Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes.

It is well known that steric stabilization of the surface of liposomes by a polyethyleneglycol (PEG) conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended circulation times of the liposomes (t1/2 approximately 20 h in rat). Recently, we reported on the "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. We also reported that abundant binding of IgM, secreted into the blood stream after the first dose and, to PEGylated liposomes, plays an essential role in the induction of the ABC phenomenon. Spleen is well known to play a central role in the immune reaction and to produce IgM following a bacterial infection. The aim of the present study was to determine whether spleen contributes to the induction of the ABC phenomenon and to unravel its role in the phenomenon. In rats that were splenectomized (surgical removal of spleen) prior to the first injection of liposomes (0.001 micromol phospholipids/kg), the ABC phenomenon was totally abolished. In these rats serum IgM concentrations as well as the amounts of IgM bound to PEGylated liposomes were substantially reduced. Splenectomy attenuated the ABC phenomenon when performed until 3 days post-first injection. Removal of the spleen 4 days post-first injection left the ABC phenomenon unchanged. This finding indicates that the immune reaction in the spleen against the PEGylated liposomes occurs during at least 2-3 days following the first administration and then IgM reactive to PEGylated liposomes is produced. The present study proves that the spleen plays a critical role in the induction phase of the ABC phenomenon. For effective clinical application, many liposomal drug formulations will require multiple injections. The ABC phenomenon described in this and several preceding papers therefore has important implications for the development and evaluation of therapeutically useful liposomal formulations requiring multiple-dose administration.


Inducing effect of liposomalization on the transdermal delivery of hydrocortisone: creation of a drug supersaturated state.

In order to investigate the effect of liposomal drugs on skin delivery, it was postulated that the process of liposomalization might lead the drug to an overpredicted solubility state which has far-reaching implications for drug skin permeation and accumulation. In this regard, conventional (CL) and flexible liposomes (FL) were prepared by the lipid film hydration method and the particles were downsized by sonication using hydrocortisone (HC) as a poorly water soluble model drug. The solutions derived from the whole CL and FL suspensions eluted on a Sephadex G-50 column (SG-50) demonstrated that most part of HC not only resides solely in the water phase but also it might exist in an improved solubility state. The results of the in vitro study using rat abdominal skin and occlusive application indicated that HC penetrated and accumulated much better solely than when associated with CL or FL. In regard to the penetration of the non-entrapped HC associated to liposomes bilayer fragments, a very small amount of phospholipids in the non-liposomal part eluted on SG-50 was found that could not justify by itself the penetration of HC associated to liposome bilayer fragments. It was proposed that all the steps of the liposomes preparation process might contribute for the increased HC solubility state, but definitively the presence of phospholipids played a crucial role on improving the HC solubility in the absence of sodium cholate. In comparison with commercially available ointments, the non-entrapped HC solution derived from the whole CL suspension eluted on SG-50 showed a higher concentration of HC accumulated and more uniformly distributed as well in the epidermis and dermis compartments. In addition, the thermodynamic activity of the non-entrapped HC solutions maintaining a driving force of the drug across the skin barrier pointed out that the level of HC solubility achieved during liposome preparation has far-reaching implication for drug skin permeation and accumulation in the experimental conditions used. The findings also indicated that the non-entrapped drug solutions obtained on the process of liposomalization could be useful on transdermal drug delivery systems, particularly for improving the permeation and accumulation capacity of poorly soluble drugs.


Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes.

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.


Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes.

Steric stabilization of the surface of liposomes by a PEG conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended their circulation times (t(1/2) approximately 20h in rat). Recently, we reported on the "accelerated blood clearance phenomenon", causing "invisible" PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. In addition, we reported that certain serum factor(s) secreted into the blood after the first dose of PEGylated liposomes play an essential role in the phenomenon. The aim of the present study was to identify the major serum protein(s) responsible for the phenomenon and to unravel their mode of action. The amount of protein binding to PEGylated liposomes during incubation with serum hardly correlated with the extent of the phenomenon. PEGylated liposomes incubated with serum obtained from rats pre-injected 5 days before with the same liposomes showed a much more complex pattern of bound proteins than when incubated with naïve serum, as revealed by 2D-PAGE and SDS-PAGE. Subsequent analysis with LC-MS/MS and Western blot showed that the major pre-treated serum protein binding to PEGylated liposomes was IgM. Semi-quantitative analysis showed that larger amount of IgM bound to PEGylated liposomes compared to conventional liposomes. It was further demonstrated that PEGylated liposomes could activate the complement system due to IgM binding when incubated in serum from rats pre-injected with PEGylated liposomes, while conventional liposomes were not. These findings suggest that the selective binding of IgM to the second injected PEGylated liposomes and the subsequent complement activation by IgM resulted in the accelerated clearance and enhanced hepatic uptake of the second injected PEGylated liposomes. Based on the results described here, we are drawing attention to the potential occurrence of unexpected immune reactions upon intravenous administration of PEGylated liposomes or other particles and, by extension, PEGylated proteins and DNAs.


Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection.

Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.


Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood.

We have reported that targeted, pH-sensitive sterically stabilized liposomes are able to increase the cytotoxicity of DXR in vitro against B lymphoma cells, but the rate of release of DXR in plasma was too rapid to permit the results to be extended to in vivo applications. The purpose of the study reported here is two-fold. First, to understand the mechanism of the rapid release of DXR from pH-sensitive sterically stabilized liposomes (PSL) in human plasma. Second, to reformulate the above liposomes to improve their drug retention, while retaining their pH sensitivity. The stability of the PSL formulations in human plasma was evaluated by comparing the rate of release of encapsulated DXR with that of HPTS, a water-soluble fluorescent marker. Since DXR, but not HPTS, a water soluble-less membrane permeable fluorescence marker, was rapidly released from liposomes in the presence of plasma, the rapid release of DXR is likely caused by the diffusion of DXR molecules through the lipid bilayer, not by the disruption of the membrane. In order to develop more stable PSL formulations, various molar ratios of the membrane rigidifying lipid, hydrogenated soy HSPC and/or CHOL, were added to the lipid composition and the rate of release of encapsulated solutes and pH-sensitivity were evaluated. The compositions that showed the best drug retention and pH-sensitivity were a mixture of DOPE/HSPC/CHEMS/CHOL/mPEG(2000)-DSPE at a molar ratio of 4:2:2:2:0.3 and DOPE/HSPC/CHEMS/CHOL at a molar ratio of 4:2:2:2. Our formulations, if targeted to internalizing antigens on cancer cells, may increase intracellular drug release rates within acidic compartment, resulting in a further increase in the therapeutic efficacy of targeted anticancer drug-containing liposomes.


Gene Expression in Primary Cultured Mouse Hepatocytes with a Cationic Liposomal Vector, TFL-3: Comparison with Rat Hepatocytes

We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.


Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: Effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes

We recently reported that a second dose of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG2000-liposomes) is rapidly cleared from the blood and accumulates in the liver when injected twice in the same rat or mouse at several-day intervals (referred to as the "accelerated blood clearance (ABC) phenomenon"). In the present study we observed that a high dose (5 micromol/kg) of conventional liposomes (CL: without a PEG-coating) can induce the same phenomenon, while a low lipid dose (0.001 micromol/kg) did not. The induction of the phenomenon by mPEG2000-liposomes decreased with increasing first dose (0.001-5 micromol/kg). We observed a strong inverse relationship between the dose of initially injected PEG2000-liposomes and the extent to which the ABC phenomenon was induced: the higher the dose the smaller the phenomenon. Increasing the PEG density at the liposome surface beyond 5 mol% attenuated rather than induced the induction of the phenomenon, but elongation of the PEG chain length up to M.W. 5000, had no effect. In a series of hematological, serum-biochemical and histopathological safety evaluations we observed neither acute toxicity nor any signs of hepatic damage during the induction of the ABC phenomenon. Morphological examination of the liver by transmission electron microscopy (TEM) showed extensive accumulation of the second dose of mPEG2000-liposomes in the Kupffer cells, even already after 15 min, suggesting that the PEG liposomes had somehow lost the protective effect of the surface-grafted PEG against rapid clearance. The observations reported in this paper may have a considerable impact on the design and engineering of PEGylated liposomal formulations for use in multiple drug therapy.


Influence of the physicochemical properties of liposomes on the accelerated blood clearance phenomenon in rat

We have recently reported that PEGylated liposomes (PL) are cleared rapidly from the blood circulation when they are administered twice in the same rat at certain intervals, even if the liposomes are sterically stabilized by a surface modification with PEG (referred to as the accelerated blood clearance (ABC) phenomenon, J. Control. Release, 88, 35-42 (2003)). Now we report on the influence of physicochemical properties (PEG-modification, size and surface charge) of either the first or the second dose of liposomes on the ABC phenomenon. When, for the first dose, conventional liposomes (CL; without a PEG coating) of 110-nm diameter were injected, only a very slight ABC phenomenon was observed, irrespective of the liposomal surface charge: both clearance rate and hepatic accumulation of the second injected PL were only slightly enhanced compared to those of a single dose of PL. Interestingly, when for the first injection small-size liposomes (60 nm) were used, either charged or PEG-modified, but not neutral, the ABC phenomenon was clearly manifest. Apparently, the induction of the ABC phenomenon is not only determined by the PEG coating but also by the size and surface charge of the first dose of liposomes. Also when for the second dose small-size PEGylated liposomes were used, the ABC phenomenon was observed after induction by a first injection of PL, whereas plasma kinetics and organ uptake of a second dose of negatively charged CL (NCL, 110 nm) or small-sized NCL (SNCL, 60 nm) were not altered. Apparently, the PEG coating on the second dose is essential for the liposomes to be susceptible to the ABC phenomenon. The results reported here suggest that the physicochemical properties of both the first and second dose of liposomes are important either for the induction of the phenomenon or for its expression. Our observations may have a considerable impact on the clinical application and engineering of liposomal formulations for use in multiple drug therapy.


Increased Gene Expression by Cationic Liposomes (TFL-3) in Lung Metastases Following Intravenous Injection

We recently showed that size, not surface charge, is a major determinant of the in vitro lipofection efficiency of pDNA/TFL-3 complex (lipoplex), even in the presence of serum. In this study, the effect of lipoplex size as a result of interaction with serum proteins on in vitro lipofection and the relationship of this with in vivo lipofection was examined in a murine lung metastasis model. As previously described, the pDNA to lipid ratio (P/L ratio) affected both the size and zeta potential of the lipoplex. In vitro studies also indicated that transgene expression in B16BL6 cells was largely dependent on the size of the lipoplex, both in the absence or presence (50% (v/v)) of serum. An in vivo lipofection experiment showed that predominant gene expression in lungs occurred only in tumor-bearing mice, not in normal mice. Based on the in vitro study, this tumor-related gene expression was not related to lipoplex size in the presence of serum (50% (v/v)), suggesting that the size alteration, as the result of interactions with serum proteins in the blood stream may not play an important role in the case of systemic injections. In addition, the efficient gene expression in tumor-bearing lung was not related to the progression of lung metastases. The area-specific gene expression in tumor-bearing lungs, which was largely dependent on the P/L ratio of the lipoplexes, was observed by fluorescent microscopy. Although the underlying mechanism for the area-specific transgene expression is not clear, it may be related to the interaction of lipoplexes with tumor cells, vascular endothelial cells under angiogenesis and normal cells in the lungs. The possibility that TFL-3 is a useful utility to the targeted delivery of pDNA to lungs and tumor-related lipofection is demonstrated. This result suggests that area-specific gene expression in lung metastases may be achieved by controlling the physicochemical properties of the lipoplex, i.e. the P/L ratio.


Cell type-specific gene expression, mediated by TFL-3, a cationic liposomal vector, is controlled by a post-transcription process of delivered plasmid DNA

The issue of whether the TFL-3, a recently developed cationic liposome, achieves efficient gene expression in different mammalian cell lines (NIH/3T3, LLC, A431 and HeLa cells) was examined. The issue of whether gene expression is related to the amount of plasmid DNA (pDNA) delivered in cells or nuclei following transfection was also examined. The cells were transfected for 1h with pDNA/TFL-3 lipoplexes, and the transfection efficiency was determined by means of a luciferase activity assay. The amount of intracellular and intranuclear pDNA following the transfection was also quantitatively determined. Successful transgene expressions in all cell lines we tested were observed under our experimental conditions, suggesting that the TFL-3 represents a suitable nonviral vector system for the successful gene expression in mammalian cells in vitro. The degree and rate of gene expression were dependent on the type of cells used as well as the incubation time after transfection, but these parameters were independent of the amount of gene delivered to cells and nuclei. These results suggest that TFL-3 mediated gene expression is largely controlled by the process of post-transcription of the delivered pDNA, and not by the process of cellular entry of pDNA and cytoplasmic trafficking of pDNA into nuclei, which is dependent on the cell type. Therefore, the results obtained here clearly suggest that the cell type-specific improvement in transcription efficiency of pDNA and translation of the derived mRNA, together with an improved delivery system to enhance the nuclear delivery of pDNA, is necessary to achieve efficient transgene expression in mammalian cells.


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.


An assessment of relative transcriptional availability from nonviral vectors

To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.


Culture Time-Dependent Gene Expression in Isolated Primary Cultured Rat Hepatocytes by Transfection with the Cationic Liposomal Vector TFL-3

The development of a carrier system that enables the transfer of a functional exogenous gene to non- or less frequently dividing mammalian cells is essential for increasing the available options for the treatment of various diseases. The issue of whether TFL-3, a recently developed cationic liposome, can be successfully used to achieve gene expression in primary cultured rat hepatocytes was examined. The hepatocytes were transfected for 4 h with plasmid DNA (pDNA) in TFL-3 at various time points after 4-h preculture. The transfection efficiency was determined at various times posttransfection with pDNA coding for chloramphenicol acetyltransferase (CAT), luciferase, or beta-galactosidase. The amount of intranuclear pDNA present, as a consequence of the lipofection, was also quantitatively determined. Successful lipofections were observed for all pDNA tested, and the efficiencies were superior to that of commercially available LIPOFECTAMINE under our experimental conditions. The degree and rate of gene expression were dependent on incubation time prior to lipofection as well as on the density of the cells per dish, but this relationship did not hold for the amount of gene delivered to the nuclei. These results indicate that TFL-3 could be a useful vector for achieving sufficient gene expression in rat hepatocytes and suggest that the culture time prior to and following lipofection, which is related to the biological condition of the cells, may be one major factor affecting efficient gene expression in nondividing cells.


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


Cationic liposome-mediated gene delivery:Biophysical study and mechanism of internalization Arch

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 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.


Lipoplex size determines lipofection efficiency with or without serum.

In order to identify factors affecting cationic liposome-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 lipofection 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.


HEPC-based liposomes trigger cytokine release from peripheral blood cells: effects of liposomal size, dose and lipid composition


Quantitative Analysis of Correlation between Nunber of Nuclear Plasmids and Gene Expression Activity after Transfection with Cationic Liposomes

A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.


Effect of Cationic Liposomes in an in Vitro Transcription and Translation System

The effects of cationic liposomes complexed with plasmid DNA on the process of transcription was examined using a recently developed rapid cell free translation system. The findings indicate that the liposome itself inhibited the process when the ratio of DNA/liposome typically used in transfection studies was used.


Encapsulation of an antivasospastic drug, fasudil, into liposomes, and in vitro stabiliy of the fasudil-loaded liposomes gradient.

The objectives of this work were to develop a liposomal fasudil, an antivasospastic drug, as a possible means to deliver the encapsulated drug to the brain, and to characterize the stability of the liposomal formulation in vitro. Transmembrane electrochemical gradients of H+ or ammonium sulfate were created, and their effect on the uptake of fasudil into preformed hydrogenated soy phosphatidylcholine/cholesterol (HSPC/CHOL) liposomes were examined. Fasudil was successfully loaded into preformed liposomes in response to sulfate ion (SO4(2-)) and, in part, by H+. Encapsulation levels approaching 100% could be achieved up to a drug to lipid ratio of 0.364 (mol/mol). A stability study of the fasudil-loaded liposomes was performed by storage at 4 degrees C in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffer (pH 7.4) and by incubation in human cerebrospinal fluid (CSF) at 37 degrees C. The formulations were stable with respect to drug retention as well as size alteration, for the period studied. A leakage study clearly showed the sustained release properties of the fasudil-loaded liposomes in human CSF. We recently reported that the intrathecal administration of liposomal fasudil significantly decreased ischemia, with no obvious adverse effect in a rat model [Neurol. Med. Chir. 41 (2001) 109]. Taken together, efficient encapsulation of fasudil into preformed liposomes, their long-term stability at 4 degrees C and the sustained release characteristics in CSF indicate that fasudil-loaded liposomes could be potential candidates for further clinical evaluation.


Comparative Analysis of In Vitro and In Vivo Pharmacokinetic Parameters Related to Individual Variability of GTS-21 in Canine

To clarify the cause of the canine individual variability in plasma concentration after oral administration of GTS-21, we evaluated in vitro the metabolism to 4-OH-GTS-21 in liver microsomes of the same individuals from in vivo pharmacokinetic study. First, we applied to the Michaelis-Menten kinetic parameters to a dispersion model, and compared hepatic availability (F(H)) and hepatic clearance (CL(H)) values from in vitro with bioavailability (F), hepatic plasma flow (Q(PH)), and plasma clearance (CL(P)) values from in vivo. The ratios of CL(H) to Q(PH) were ranged 0.74 to 0.94, suggesting that GTS-21 is a hepatic plasma flow-limiting drug. A significant correlation of F(H) and F in the four dogs (r=0.995, p=0.005) indicates that the variability is predominantly caused by GTS-21 O4-demethylase activity. Second, we specified the cytochrome P450 (CYP) enzymes that are involved with the metabolism by chemical inhibition. alpha-Naphthoflavone, furafylline, quinidine, quinine, and troleandomycin significantly inhibited GTS-21 O4-demethylase activity. Thus CYP1A, CYP2D15, and CYP3A12 were involved with O4-demethylation. The variability in control activity decreased on addition of alpha-naphthoflavone and furafylline. Third, we quantified the contents of CYP1A and CYP3A12 by enzyme-linked immunosorbent assay. The content of CYP1A was consistent with GTS-21 O4-demethylase activity. We concluded that canine liver CYP1A causes the individual variability in GTS-21 plasma concentration after oral administration.


First-pass of GTS-21 on canine gut wall and liver determined by portal-systemic concentration difference

To clarify the cause of the canine individual variability of plasma concentration after oral administration of GTS-21 [(E)-3-(2,4-dimethoxybenzylidene)-3,4,5,6-tetra-hydro-2,3'-bipyridine dihydrochloride], we evaluated the absorption ratio (F(A)), intestinal availability (F(G)), and hepatic availability (F(H)). The bioavailability (F) was evaluated from the ratio of the area under the plasma concentration versus time curves after oral and intravenous administration. Three isoflurane anaesthetised dogs were fitted with an electromagnetic flow probe attached to the portal vein and cannulated through the portal and the femoral veins. After intraduodenal administration of GTS-21, both plasma concentrations were determined simultaneously. F(A) x F(G) was calculated from the portal-systemic concentration difference taking into consideration the blood-plasma partition ratio. F(A) was calculated from the residual drug contents of the small intestine. F(H) was calculated by dividing F by F(A) x F(G). The F values were 0.072, 0.021, and 0.037, indicating an individual variability of ca. threefold. The F(A) values were close to 1, and the F(G) values ranged from 0.449 to 0.461. Accordingly, the F(H) values were estimated at 0.170, 0.047, and 0.083. GTS-21 was completely absorbed but lost by first-pass effects of passage through the gut wall and liver. The first-pass effect of liver is larger than that of the gut wall, and dominates the individual variability in plasma concentration.

European Journal of Pharmaceutical Sciences


Effect of cholesterol content in activation of the classical versus the alternative pathway of rat complement system induced by hydrogenated egg phosphatidylcholine-based liposomes

Liposomes composed of hydrogenated egg phosphatidylcholine (HEPC) and cholesterol (CHOL) were found to activate the rat complement (C) system in a CHOL content-dependent manner. Liposomes containing 22 or 33 mol% CHOL activated the C system in a Ca(2+)-dependent manner, suggesting that C activation occurred via the classical pathway. Liposomes containing 44 mol% CHOL activated the C system in a Ca(2+) independent manner, suggesting that C activation occurred via the alternative pathway. The CHOL content appeared to dictate the pathway by which the C system was activated. This C activation was inhibited by removal of serum component(s), which adsorb to the liposomes. Activation of the alternative pathway, induced by the liposomes, was reduced by the depletion of IgG and IgM, whereas the classical pathway activation was reduced by the depletion of IgG, but not IgM. In addition, the removal of adsorbed serum component(s) by treatment with 44 mol% CHOL-containing liposomes decreased serum IgG and IgM levels that adsorb to the same liposomes, whereas the removal of adsorbed serum component(s) by treatment with 22 mol% CHOL-containing liposomes only slightly decreased serum IgG levels, which adsorbs to the same liposomes. Collectively, both IgG and IgM, which are specifically adsorbed to the liposomes in a CHOL-content dependent manner, were responsible for C activation via the alternative pathway induced by the 44 mol% CHOL containing liposomes. IgG alone would be partially responsible for C activation via the classical pathway induced by 22 or 33 mol% CHOL-containing liposomes. The discovery of this unique C-activating property of liposomes will be of value in attempts to decipher the underlying mechanism of C activation by providing a useful model membrane system.


Species Difference in Correlation between in Vivo/ in Vitro LiposomeComplement Interactions

The objective of this study was to investigate the correlation between in vitro and in vivo liposome-complement interactions. Third component of the complement (C3) fragments associated with hydrogenated egg phosphatidylcholine (HEPC)-based liposomes in vivo and complement-dependent destabilization in vitro were determined as an indication of liposome-complement interaction in vivo and in vitro, respectively. C3 fragments on the liposomes were detected in both rats and guinea pigs. Pretreatment with K76COOH (K76), a complement inactivating agent, reduced the binding of C3 fragments. These findings indicated that the liposomes remarkably activated the complement system in both animals in vivo. Interestingly, significant complement-dependent liposome destabilization was observed in rat serum, but not in guinea pig serum, indicating that the liposomes activated the complement system in rats, but not in guinea pigs in vitro. Taken together, it is apparent that in vitro complement activation by the liposomes is not in agreement with in vivo complement activation in ginea pigs. This discrepancy in the liposome-complement interaction would suggest the need for further investigation to utilize the information obtained from the liposome-complement interaction to predict in vivo behavior of the liposomes.


Efficacy of Intrathecal Liposomal Fasudil for Experimental Cerebral Vasospasm after Subarachnoid Hemorrhage

To investigate the safety and efficacy of liposomal fasudil in a sustained-release form for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). Eighteen rats were divided into three groups, each of which received 2.5 mg/kg of liposomal fasudil, 5 mg/kg of liposomal fasudil, or drug-free liposomes after SAH. Next, experimental SAH was induced in 15 dogs by injection of autologous arterial blood into the cisterna magna twice after baseline vertebral angiography. In six dogs, 0.94 mg/kg of liposomal fasudil was injected into the cisterna magna (treatment group). In four dogs, drug-free liposomes were similarly injected (placebo group), and the remaining five dogs were not treated with liposomal injection after SAH (control group). Angiography was repeated on Day 7, and cerebrospinal fluid was collected before the dogs were killed. A high dose of liposomal fasudil caused no significant changes in mean arterial blood pressure and did not induce seizures during the observation period. Gross and microscopic examination of the brains revealed no abnormalities, but severe vasospasm was noted in the rat basilar artery, mainly in the group treated with drug-free liposomes. Likewise, in the canine placebo and control groups, significant vasospasm occurred in the basilar artery on Day 7. In the treatment group, vasospasm in the basilar artery was significantly ameliorated (P < 0.01). In vivo, 90% of fasudil was released from liposomes in the cerebrospinal fluid. A single injection of intrathecal liposomal fasudil is safe and effective for the prevention of vasospasm in experimental SAH.


The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig

In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.


Biodistribution of liposomes and C3 fragments associated with liposomes:evaluation of their relationship

The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.


Optimization of antitumor effect of liposomally encapsulated doxorubicin based on simulations by pharmacokinetic/pharmacodynamic modeling

It has been reported that long circulating liposomes enhanced the antitumor effect of doxorubicin (DOX) by increasing delivery of DOX to tumor tissues. However, there is no quantitative information on the relationship between the antitumor effect and liposomal characteristics governing the release rate of entrapped drugs, although the importance of drug release-rate control from liposomes has been pointed out. Here, we developed a physiological model for free and liposomal DOX to calculate the time course of free DOX in the extracellular space and linked this with a cell kill kinetic model to quantify the antitumor effect of DOX. Simulations were performed to clarify the relationship between antitumor effect and pharmacokinetic or physicochemical parameters of liposomes, as well as pharmacological or physiological parameters of tumor tissues. The importance of long circulation time of liposomes was confirmed. The optimum rate of drug release from long circulating liposomes was found at the release rate constant of around 0.06 h(-1). This optimum value was not dependent on the tumor proliferation time, sensitivity of tumor cells to DOX, or the tumor blood flow-rate. This simulation indicated that the optimization of the delivery to tumor tissue by long circulating liposomes could be possible by changing the release rate of DOX for the maximum antitumor effect.


Development of a pharmacokinetic/pharmacodynamic(PK/PD)-simulation system for doxorubicin in long circulating liposomes in mice using peritoneal P388

The objective of this study was to develop a simulation system that optimizes the pharmacokinetic parameters of drug carriers for anticancer agents in order to maximize their anticancer effects. The pharmacokinetic/pharmacodynamic (PK/PD) model of doxorubicin (DOX) encapsulated into liposomes has been developed for mice and each parameter required for simulations was obtained in the peritoneally inoculated P388 leukemia model in mice. PK parameters, which describe the dispositions of free and liposomally encapsulated DOX, were obtained by kinetic analysis of experimental data in this study, as well as from literature. PD parameters, which describe the growth and death rate of cancer cells in vivo, were also determined. The PK/PD model developed in this study is capable of simulating the time course of the number of cancer cells quantitatively and evaluating the significance of each parameter on the carrier system for DOX. Simulations based on the PK/PD model predict the optimum rate of drug release from long circulating liposomes as 0.06 h(-1) for maximum anticancer effect. Thus, this simulation system provides useful information relative to the optimization of drug carriers for DOX.


In Vivo Studies on the Role of Complement in the Clearance of Liposomes in Rats and Guinea Pigs

The ability of complement (C) system to remove liposomes from blood circulation was examined in vivo using rat and guinea pig as models. Although the liposomes were not degraded in guinea pig serum in vitro, they were degraded remarkably in guinea pig circulation, as assessed by the urinary excretion of [3H]inulin released from liposomes. The suppression of rat C system to 64% normal C hemolytic activity by treating animals with K76COOH agent resulted in a significant decrease in both the uptake of liposomes by liver and the release of [3H]inulin, providing in vivo evidence for C-mediated clearance of liposomes in rats via uptake by macrophages and degradation in blood circulation, respectively. On the other hand, the K76COOH-induced suppression of C (70% normal hemolytic activity) in guinea pigs slightly increased both the hepatic uptake and the release of [3H]inulin. In addition, the hepatic uptake and in vivo degradation in guinea pigs varied in an opposite manner when the animals were preloaded by empty liposomes or when the liposome size and cholesterol content varied. These results suggest there is a difference between the factors involved in liposome degradation and the factors involved in hepatic uptake and also support the likelihood that there is no C-mediated degradation in guinea pigs.


Design and synthesis of cholestane derivatives bearing a cascade-type polyol and the effect of their property on a complement system in rat serum

Bioorganic & Medicinal Chemistry Letters


血中滞留性リポソーム封入ドキソルビシンの腹腔癌における抗腫瘍効果防御システムの構築


Intracellular Regulation of Macromolecules using pH-senstive Liposomes and Nuclear Localization Signal : Qualitative and Quantitative Evaluation of intracellular Trafficking.

The objective of this study is to present a rational strategy to target macromolecules to the nucleus via the endocytic pathway. The two major barriers in this route to the nucleus are known as endosomal escape and nuclear transport. pH-sensitive liposomes were used in order to achieve endosomal escape under the conditions of low pH in endosomes. Bovine serum albumin (alb) served as a model compound to be delivered to nucleus and was encapsulated into the pH-sensitive liposomes. The liposomes are composed of dioleoyl phosphatidyl ethanolamine: cholesterylhemisuccinate. They were taken up by rat peritoneal macrophages via endocytosis and subsequently underwent degradation, principally by lysosomal enzymes. By using pH-sensitive liposomes, intracellular degradation was reduced by a significant extent, as expected, via endosomal escape. Cytosolic delivery of FITC-labelled alb was also detected by confocal microscopy. Selective targeting to the nucleus was performed by adding the nuclear localization signal (NLS) of the SV-40 large T antigen to the FITC-alb, which were then encapsulated into the pH-sensitive liposomes. Confocal microscopy revealed that FITC-alb, in the presence of NLS was successfully delivered into nucleus, while no transport was observed in the absence of NLS. These results provide a useful strategy for the nuclear targeting of macromolecules using pH-sensitive liposomes in conjunction with NLS.

Biochemical and Biophysical Research Communications


Complement Dependent and Independent Liposome Uptake by Peritoneal Macrophages:Cholesterol Content Dependency

The uptake mechanisms of liposomes by rat peritoneal macrophages (PMs) were investigated. Incubation of liposomes with fresh rat serum enhanced the uptake of liposomes depending on the liposome size and cholesterol (CH) content. The binding of liposomes was also enhanced by serum, and this increase depended on the size and CH content as in the case of liposome uptake, which suggested that the binding of opsonized liposomes with PMs govern the extent in liposome uptake. The rate constant for the internalization (k(int)) was calculated by measuring both uptake and binding. The k(int) cannot explain the variation of liposome uptake for different sizes and CH contents. The kint values for liposomes with high (44%) and medium (33%) CH contents were constant (2.5 h(-1)) , while those for liposomes with low (22%) CH content were significantly elevated (5-9 h(-1)). These results indicate the presence of at least two kinds of uptake mechanisms of liposomes. Treatment of serum with anti-C3 antibody completely inhibited the enhanced uptake of CH-high, large liposomes, which suggested that complement receptor-mediated phagocytosis may be an uptake mechanism for CH-high and -medium liposomes. In addition, complement-independent enhanced uptake was suggested for CH-low liposomes, since no inhibition was observed for CH-low liposomes by anti-C3 antibody and these liposomes were disintegrated in serum via complement-independent pathway. These results provided evidence that PMs take up liposomes via complement-dependent and independent mechanisms depending on the CH content of the liposomes.


Kinetic Analysis of the Interaction between Liposomes and the Complement System in Rat Serum:Re-evaluation of Size-Dependency

The size of liposomes is considered to be an important factor in determining the liposome-complement interaction. In this study, the release of carboxyfluorescein (CF) from liposomes was measured continuously for three different diameters (800, 400 and 200 nm) by changing the liposome concentration from 1 to 1000 nmol/ml. At a low liposome concentration range (1-10 nmol/ml), small liposomes (200 nm) released CF to a similar extent (approximately 35%) as in the medium (400 nm) and large (800 nm) liposomes. The affinity (Km) and capacity (Lmax) of a complement system to release liposomally encapsulated CF were estimated by kinetic analysis of the liposome-complement interaction. Surprisingly, there was no remarkable size dependency in the Km and Lmax in terms of liposome number, although these parameters depended on the size of liposomes in terms of lipid concentration. These results indicated the possibility that the complement system does not discriminate according to liposome size.


Mechanism of initial distribution of blood-borne colon carcinoma cells in the liver

The distribution characteristics of a human colon carcinoma cell line, KM12-HX cells, were examined. After intraportal vein (i.p.v.) or intravenous (i.v.) injection into rats, almost all the injected tumor cells are distributed to liver or lung, respectively, both after 30 s and 30 min. Our previous kinetic analysis of the fate of tumor cells revealed that the cumulative amount of tumor cells distributed in the liver is a factor determining the degree of metastasis. Thus, we examined the mechanism of initial efficient trapping of tumor cells by the liver in more detail. Thirty minutes after tumor cells were injected into the left ventricle of the heart, the distribution of tumor cells was more restricted in several tissues (kidney, small intestine, large intestine and spleen), as compared with the distribution of microspheres undergoing 100% extraction, indicating that the first-pass extraction of KM12-HX cells is incomplete in these organs. The hepatic first-pass distribution of these tumor cells was unaffected by pretreatment of liposomes, such that the preinjected amount was sufficient to saturate the phagocytotic function of macrophages. Thus, the mechanism of initial distribution of the tumor cells to the liver is different from the mechanism of liposome uptake by macrophages. Considering that the diameter of microvessels in sinusoid and KM12-HX cells is approximately 7 and 12 microm, respectively, it is possible that these tumor cells are trapped physically in hepatic microvessels. In fact, after i.p.v. injection of microspheres 5 microm in diameter, only 20% of the dose was distributed to liver and the rest to other tissues. In contrast, almost 100% of microspheres 10 microm in diameter were distributed to the liver. These results support the hypothesis that the initial organ distribution of blood-borne tumor cells is determined by mechanical and physical properties of the cells.


A novel plasma factor initiating complement activation on cetylmannoside-modified liposomes in human plasma


Characterization of Bovine Serum Factor Triggering the Lysis of Liposomes via Complement Activation


リポソーム膜中のコレステロール含量が及ぼす補体第3成分(C3)の結合と体内動態への影響


Enhancing effect of cholesterol on the elimination of liposomes from circulation is mediated by complement activation


Effect of Serum Components from Different Species on Destabilizing Hydrogenated Phosphatidylcholine-Based Liposomes


セチルマンノシド修飾リポソームへのC3フラグメントの結合における粒子径の影響

セチルマンノシド修飾リポソームへのC3フラグメントの結合における粒子径の影響


リポソームによる抗癌剤の抗腫瘍効果の最適化に関する速度論的研究

リポソームによる抗癌剤の抗腫瘍効果の最適化に関する速度論的研究


In vivo実験系による異種リポソーム間の肝取込における競合機構の解明

In vivo実験系による異種リポソーム間の肝取込における競合機構の解明


Biopharmaceutical evaluation of the liposomes prepared by rehydration of freeze-dried empty liposomes(FDELs)with an aqueous solution of a drug

We have evaluated a method for preparation of a dispersion of liposomes encapsulating a drug, namely rehydration of freeze-dried empty (not containing drug) liposomes with an aqueous drug solution (FDEL method). In the present study, we characterized and compared this method with the conventional method using a lipid composition of DPPC-DPPG-cholesterol in a molar ratio of 27:3:20. Two hydrophilic compounds, [3H]-inulin and [3H]-mannitol, were used as model drugs. Liposomal preparations by the FDEL method had an encapsulation efficiency of 2.9 and 6.7% for [3H]-inulin and [3H]-mannitol, respectively, when rehydrated and incubated at 70 degrees C. Since non-specific adsorption of these markers to liposomal membrane is negligible, this method produces liposomes which encapsulate a drug in the intravesicular space. One-tenth of the marker encapsulated in the liposomes prepared by the FDEL method (F-liposomes) was released very rapidly on incubation with rat plasma, followed by the slow release of the remaining fraction thereafter. No such rapid-release phase was observed for the liposomes prepared by the conventional method (C-liposomes). This suggests the existence of two types of encapsulation, loose encapsulation and tight encapsulation, in F-liposomes at least. Pharmacokinetic parameters of marker encapsulated tightly in F-liposomes were comparable to those in C-liposomes. It is likely that amphipathic drugs such as doxorubicin are incorporated into liposomes more easily than inulin and mannitol when formulated by the FDEL method. These results therefore suggest that the FDEL method is useful in the preparation of a liposomal formulation of a drug.


Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate

Kupffer cells were selectively eliminated in mice by the intravenous administration of liposome-entrapped dichloromethylene diphosphonate. At 5 days, small peroxidase-negative and acid-phosphatase-weakly-positive macrophages appeared, increased in number, and differentiated into peroxidase- and acid-phosphatase-positive Kupffer cells. Repopulating small macrophages actively proliferated, and the number of Kupffer cells returned to the normal level by day 14. The numbers of macrophage precursors in the liver as detected by the monoclonal antibodies ER-MP20 and ER-MP58 increased after liposome-entrapped dichloromethylene diphosphonate injection. ER-MP58-positive cells proliferated and differentiated into ER-MP20-positive cells and eventually into BM8-positive Kupffer cells in the liver. Bone-marrow-derived ER-MP58-positive cells were also detectable in the liver and differentiated into ER-MP20-positive cells, but they did not become BM8-positive macrophages. Macrophage colony-stimulating factor mRNA expression was enhanced in the liver 1 day after injection. The administration of macrophage colony-stimulating factor did not shorten the period of Kupffer cell depletion but increased the number and the proliferative capacity of repopulating Kupffer cells. These findings implied that repopulating Kupffer cells are derived from a macrophage precursor pool in the liver rather than from bone-marrow-derived monocytes. Local production of macrophage colony-stimulating factor in the liver plays a crucial role in the differentiation, maturation, and proliferation of Kupffer cells.


Synergistic Effect Between Size and Cholesterol Content in the Enhanced Hepatic Uptake Clearance of Liposomes Through Complement Activation in Rats


Size-dependent release of carboxyfluorescein from cetylmannoside-modifide liposomes in human plasma


Species difference in the disposition of liposomes among mice, rats, and rabbits: Allometric relationship and species dependent hepatic uptake mechanism

The species difference in the pharmacokinetics of liposomes was investigated in mice, rats and rabbits. Liposomes were intravenously injected at doses of 1, 10 and 100 (nmol/g body weight), and the time courses of liposomes in blood, liver and spleen were measured. Pharmacokinetic parameters were regressed as a function of body weight (BW) and dose of liposomes (D). The uptake mechanism of liposomes was also examined with the isolated perfused liver between rats and mice. Mean residence time increased with the increase of BW and D of liposomes. This increase of mean residence time resulted from the decreased total body clearance, which was principally explained by the species difference in the hepatic uptake clearance (CLh) of liposomes. The parameter CLh was regressed well by a multiple regression as a function of BW and D. In this analysis, an exponent for BW was around 0.5, which clearly indicates that smaller animals have higher uptake clearance per unit BW. Immunohistochemical analysis revealed that there was no significant difference in the density of Kupffer cells among these species. This suggest that the species difference in CLh resulted not from the density of Kupffer cells but from the uptake ability of Kupffer cells among species. In the isolated perfused liver, the hepatic uptake of liposomes was mainly explained by opsonin dependent uptake in rats, while opsonin independent uptake in mice. These quantitative and qualitative information on the species difference of liposome disposition will provide an useful information for constructing a drug delivery system using liposomes.


Is control of distribution of liposomes between tumors and bone marrow possible?

The objective of this study is to clarify to what extent the accumulation of liposomes from the blood into the tumor and bone marrow can be controlled by liposome size and membrane fluidity. Liposomes with different diameters (50-400 nm) and different membrane fluidity were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol (Ch) and dicetylphosphate in various molar ratios. These liposomes were injected intravenously into rats bearing Yoshida sarcoma, and the ratios of the accumulation of liposomes in the tumor to those in the bone marrow, liver and spleen were compared. The tumor-to-bone marrow accumulation ratio increased with the decrease in liposome size from 400 to 50 nm. This ratio was greater than those for the liver and spleen at all sizes. Although tumor-to-liver accumulation ratios of 50- and 100-nm HEPC-containing liposomes were higher than those of EPC-containing liposomes, no obvious difference in tumor-to-bone marrow or tumor-to-spleen accumulation ratios was found between these liposomes. Tumor-to-bone marrow accumulation ratio of HEPC-containing liposomes increased remarkably with the decrease in Ch content from 40 to 30 or 20 mol% compared with ratios for the liver and spleen. Interestingly, the tumor uptake clearance of liposomes of the same size was constant regardless of their membrane fluidity. These findings show that the increases in these accumulation ratios are due to their decreased uptake clearance by the bone marrow. Furthermore, the uptake of 50-nm HEPC-containing liposomes by the bone marrow was specifically inhibited by preinjection of other liposomes, but not when they were exposed in advance to in vivo components. These observations suggest the involvement of in vivo component(s) in the uptake of these liposomes by the bone marrow. We conclude that small HEPC-liposomes with low Ch content show their significantly decreased uptake by the bone marrow due to their decreased recognition by this tissue.

Biochimica et Biophysica Acta (BBA) - Biomembranes


リポソームの細胞内分解過程に及ぼす投与量の影響

リポソームの細胞内分解過程に及ぼす投与量の影響


リポソーム封入クロドロネートによるKupffer細胞の障害機序

リポソーム封入クロドロネートによるKupffer細胞の障害機序


Effects of the size and fluidity of liposomes on their accumulation in tumors:A presumption of their interaction with tumors


Effect of vesicle size on in vivo release of daunorubicin from hydrogenated egg phosphatidylcholine-based liposomes into blood circulation

The effect of the vesicle size on in vivo release of daunorubicin, an anthracycline anticancer drug, from liposomes into the circulation was studied in rats. The liposomes were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol and dicetylphosphate at a molar ratio of 5:4:1, and their mean vesicle sizes were adjusted to about 50 and 100 nm. The drug retained in the liposomes and the drug that leaked were separated from plasma by gel filtration. On the assumption that the lipid content does not change, the drug release from each liposome preparation was estimated from the latency (%) calculated from the drug/lipid concentration ratio of the liposome preparation. From HEPC-liposomes with a diameter of 50 nm, the drug was released gradually after intravenous administration, and the cumulative percent release of the drug reached 40% after 240 min. However, from EPC-liposomes with the same size, 50% of the drug was released within 5 min, and more than 90% of the drug was released within 60 min. From HEPC-liposomes with a diameter of 100 nm, no drug release was observed for 240 min after administration. These findings indicate that the vesicle size and the lipid composition of liposome preparation affect the in vivo drug release.


Effects of Membrane Fluidity on Accumulation of Small Unilamellar Vesicles in Liver and Tumor

小さい一枚膜リポソームの肝臓回避と癌組織集積に及ぼす膜流動性の影響


Effect of cetylmannoside modification on the alternative complement pathway activation by liposomes in rat serum

Complement activation is important for removing foreign substances by the mononuclear phagocyte system in vivo. The interaction between liposomes and complement components is considered to affect the clearance of liposomes from the circulation. It has been previously demonstrated in our laboratory that multilamellar vesicles (MLV) with surfaces modified by cetylmannoside (Man) were eliminated from the circulation rapidly and showed an approximately 2-fold higher hepatic accumulation compared with control MLV (PC-MLV) (Yamashita et al., Int. J. Pharmaceut., 70, 225, 1991). In this study, we investigated the effect of Man-modification on complement system activation. As far as elimination from the blood is concerned, the initial values of blood liposome concentration were decreased and liposomes were removed from the circulation rapidly in accordance with the extent of the Man content into their membranes. The Man-modification also affected the organ distribution of injected liposomes and their stability in rat serum. Except for MLV containing 50 mol% Man, it was observed that the hepatic uptake of liposomes was enhanced according to the increasing Man content, whereas splenic uptake was decreased and the splenic clearance was comparable. The stability of liposomes in rat serum decreased with increasing Man content. Liposomal instability in rat serum was significantly reduced by preheating the serum at 56 degrees C for 30 min, the treatment with anti-C3 antiserum and with EDTA but not abolished in serum treated with EGTA/MgCl2. Thus, it is considered that the activation of the complement system through the alternative pathway is facilitated as a result of increasing the Man content in the liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetic modelling of liposome degradation in peritoneal macrophages

The objective of this study was to quantify and model the degradation process of liposomes in peritoneal macrophages (PMs). Iodinated albumin (125I-alb) was chosen to be the marker of liposome degradation. The time course of the degradation of free 125I-alb after pinocytosis by PMs followed first-order kinetics with a half-life of 23 min. The degradation of liposomally encapsulated 125I-alb was also quantified. Kinetic modelling of liposome degradation indicated the existence of two kinetically different processes, one with a half-life of 13 min and the other with a half-life of 7.5 h. Comparing the degradation of liposomal and free 125I-alb suggested that 125I-alb was delivered to lysosomes much faster through phagocytosis than pinocytosis. These results indicate that the intracellular degradation kinetics of pinosomes and phagosomes is different. This method can quantify the rate and extent of liposomal degradation in macrophages and provide kinetic information on the intracellular destiny of liposomally encapsulated compounds.


Size dependent liposome degradation in blood:In vivo/in vitro correlation by kinetic modeIing

The degradation of liposomes in blood circulation is important in regulating the releasing rate of encapsulated compounds. In this study, the effect of liposome size--one of the principal determining factors in liposome disposition--on their degradation in serum/blood was evaluated quantitatively both in vitro and in vivo. In the in vitro study, the time courses of the degradation of liposomes in fresh rat serum were measured continuously using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker and were described by the kinetic model with the lag time (tau), first order degradation rate constant (k), and the maximum degradation (alpha). Both k and alpha increased with the increase of liposome size, which indicated a higher affinity of larger liposomes for complement activation. In the in vivo study, the degradation of liposomes was evaluated sensitively by a first order degradation rate constant (kd) in blood circulation. The kd was obtained by kinetically modeling the liposome degradation in vivo using 3H-inulin as an aqueous phase marker. The size dependent kd correlated well with the hepatic uptake clearance, which suggests an underlying complement activation mechanism common to both degradation and hepatic uptake of liposomes. There was a good correlation in the degradation rate constant between in vitro and in vivo trials. These kinetic analyses validate the quantitative evaluation of liposome degradation in blood circulation and provide a useful way to predict the degradation of liposomes in vivo from in vitro experiments.


Solubilization by Triton X-100 makes possible complete recoverery of lipids from liposomes in enzymatic assay

We have developed an accurate and sensitive method for enzymatically determining phosphatidylcholine (PC) and cholesterol (CHOL) in liposomes. Solubilizing liposomes with a high concentration (80%) of Triton X-100 at 65 degrees C for 5 min led to the complete recovery of the lipids by current assay using commercial kits. The method had good linearity in a range of 0.004-0.4 mumol PC. Using this method, PC and CHOL were completely recovered from various liposomes. We conclude that PC and CHOL in liposomes can be determined accurately and sensitively by this method.


Effects of ascorbic acid on iproniazid-induced hepatitis in phenobarbital-treated rats

The effects of ascorbic acid (AA) on hepatic injury induced by iproniazid (IPN) in phenobarbital-treated rats were investigated by the evaluation of hepatic function using the clearance of aminopyrine (AM). Either IPN or isopropylhydrazine (IP-Hy), a potent toxic metabolite of IPN, were administered as a pretreatment to rats with or without AA. After i.v. injection of AM, the blood concentration of AM was determined by capillary gas chromatography by isotope dilution analysis using deuterium-labeled AM (AM-d9) as the internal standard. The kinetic parameters of AM, Vd, kel and total body clearance, were estimated from the time course of blood concentration. Pretreatment with IPN with AA led to a marked increase in the kel and in the clearance compared with pretreatment using IPN alone. A significant increase in the kel and the clearance was also found in the case of combined pretreatment using IP-Hy with AA. The effects of AA on the hepatic injury induced by IPN were studied according to its histological aspects. In the specimens obtained following the administration of IPN or IP-Hy with AA, the degree of cell necrosis was remarkably lowed both quantitatively and qualitatively. The present results clearly demonstrate that AA was effective in reducing IPN-induced hepatitis.


Effects of Benidipine Hydrochloride(Coniel*),a new Calcium Antagonist,on the Cardiac Output,Regional blood Flow and Vascular Resistance in Conscious,Spontaneously Hypertensive Rats

Benidipine hydrochloride is a calcium antagonist with a 1,4-dihydropyridine derivative structure, and exhibits long-lasting antihypertensive effects by inhibiting the voltage-dependent Ca2+ channels. This study was undertaken to examine the effect of benidipine on central haemodynamics and regional blood flow (RBF) after intravenous administration of benidipine in conscious, spontaneously hypertensive rats. The microsphere method was used to measure cardiac output and RBF before and after the drug administration, using microspheres labelled with 57Co and 51Cr. Thirty minutes after the intravenous administration of benidipine (3 micrograms kg-1), the mean arterial pressure fell by 15% without significantly increasing the heart rate. The cardiac output increased by 41% and the systemic resistance decreased by 39%. Benidipine significantly increased RBF by 37, 35, and 22% in kidney, heart, and small intestine, respectively, and decreased vascular resistance by 38, 38, and 32%, respectively. We concluded that benidipine reduced blood pressure by increasing RBF in the kidney and heart, while keeping RBF in other organs at a normal level. These results will provide a fundamental basis in support of the clinical benefits of benidipine for hypertensive patients, particularly those with renal failure.


Effects of Fluidity and Vesicle Size on Antitumor Activity and Myelosuppresive Activity of Liposomes Loaded with Daunorubicin

The effects of fluidity and vesicle size on the antitumor activity and myelosuppressive activity of liposomes loaded with daunorubicin, an anthracycline antitumor drug, were investigated in Yoshida sarcoma-bearing rats. Liposomes composed of egg phosphatidylcholine (EPC) or hydrogenated egg phosphatidylcholine (HEPC), cholesterol and dicetyl phosphate in a molar ratio of 5:4:1 were injected intravenously into rats 5 d after subcutaneous inoculation of Yoshida sarcoma. At non-effect dosage in free drug, HEPC-liposomes with a diameter of 58 or 142 nm showed the greatest inhibitory effect against Yoshida sarcoma among liposomes tested, whereas larger ones (272 nm) had weaker effect. Small EPC-liposomes (57 nm) had no effect. Larger HEPC-liposomes (especially 142 nm) greatly decreased the number of peripheral white blood cell compared with free drug at the same dose, indicating relatively strong myelosuppressive toxicity. However, small EPC- and HEPC-liposomes with a diameter of 57 and 58 nm, respectively, showed toxic effects comparable to that of free drug. Examination of the dose-dependency of therapeutic effects and toxicity indicated encapsulation of daunorubicin in the small HEPC-liposomes to enhance the therapeutic index about 3 times that of free drug. These findings indicate the possibility of using small HEPC-liposome as a drug carrier for targeting solid tumors.


Uptake of Liposoes Surface-Modified with Glycyrrhizin by Primary Cultured Rat Hepatocytes

Previously, we synthesized 30-stearyl glycyrrhizin (GLOSt) and reported that small unilamellar liposomes containing GLOSt (GLOSt-SUV) accumulated in the liver several times more than the control liposomes (control-SUV). In the present study, to determine the interaction between GLOSt-SUV and hepatocytes, in vitro uptake experiments were achieved with primary cultured rat hepatocytes. The uptake amount of GLOSt-SUV by rat hepatocytes was considerably higher compared to the control-SUV, while GLOSt-SUV showed about a 10-fold higher uptake level than the control-SUV during 2 h of incubation. It was assumed that GLOSt-SUV not only bind to the surface of the hepatocytes but are internalized and degraded in the cells, because at 37 degrees C, GLOSt-SUV were taken up and the level of the degradable marker was lower than the inert marker, and this did not occur at 4 degrees C. Since the uptake of GLOSt-SUV was inhibited by glycyrrhizin (GL), it was suggested that a binding-site for GL is present on the surface of hepatocytes, and GLOSt-SUV are likely to be internalized via this site by the hepatocytes. Furthermore, it was confirmed that the efficacy of GLOSt on liposomes is not affected by the fluidity of the liposomal membrane.


Kinetic modeling of liposome degradation in serum:Effect of size and concentration of liposomes in vitro

The purpose of this study is to propose a new method for quantitative evaluation of liposome degradation in serum. The time course of liposome degradation in rat serum was monitored continuously, using 6(5)-carboxyfluorescein as an aqueous phase marker. The degradation curves exhibited three characteristic phases: lag time, degradation, and plateau. This curve was described by a kinetic model with three parameters: lag time (tau), first-order degradation rate constant (k), and maximum degradation (alpha). The rate and extent of the degradation of liposomes were evaluated separately in terms of k and alpha, respectively. The effects of size and concentration of liposomes on their degradation kinetics were examined using this method. Both k and alpha increased with increasing liposomal size. The increased affinity of larger liposomes for complement was suggested to increase both k and alpha. On the other hand, alpha decreased with increasing liposomal concentration without altering k. The decreased extent of degradation was considered to result from the depletion of complement components. There was no significant effect of size and concentration of liposomes on tau. Quantitative evaluation of the rate and extent of degradation of liposomes will provide deeper insights into the interaction between liposomes and serum components, and basic information on liposomes as potential drug carriers.


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 degrees 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.


抗ヒトC3モノクローナル抗体を用いたリポソーム表面上のC3フラグメント定量

抗ヒトC3モノクローナル抗体を用いたリポソーム表面上のC3フラグメント定量


Plasma Factor Triggering Alternative Complement Pathway Activation by Liposomes


ヒドラジン遊離薬物からのフリーラジカルの生成とアスコルビン酸の効果

ヒドラジン遊離薬物からのフリーラジカルの生成とアスコルビン酸の効果


The Complement- but not Mannos Receptor-Mediated Phagocytosis is Involved in the Hepatic Uptake of Cetylmannoside-Modified Liposomes in Situ

In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These hepatic uptakes of liposomes were obviously inhibited by preheating the serum at 56 degrees C for thirty minutes or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.


キャピラリーガスクロマトグラフィによるアミノピリンの重水素標識体分離と肝機能評価への応用


Kinetic modeling of liposome degradation in blood circulation

The aim of this study is to develop a kinetic model for the quantitative evaluation of, and to examine dose dependency in liposome degradation in blood circulation in vivo. Multilamellar liposomes labeled with 3H-inulin were administered intravenously into rats and the time courses of blood concentration and urinary excretion of 3H-inulin were measured. The dosages of liposomes were fixed at 1, 5, and 100 mumolPCkg-1. Remarkable saturation was found in the time courses of both blood concentration and urinary excretion. Then a kinetic model for the degradation of liposomes in blood was developed, assuming that the degradation follows the first order rate process for each dose. The model fitted the observed time courses of excreted 3H-inulin well, and dose dependency could be observed in the rate constants for liposome degradation, which are more sensitive than urinary excretion of 3H-inulin. The degradation rate constant correlated well with the uptake rate constant, which suggests the same underlying mechanism for both uptake and degradation. These results indicate the usefulness of kinetic modeling in the quantitative evaluation of liposome degradation in blood circulation in vivo.


Distinction between the depletion of opsonins and the saturation of uptake in the dose-dependent hepatic uptake of liposomes

Opsonins play a role in the hepatic uptake of particles such as bacteria, lipid emulsion, and liposomes. The objective of this study was to distinguish between opsonin depletion and uptake saturation in the dose-dependent hepatic uptake of liposomes. The uptake of opsonized and unopsonized liposomes was determined in the isolated perfused liver. Serum (2.9 mL) was required to opsonize 1 mumol liposomes fully, indicating that a rat (250 g with 10 mL of serum) can opsonize 3.5 mumol liposomes. Next the dose effect on hepatic uptake of opsonized and unopsonized liposomes was examined. Saturation of uptake was found only for the opsonized liposomes. On the other hand, the hepatic uptake clearance decreased dose dependently from 4.31 to 0.79 (mL/min), with increasing doses from 0.075 to 17 mumol/250 g, respectively, after i.v. administration. Thus, the decrease in the hepatic uptake clearance at the medium dose was due to the saturation of uptake alone, and at the high dose it was due to opsonin depletion as well. These results show that the saturation of liposomal uptake in the liver and the depletion of opsonins occurred at different liposome dosage levels.


Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats(2)


Kinetic analysis of AUC-dependent saturable clearance of liposomes:Mathematical description of AUC dependency

Journal of Pharmacokinetics and Pharmacodynamics


In vivo evaluation of the effect of the size and opsonization on the hepatic extraction of liposomes in rats:An application of oldendorf method

In the hepatic uptake of large particles such as liposomes, a serum component called opsonin plays an important role. In this study, the 'Oldendorf method' is introduced to evaluate the hepatic extraction under the condition of single passage, which enabled examination of the effect of opsonization on liposome uptake by the intact liver. 14C-labelled liposomes and, an internal reference, 3H-H2O were injected as a bolus into portal vein. Liver uptake index (LUI) was calculated from the ratio of the extraction of 14C to that of 3H. The effect of liposome size (mean diameter of 0.8, 0.4, 0.2, and 0.05 micron) and opsonization (preincubation with fresh blood for 5 min) on liposomal hepatic uptake were investigated using this method. LUI increased with size significantly (p < 0.001), and opsonization enhanced LUI only for the large liposomes (0.8 micron). This result suggests that the critical diameter of opsonization for these liposomes lies between 0.4 and 0.8 micron.


Non-Michaelis-Menten type hepatic uptake of liposomes in the rat

The objective of this study was to verify the methodology for measuring uptake clearance of liposomes and to characterize kinetically the saturable hepatic uptake of liposomes-through phagocytosis. The correction of vascular space was important in the evaluation of hepatic uptake. The efflux of liposomes from liver was shown to be negligible, by a repeated dose study, and thus, hepatic clearance can be obtained by the hepatic uptake divided by the area under the blood concentration-time curve (AUC). The determinant parameter which describes the saturability of uptake clearance of liposomes, independent of infusion rate, was investigated, using the data of an in-vivo constant infusion study, where infusion rate-dependent saturable hepatic clearance was observed. The mean blood concentration failed to obtain an infusion rate-independent function. On the other hand, the AUC could explain the saturability of hepatic clearance for every infusion rate by a unique relationship. The hepatic uptake amount could also explain this saturability, independent of infusion rate. These kinetic characteristics are inconsistent with Michaelis-Menten type kinetics, therefore a new model is required to describe the saturable hepatic clearance in the disposition of liposomes.

The Journal of Pharmacy and Pharmacology


シメチジンのpH変化における物性の安定性

シメチジンのpH変化における物性の安定性


Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats:I

The purpose of this study was to perform a kinetic analysis of the tissue distribution of doxorubicin (DXR) and liposomes separately after intravenous administration of DXR entrapped in liposomes in rats. Liposomes were double labeled with 14C-DXR (L-DXR) and 3H-inulin (L-INU). Blood and tissues were sampled at specified times until 120 min. Blood clearance of L-DXR was similar to that of L-INU. Distribution of both L-DXR and L-INU into the liver was parallel and extensive, while in the heart, the pattern of distribution differed between L-DXR and L-INU after peak concentration. Time courses of tissue concentration were explained well by dividing tissue into a shallow compartment with efflux and a deep compartment without efflux. In the liver, pharmacokinetic parameters of L-DXR and L-INU were similar, and the two kinetically different compartments may correspond to different uptake processes in hepatic endocytosis. In the heart, the shallow compartment was considered to correspond to the cardiac vascular space, and the intercompartmental rate constant (k3) for L-DXR was much larger than that for L-INU. The estimated half-life for this process was 20 min. The half-life for the degradation of liposomes in blood circulation was also estimated at 20 min from data on the urinary excretion of released 3H-inulin. These results suggest that the release of DXR from liposomes may be the rate-limiting process in the tissue distribution of DXR to the heart.


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.

Biochimica et Biophysica Acta (BBA) - Biomembranes


マンノース修飾リポソームの安定性に及ぼす種差の影響

マンノース修飾リポソームの安定性に及ぼす種差の影響


イプロニアジドの体内動態並びにフリーラジカル中間代謝物に及ぼすアスコルビン酸の影響

Journal of the Pharmaceutical Society of Japan


[Effects of ascorbic acid on the free radical formations of isoniazid and its metabolites].

イソニアジドのフリーラジカル中間代謝物に及ぼすアスコルビン酸の影響

By the use of electron spin resonance (ESR) spectroscopy and of spin-trapping technique, the effects of ascorbic acid on the formation of the free radical intermediates due to isoniazid (INAH) and its metabolites were investigated with a microsomal system. When alpha-(4-pyridyl 1-oxide)-N-tert butylnitrone (4-POBN) was used as a spin trapping agent, the ESR signal due to hydrazine (Hy) was formed to be most intensive among others. Therefore, it was presumed that Hy is a potent intermediate to cause an INAH-induced hepatic injury. In the presence of ascorbic acid (AA), the free radical formation of Hy, INAH and acetyl hydrazine was significantly inhibited, suggesting that AA may affect the INAH-hepatitis. By the addition of inhibitors of cytochrome P-450 like metyrapone and CO, the generation of the radical from Hy decreased, confirming that the radical is formed by the cytochrome P-450 dependent microsome systems. The 4-POBN-trapped radical species generated from Hy was presumed to be the hydrazyl radical by the results of mass spectrometry.


Potential Value of Cetylmannoside-modified Liposomes as Carriers of Macrophage Activators to Human Blood Monocytes

The present study was undertaken to examine the potential value of cetylmannoside-modified multilamellar liposomes (Man-MLV) as carriers for transfer of macrophage activators to blood monocytes. Highly purified blood monocytes were isolated by centrifugal elutriation from healthy donors under endotoxin-free conditions. Freshly prepared monocytes phagocytosed Man-MLV to a lesser extent than monocyte-derived macrophages, but they took up Man-MLV much more effectively than control liposomes without cetylmannoside (control MLV). Phagocytosis of Man-MLV, but not control MLV by monocytes was inhibited by addition of D-mannose, but not of D-galactose. Desmethyl-muramyl dipeptide (norMDP) entrapped in Man-MLV was far more effective than norMDP entrapped in MLV in activating monocytes to the tumoricidal state. The effect of encapsulation of recombinant human macrophage colony-stimulating factor (M-CSF) in Man-MLV on prolongation of survival of monocytes was examined. Blood monocytes that had been incubated for up to 21 days with Man-MLV containing 5-20 U of M-CSF per ml were effective in prolonging monocyte survival, but monocytes that had been incubated in medium with less than 50 U/ml of M-CSF or with control MLV containing 5-10 U of M-CSF showed no increase of monocyte survival over that in medium alone. Addition of rabbit anti-M-CSF antiserum did not affect survival prolongation of monocytes by M-CSF encapsulated in Man-MLV. We conclude that liposomes modified with cetylmannoside are far more effective than unmodified liposomes as a carrier to deliver biological response modifiers to human blood monocytes.


Enhancing effect of cetylmannoside on targeting of liposomes to Kupffer cells in rats


Targeting of lipsomes surface-modified with glycyrrhizin to the liver I Preparation and biological disposition

We consider glycyrrhizin to be a new ligand for liposomes to the liver because it is known that about 80% of glycyrrhizin is excreted into the bile after intravenous administration in rats. In order to modify the liposomal surface with glycyrrhizin, 30-stearyl glycyrrhizin (GLOSt), one of the lipophilic glycyrrhizin derivatives, was synthesized. The structure of this new compound was identified by nuclear magnetic resonance (NMR), infrared (IR) and mass spectra (MS). Sonicated liposomes were prepared from hydrogenated egg phosphatidylcholine-cholesterol-GLOSt or dicetyl phosphate (DCP) (4:4:1) and were labelled with [3H]inulin as an aqueous marker. It was confirmed by measuring the encapsulation efficiencies and the mean diameters that GLOSt-containing sonicated liposomes (GLOSt-SUV) were SUV-type as well as DCP-containing control liposomes (control-SUV). Four hours after intravenous injection into rats at a dose of 90 mumol as total lipid per kg of rat body weight, GLOSt-SUV showed 4-fold more accumulation (42.4%) in the liver than control-SUV. Therefore, glycyrrhizin is considered to be a useful new ligand on liposomes for targeting to the liver.


Saturable,non-Michaelis-Menten uptake of liposomes by the reticuloendothelial system

Multilamellar vesicles (300-350 nm) were infused into the rat femoral vein at the rate of 4, 40 and 400 nmol phosphatidycholine min-1 for 6 h using [3H]inulin as an aqueous marker. The time courses of blood concentration of vesicles, normalized for infusion rate, were not superimposable, showing the non-linearity of liposome disposition in the blood circulation. These time courses of blood concentration were well fitted by a single Michaelis-Menten equation. On the other hand, the time courses of tissue content could not be so accommodated. Additionally, the observed relationship between the uptake of liposomes by the liver and their clearance from it and other organs differed essentially from a simulation based on Michaelis-Menten type saturable kinetics. Therefore, it is suggested that there is a time-dependent non-Michaelis-Menten type process in the phagocytosis of macrophages in the reticuloendothelial system.

The Journal of Pharmacy and Pharmacology


In vivo実験系における血中でのリポソームの安定性の定量的評価法

In vivo実験系における血中でのリポソームの安定性の定量的評価法


Effects of glucose and ascorbic acid on absorption and first pass metabolism of isoniazid in rats


マクロファージコロニー刺激因子含有マンノース修飾リポソームによるヒト単球の生存率増強効果