Hiroshi Kiwada

The size of liposomes: a factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs

The size of liposomes has been shown to be an important factor in the efficient delivery of an antitumor agent to a tumor. In this paper, the effects of the size of liposomes on the pharmacokinetics of liposomes and liposome-encapsulated drugs are discussed with reference to: (1) the circulation amount and residence time of liposomes in the blood, (2) the accumulation of liposomes in the tumor, and (3) in vivo drug release from liposomes. In addition, the effect of size on therapeutic activity (antitumor efficacy and toxicity) of a liposomal anticancer preparation is discussed. Finally we discuss the importance of liposome size in the design of a more effective liposomal antitumor preparation.

Enhanced Hepatic Uptake of Liposomes Through Complement Activation Depending on the Size of Liposomes

The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56°C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.

Accelerated clearance of PEGylated liposomes in rats after repeated injections

Polyethylene glycol-modified liposomes (PEGylated liposomes) represent promising carrier systems for therapeutic agents. Herein, we report on a study of the effect of repeated injection of PEGylated liposomes on their pharmacokinetics in rats. The first dose resulted in a reduction in the circulation time and an increase in hepatic accumulation of the second dose in a time-interval of injection-dependent manner. No significant increases in the number of Kupffer cells were detectable, although the liver most likely played an important role in the accelerated clearance. Interestingly, the acceleration in clearance became less pronounced, when the third dose was injected at 4, 7 or 14 days after the second injection (the second dose was given 5 weeks after the first injection). An accelerated clearance was evoked in normal rats by the transfusion of serum from rats that had received PEGylated liposomes 5 days earlier, indicating that humoral serum factor(s) are also involved in causing the accelerated clearance. A complement consumption assay indicated that the complement system is not the factor. In summary, multiple injections of PEGylated liposomes clearly altered their pharmacokinetic behavior in rats. It is likely that cellular immunity (Kupffer cells) and humoral immunity are required to cause the phenomenon. The results reported here have a considerable impact in and important implications on the clinical application, design and engineering of PEGylated liposomes for use in repeated intravenous administration.

Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization.

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.

Accelerated clearance of a second injection of PEGylated liposomes in mice.

We recently reported that the firstly injected PEGylated liposomes dramatically affected the rate of blood clearance of secondly injected PEGylated liposomes in rats in a time interval of injection dependent manner [J. Control. Release (2003)]. Mice are frequently used in evaluations of the therapeutic efficacy of PEGylated liposomal formulations, but the pharmacokinetics of repeatedly injected PEGylated liposomes in mice is not fully understood. In this study, therefore, we examined in mice the effect of the repeated injection of PEGylated liposomes on their pharmacokinetics. An intravenous pretreatment with PEGylated liposomes produced a striking change in the biodistribution of the second dose which was given several days after the first injection. The first dose resulted in a reduction in the circulation half-life of the second dose. The degree of alteration was dependent on the time interval between the injections. The rapid clearance of the second dose was strongly related to hepatic clearance (CLh). This finding suggests that a considerable increase in hepatic accumulation accounts for this phenomenon. But, no liver injury or an increase in the number of Kupffer cells were detected in histopathological evaluations. Collectively, although the multiple injections of the PEGylated liposomes had no obvious physical effects, such as inflammation, their pharmacokinetic behavior was clearly altered in mice. The results obtained here have important implications not only with respect to the design and engineering of liposomes for human use, but for evaluating the therapeutic efficacy of liposomal formulations in experimental animal models as well.

Particle size-dependent triggering of accelerated blood clearance phenomenon.

A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2 nm in diameter) or PEGylated liposomes (119, 261 or 795 nm) were preadministered into BALB/c mice. Three days after the preadministration [(3)H]-labeled PEGylated liposomes (127 nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2-795 nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7-31.5 nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.

Lipoplex size determines lipofection efficiency with or without serum.

In order to identify factors affecting cationic Iiposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining Iipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.

T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes

Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [(3)H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [(3)H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.

Contribution of complement system on destabilization of liposomes composed of hydrogenated egg phosphatidylcholine in rat fresh plasma

Large multilamellar vesicles (MLV) composed of hydrogenated egg phosphatidylcholine (HEPC), cholesterol (CH), and dicetyl phosphate (DCP) rapidly release part of an entrapped aqueous marker when incubated with fresh rat plasma and thus have severely limited usefulness as drug carriers. The mechanisms causing the instability of liposomes in plasma were investigated in this study. The leakage of liposomal constituents was completely inhibited by pre-heating at 56 degrees C for 30 min with plasma or by treating with EDTA, K-76COOH, or anti-C3 antiserum but was not inhibited with EGTA/MgCl2. These results indicated that the destabilization of liposomes in fresh rat plasma was induced by activation of the alternative complement pathway (ACP). Furthermore, the complement third component (C3) was detected from the liposomes incubated with fresh plasma by SDS-PAGE followed by Western blotting and immune detection. The C3b deposited on the liposomal surface via ACP was rapidly cleaved to iC3b. The results obtained in the present study suggest a possibility that the liposomes composed of HEPC (without any surface modification) may be effective carriers for macrophages because C3b and its degradative products, iC3b are related to the opsonic function on phagocytosis of foreign particles by macrophages.

Evasion of the Accelerated Blood Clearance Phenomenon by Coating of Nanoparticles with Various Hydrophilic Polymers

The accelerated blood clearance (ABC) phenomenon is induced upon repeated injections of poly(ethylene glycol) (PEG)-coated colloidal carriers. It is essential to suppress this phenomenon in a clinical setting because the pharmacokinetics must be reproducible. In this study, we evaluated the induction of the ABC phenomenon using nanoparticles coated with various hydrophilic polymers instead of PEG. Nanoparticles encapsulating prostaglandin E1 were prepared by the solvent diffusion method from a blend of poly(lactic acid) (PLA) and block copolymers consisting of various hydrophilic polymers and PLA. Coating of nanoparticles with poly(N-vinyl-2-pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) led to extended residence of the nanoparticles in blood circulation in rats, although they had a shorter half-life than the PEG-coated nanoparticles. The ABC phenomenon was not induced upon repeated injection of PVP-coated nanoparticles at various time intervals, dosages, or frequencies, whereas it was elicited by PEG-coated nanoparticles. In addition, anti-PVP IgM antibody, which is estimated to be one of the crucial factors for induction of the ABC phenomenon, was not produced after injection of PVP-coated nanoparticles. These results suggest that the use of PVP, instead of PEG, as a coating material for colloidal carriers can evade the ABC phenomenon.