somewhat with increasing molar content of PEG derivative and PEG chain length. These findings clearly suggest that the PEGylation of liposomes are not the major cause of the ABC phenomenon but, rather, played a role in preventing it. In addition, increasing the lipid dose in a prior dose of mPEG(2000)-liposomes (0-25 micromol/kg) increased the induction of the phenomenon in a sigmoid manner. The surface charge and size of the liposomes were not critical for the induction of the phenomenon, although generally these serve as determinants in the biodistribution of liposomes. The results reported here clearly indicate that the physicochemical properties of a prior dose of liposomes strongly affect the pharmacokinetic behavior of a subsequent injection of mPEG(2000)-liposomes: The extent of PEGylation and the lipid dose had an effect, but the surface charge and size did not. The results reported herein have a considerable impact on the design and engineering of liposomal formulations for use in multiple drug therapy as well as in therapy that involves the use of liposomal drugs.     

Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes

Sterically stabilized liposomes are considered promising carriers of therapeutic agents because they can facilitate controlled release of the drugs, thereby reducing drug-related toxicity and/or targeted delivery of drugs. Herein, we studied the pharmacokinetics and biodistribution of repeated injections of radiolabeled polyethyleneglycol (PEG) liposomes. Weekly injections of (99m)Tc-PEG liposomes dramatically influenced the circulatory half-life in rats. Biodistribution 4 h after the second dose showed a significantly reduced blood content (from 52.6 +/- 3.7 to 0.6 +/- 0.1% injected dose (ID), P <.01) accompanied by a highly increased uptake in the liver (from 8.1 +/- 0.8 to 46.2 +/- 9.8%ID, P <.01) and in the spleen (from 2.2 +/- 0.2 to 5.3 +/- 0.7%ID, P <.01). At subsequent injections the effect was less pronounced: after the fourth dose, the pharmacokinetics of the radiolabel had almost returned to normal. The same phenomenon was observed in a rhesus monkey, but not in mice. The enhanced blood clearance of the PEG liposomes also was observed in rats after transfusion of serum from rats that had received PEG liposomes 1 week earlier, indicating that the enhanced blood clearance was caused by a soluble serum factor. This serum factor was a heat-labile molecule that coeluted on a size exclusion column with a 150-kDa protein. In summary, i.v. administration of sterically stabilized PEG liposomes significantly altered the pharmacokinetic behavior of subsequently injected PEG liposomes in a time- and frequency-dependent manner. The observed phenomenon may have important implications for the repeated administration of sterically stabilized liposomes for targeted drug delivery.     

Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors

The folate receptor is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anticancer agents to folate receptor-expressing tumors. This study examines folate-lipid conjugates as a means of enhancing the tumor selectivity of liposome-encapsulated drugs in a mouse lymphoma model. Folate-derivatized polyethylene glycol (PEG3350)-distearoyl-phosphatidylethanolamine was post-loaded at various concentrations into the following preparations: radiolabeled PEGylated liposomes, PEGylated liposomes labeled in the aqueous compartment with dextran fluorescein, and PEGylated liposomal doxorubicin (PLD, Doxil). We incubated folate-targeted radiolabeled or fluorescent liposomes with mouse J6456 lymphoma cells up-regulated for their folate receptors (J6456-FR) to determine the optimal ligand concentration required in the lipid bilayer for liposomal cell association, and to examine whether folate-targeted liposomes are internalized by J6456-FR cells in suspension. Liposomal association with cells was quantified based on radioactivity and fluorescence-activated cell sorting analysis, and internalization was assessed by confocal fluorescence microscopy. We found an optimal ligand molar concentration of approximately 0.5% using our ligand. A substantial lipid dose-dependent increase in cell-associated fluorescence was found in folate-targeted liposomes compared with nontargeted liposomes. Confocal depth scanning showed that a substantial amount of the folate-targeted liposomes are internalized by J6456-FR cells. Binding and uptake of folate-targeted PLD by J6456-FR cells were also observed in vivo after i.p. injection of folate-targeted PLD in mice bearing ascitic J6456-FR tumors. The drug levels in ascitic tumor cells were increased by 17-fold, whereas those in plasma were decreased by 14-fold when folate-targeted PLD were compared with nontargeted PLD in the i.p. model. Folate-targeted liposomes represent an attractive approach for the intracellular delivery of drugs to folate receptor-expressing lymphoma cells and seem to be a promising tool for in vivo intracavitary drug targeting.     

Polymer vesicles in vivo: correlations with PEG molecular weight

PEG-modified lipid vesicles have already shown considerable utility in delaying vesicle clearance from the circulation. They are, however, limited in their ability to stably integrate high molar ratios of PEG–lipid due to the high curvature and micellar preference of the very large hydrophilic PEG chain. Polymersomes, by contrast, are vesicles composed entirely of PEG-based block copolymer amphiphiles that are not only more proportionately designed, but also have already been shown to considerably broaden the range of vesicle properties (e.g. stability). Here, polymersomes composed of varying length copolymer chains were injected into rats and found to have in vivo circulation times, τ_1/2, up to about two-fold longer than PEGylated, or Stealth, liposomes. The dependence of τ_1/2 on PEG molecular weight is nonetheless limited by uptake into the liver and spleen—as with liposomes. In vitro incubations of polymersomes in plasma indicate gradual opsonization through plasma protein adsorption, such that, when vesicles are held in an optical trap and presented to a phagocyte, rapid engulfment occurs only after incubation times of similar magnitude to τ_1/2. The stealthiness introduced to liposomes through PEGylation is thus extended here with completely synthetic polymersomes.