Liposomes with prolonged blood circulation and selective localization in Klebsiella pneumoniae-infected lung tissue.

Studies of two types of small liposomes, differing with respect to their lipid composition in terms of bilayer fluidity, charge, and hydrophilicity of the liposomal surface, were done to evaluate their usefulness for delivery of encapsulated therapeutic agents to sites of infection. The liposomes showed substantial localization in infected lung tissue after intravenous administration. This was demonstrated in a model of unilateral Klebsiella pneumoniae pneumonia in rats, in which the left lung was infected but the right lung of the same animal developed no infection. The degree of localization in the infected left lung was different for the two types of liposomes. For the liposome type with the longer blood residence time, containing a surface coating of polyethylene glycol, localization in the infected left lung was dependent on the liposomal dose, correlated with the intensity of infection, and reached 9% of the injected liposomal dose in severely infected rats.

Liposomes as carriers of antimicrobial agents or immunomodulatory agents in the treatment of infections.

Targeting of antimicrobial agents by means of liposomes is under investigation and may be of importance in the treatment of infections that prove refractory to conventional forms of antimicrobial treatment. The ability to achieve a significantly longer residence time of liposomes in plasma and limited uptake of liposomes by the mononuclear phagocyte system opens up new areas of investigation and potential therapeutic application. By manipulating the liposomal composition, rates of uptake and intracellular degradation can be influenced and thereby the rates at which liposome-encapsulated agents are released and become available to exert their therapeutic action. With respect to the targeting of macrophage modulators at the mononuclear phagocyte system by means of liposomes for maximal stimulation of the nonspecific antimicrobial resistance, experimental evidence is now available of the potential usefulness of liposomes as carriers of these agents. This approach may also be of importance for the potentiation of treatment of severe infections.

Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue.

In an experimental model of unilateral pneumonia caused by Klebsiella pneumoniae in rats we investigated whether intravenous administration of liposomes with prolonged blood circulation time resulted in significant localization of liposomes in infected lung tissue. Liposomes (100 nm) composed of hydrogenated phosphatidylinositol:hydrogenated phosphatidylcholine:cholesterol (molar ratio, 1:10:5) radiolabeled with gallium-67-deferoxamine showed relatively long blood circulation time. The degree of localization of these long circulating liposomes in the infected left lung was significantly higher compared to that of localization of 110 nm egg phosphatidylglycerol:egg phosphatidylcholine:cholesterol (molar ratio, 1:10:5) liposomes which exhibited relatively short blood circulation time. At 16 h after administration of the long circulating liposomes (when 10% of the injected dose was still present in the bloodstream) localization of liposomes in the infected left lung was increased up to 10-fold compared to the left lung of uninfected rats, and appeared to be highly correlated with the intensity of the infection. In the uninfected right lung the localization of long circulating liposomes was not increased. The degree of localization of liposomes in the infected tissue is dependent on the residence time of liposomes in the blood compartment. The extent of localization of long circulating liposomes in infected tissue appeared to be dependent on the liposomal dose administered.

Liposomes and lipid carriers in the treatment of microbial infections.

Since antibiotic treatment of severe infections is not always successful, intensification of the antibiotic treatment is needed. Targeting of antibiotics to infected tissues or cells by encapsulation in liposomes is under investigation and may be of importance in the treatment of infections that prove refractory to conventional forms of antibiotic therapy. In animal models of intracellular infections involving the mononuclear phagocyte system--parasitic, fungal, bacterial and viral infections--an improved therapeutic index and reduced toxicity resulting from encapsulation of the antibiotics in liposomes have been demonstrated. By varying the lipid composition of the liposomes it is possible to manipulate their intracellular degradation and thereby the intracellular release and therapeutic availability of the antibiotic. Efficacy of liposome-encapsulated antibiotics in the treatment of infectious diseases outside the mononuclear phagocyte system may be realized by manipulation of the liposome composition. Evidence for this is found in the treatment of systemic fungal infections, in which liposome appear to be very effective as a carrier of amphotericin B. The most advanced application of liposome-based therapy is in this field, and clinical studies with liposome-encapsulated amphotericin B have been in progress for several years.

Increased efficacy of ganciclovir and foscarnet inhibition of cytomegalovirus replication in vitro by encapsulation in liposomes.

The antiviral effect of ganciclovir and foscarnet encapsulated in liposomes was studied against cytomegalovirus in human embryonic lung fibroblast cells. Substantially greater activity (3.5-fold and 38-fold respectively) in terms of inhibition of virus replication intracellularly was found compared to that seen with non-encapsulated antiviral agents, and liposome encapsulation did not result in increased cytotoxicity.

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.

Effect of free or liposome-encapsulated muramyl dipeptide on uptake and intracellular survival of Listeria monocytogenes in mouse peritoneal macrophages in vitro.

The effect of free versus liposome-encapsulated muramyl dipeptide (MDP) on the uptake and intracellular survival of Listeria monocytogenes in mouse peritoneal macrophages in vitro was investigated. Macrophages in monolayer culture were exposed to free MDP at various concentrations during different time periods before incubation with Listeria monocytogenes. An increase in bacterial uptake dependent on the concentration of MDP and the length of exposure was observed. Exposure of macrophages to 200 micrograms of free MDP per milliliter for 15 h led to a threefold increase in bacterial uptake, resulting in an average of 15 bacteria per macrophage after 30 min of incubation. In addition, intracellular bacteria were killed in the MDP-exposed macrophages, in contrast to the intracellular multiplication of Listeria monocytogenes in macrophages not exposed to MDP. Encapsulation of MDP within liposomes resulted in a significant enhancement of its activity: liposomal encapsulation led to a 1,000-fold reduction in the amount of MDP required to obtain these effects on bacterial uptake and intracellular killing, whereas empty liposomes had no effect at all. Liposomal encapsulation may be an appropriate means of increasing delivery of the muramyl peptides to the macrophages.

Effect of lipid composition on activity of liposome-entrapped ampicillin against intracellular Listeria monocytogenes.

The effect of lipid composition on the intracellular antibacterial activity of ampicillin-containing liposomes was studied in vitro by using mouse peritoneal macrophages infected with Listeria monocytogenes. Two types of liposomes, a fluid type, consisting of cholesterol-phosphatidylcholine-phosphatidylserine (5:4:1), and a solid type, consisting of cholesterol-distearoylphosphatidylcholine-dipalmitoylphosphatidylglyc ero l (10:10:1), were used. Although the cellular uptake of both types of liposomes was similar, they differed with respect to the rate of intracellular degradation. A correlation was found between the relatively slow degradation of the solid liposomes and a delayed intracellular release of the encapsulated ampicillin, as reflected in absent or delayed intracellular killing of L. monocytogenes.

Liposome-encapsulated ampicillin against Listeria monocytogenes in vivo and in vitro.

In an experimental infection caused by Listeria monocytogenes in mice a considerable enhancement (90-fold) of the therapeutic activity of ampicillin resulting from liposomal encapsulation was observed. The mechanism by which liposomes improved the therapeutic index of ampicillin in this infection appeared to be an increased delivery of the antibiotic to the site of infection, i.e. the liver and spleen. Substantial amounts of liposomal ampicillin were recovered from isolated Kupffer cells, the target cells of L. monocytogenes after intravenous inoculation. In addition, in studies on the survival of L. monocytogenes within murine peritoneal macrophages in vitro it was found that liposomal encapsulation of ampicillin resulted in an increased availability of the antibiotic for the intracellular bacteria: liposomal ampicillin killed 90% of the intracellular bacteria, whereas a similar concentration of free ampicillin plus empty liposomes had no effect upon the intracellular bacteria.

Effect of liposome-entrapped ampicillin on survival of Listeria monocytogenes in murine peritoneal macrophages.

The effect of liposomal encapsulation of ampicillin on the antibacterial activity against intracellular Listeria monocytogenes was studied by comparing survival of L. monocytogenes within peritoneal mouse macrophages in the presence of free ampicillin alone or in combination with liposome-entrapped ampicillin. In the presence of 50 micrograms of free ampicillin per ml of the incubation medium, intracellular growth of L. monocytogenes was still observed, although less as compared with intracellular growth in the absence of ampicillin. At a concentration of 50 micrograms of free ampicillin plus 100 micrograms of liposome-entrapped ampicillin per ml, 99% of the intracellular bacteria were killed. On the other hand, a concentration of 150 micrograms of free ampicillin per ml plus empty liposomes only inhibited intracellular bacterial growth, and the bacteria were not killed. In addition, empty liposomes at a concentration of 1 mumol of lipid per ml had no effect on intracellular bacterial growth. In broth, liposome-entrapped ampicillin at a concentration of 100 micrograms/ml was not bactericidal for L. monocytogenes, indicating that significant leakage of ampicillin from the liposomes with subsequent killing of the bacteria by the free drug did not occur. Therefore, we concluded that liposomal encapsulation of ampicillin results in an increased availability of the antibiotic for the intracellular bacteria.

Free versus liposome-entrapped ampicillin in treatment of infection due to Listeria monocytogenes in normal and athymic (nude) mice.

Efficacy of liposomal as compared with free ampicillin in treatment of infection due to Listeria monocytogenes was studied in normal and congenitally athymic (nude) mice. After intravenous injection the multilamellar vesicles used were taken up mainly by liver and spleen, the target organs of L. monocytogenes. Injection of empty liposomes in two doses of 2 mumol of vesicle lipid each, administered at 40 and 112 hr after bacterial inoculation, did not influence the course of listerial infection in normal and nude mice. In normal mice liposomal entrapment of ampicillin resulted in significant improvement in the antibiotic's therapeutic index. The use of liposomal ampicillin at a total dose of 0.54 mg (two doses of 0.27 mg each) resulted in a therapeutic effect similar to that resulting from a total dose of 48 mg of free ampicillin (eight doses of 6 mg each). However, neither ampicillin treatment schedules cured infections in nude mice.