In vitro efficacy of a sterically stabilized immunoliposomes targeted to membrane type 1 matrix metalloproteinase (MT1-MMP).

The poor selective cytotoxicity of anticancer drugs lead to dose-limiting adverse effects which compromise the clinical outcome. Solid tumors recruit new blood vessels to support their growth, and epitopes that are uniquely expressed on tumor cells and tumor endothelial cells (ECs) can function as targets for immunoliposomal anticancer drugs. Membrane type 1 matrix metalloproteinase (MT1-MMP), an important protein related to tumor growth and angiogenesis, is expressed on malignant tumor cells and is activated ECs. Selective delivery could be achieved by targeting MT1-MMP, as well as other angiogenic ECs. In this regard, an anti-MT1-MMP Fab' antibody was used to prepare a MT1-MMP targeted sterically stabilized immunoliposomes (SIL[anti-MT1-MMP(Fab')]). The binding and intracellular distribution of SIL[anti-MT1-MMP(Fab')] and a non-targeted sterically stabilized liposomes (SL) were examined using human fibrosarcoma HT-1080 cells. SIL[anti-MT1-MMP(Fab')] was taken up by the cells in a lipid concentration, temperature, and time dependent manner, ultimately accumulating in the lysosomes. The cytotoxicity of doxorubicin (DXR)-containing SIL[anti-MT1-MMP(Fab')] (DXR-SIL[anti-MT1-MMP(Fab')]) was significantly higher than that of DXR-containing SL. The cellular internalization of SIL[anti-MT1-MMP(Fab')] was inhibited by endocytosis inhibitors, suggesting that their internalization was mediated via clathrin- or caveolae-dependent endocytosis. Furthermore, the efficient binding of SIL[anti-MT1-MMP(Fab')] was observed on human umbilical vein endothelial cells (HUVEC). Based on these results, it would be expected that DXR-SIL[anti-MT1-MMP(Fab')] may achieve direct tumor cell kill and indirect tumor cell kill via the destruction of the tumor endothelium in vivo. This strategy may have the potential for overcoming some major limitations in conventional chemotherapy in vivo.


Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes.

Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.


The gene-silencing effect of siRNA in cationic lipoplexes is enhanced by incorporating pDNA in the complex.

Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.


Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes.

Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the liposomes remain to be determined, the immunogenicity of 'empty' liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and efficiency of liposomes currently under development for clinical use.


Efficient gene expression in Megakaryocytic cell line using Nucleofection.

To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.


Effect of Transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells.

The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.


Accelerated blood clearance of PEGylated liposomes upon repeated injections: Effect of doxorubicin-encapsulation and high-dose first injection.

The "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection, has been reported to occur in rodents and rhesus monkeys. This rapid clearance was reported to be caused by the binding of PEG-specific IgM, which was generated by the first dose of injected liposomes, to the second dose of liposomes and the subsequent activation of complement, serving in turn as an opsonin. Although there are several PEGylated liposomal formulations, such as Doxil/Caelyx loaded with doxorubicin (DXR), in clinical use, the rapid clearance phenomenon has never been reported for such formulations. In the present article, we report that a first injection of PEGylated liposomes containing encapsulated DXR failed to induce the ABC phenomenon. Likewise, no rapid clearance of the test dose was observed when the first dose of "empty" PEGylated liposomes (without DXR) exceeded 5 micromol phospholipids/kg. By contrast, "empty" PEGylated liposomes at a low dose (1 micromol phospholipids/kg) induced the phenomenon as before. Western blot analysis revealed abundant binding of IgM to PEGylated liposomes when these were incubated in serum from rats that had received "empty" PEGylated liposomes. Substantially less binding of IgM was found when the liposomes were incubated in serum from rats treated with DXR-loaded PEGylated liposomes. For both the empty and the DXR-containing liposomes the amounts of IgM binding to the liposomes decreased with an increasing dose of injected liposomes. Serum obtained from rats following injection of empty PEGylated liposomes caused complement activation by addition of PEGylated liposomes in an inversely dose-dependent manner: the lower the dose, the higher the complement activation. By contrast, no complement activation was detected with serum from rats that had been treated with DXR-loaded PEGylated liposomes. These findings suggest that encapsulation of DXR as well as a relatively high lipid dose abrogate the immune response against PEGylated liposomes which is observed with the same liposomes but without DXR and at low doses. Our observations may thus have important implications for the development, evaluation and therapeutic use of liposomal cytotoxic drug formulations requiring multiple injection schemes.


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.


Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.

American Journal of Physiology, Heart and Circulatory Physiology


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.


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.


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.


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


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.


Use of the post-insertion technique to insert peptide ligands into pre-formed Stealth liposomes with retention of binding activity and cytotoxicity

Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line. The hexapeptide antagonist G was covalently coupled via a thioether bond to the terminus of polyethylene glycol (PEG) in micelles formed from maleimide-derivatized poly(ethylene glycol) (Mr 2000) distearoylphosphatidylethanolamine followed by transfer into preformed liposomes during a one-step incubation. For cellular association, we used radiolabeled liposomes. Cytotoxicity was evaluated using the MTT in vitro proliferation assay. The postinsertion approach to the formation of peptide-targeted liposomes led to the production of PLG bearing a maximum of approximately 0.3 microg antagonist G/micromol phospholipid. These liposomes had increased cellular association to H69 cells relative to nontargeted liposomes and, when loaded with doxorubicin, they resulted in similar levels of cytotoxicity to those obtained by conventional coupling techniques. The postinsertion technique is a simple, effective means for the production of biologically active peptide-targeted liposomes.


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.


くも膜下出血後の脳血管簾縮に対する髄液内投与可能な徐放性塩酸ファスジル(エリル)の開発


Targeted delivery and triggered release of liposomal doxorubicin (DXR) enhances cytotoxicity against human B-cell lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm following subarachnoid hemorrhage in rats


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.


Neuroprotection by intrathecal application of liposome-entrapped Fasudil in a rat model of ischemia

Pharmacological treatment for cerebral ischemia cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. The objective of this study is the development of a liposomal drug delivery system that maintains effective concentrations of protein kinase inhibitors fasudil in the CSF, resulting in neuroprotection against cerebral ischemia. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. Treated rats received 0.25 mg liposome-entrapped fasudil via the cisterna magna 2 hours after ischemic insult. Control rats received drug-free liposomes. Neurological condition and the infarct size were assessed at 24 and 72 hours after ischemia. The concentration of liposome-entrapped fasudil in the CSF was measured before sacrifice. Treated animals showed significantly improved neurological outcomes after the 24-hour observation period compared to the control group (p < 0.001). Treatment with 0.25 mg liposomal fasudil resulted in a reduction in the infarct area (24 hours: 29.0 +/- 4.4%, 72 hours: 28.1 +/- 3.9% of total brain slices) compared to controls (49.6 +/- 4.6%, p < 0.001), but there was no statistical difference between 24 and 72 hours. At 24 hours post-administration, CSF concentrations of liposome-entrapped fasudil were 45.4 +/- 31.5 micrograms/ml (20% of the injected dose). A single intrathecal injection of liposomal fasudil can maintain a therapeutic drug concentration in the CSF over a period of time, significantly decreasing infarct size in a rat model of acute ischemia.


Correlations between the rate of intracellular release of endocytosed liposomal doxorubicin and cytotoxicity as determined by a new assay

Previously, we showed that liposomes with surface-attached anti-CD19 were internalized into human B lymphoma cells through receptor-mediated endocytosis, resulting in improved anti-tumor efficacy 1-2 . In order to further increase the efficacy of antineoplastic drug-containing liposomes, we have taken advantage of this internalization process by producing triggered release liposomes that rapidly release drug from the enzyme-rich, acidic environment of lysosomes. To analyze the effectiveness of these triggered-release formulations, we developed a nuclear accumulation assay for doxorubicin (DXR) that allows us to determine the rate of cytoplasmic drug delivery subsequent to drug release from the endosomal/lysosomal compartments by examining the rate of accumulation of drug in cellular nuclei. We demonstrate the usefulness of this assay by comparing the kinetics of cytoplasmic drug delivery for DXR-containing, pH-sensitive, triggered release liposomes versus DXR-containing, non-sensitive, liposomal formulations. We see a significant correlation between the rate of nuclear accumulation of DXR and its in vitrocytotoxicity. This indicates that pH-sensitive formulations traffic drug to the cytoplasm and the nucleus significantly more rapidly than do non-sensitive formulations. We conclude that the development of triggered release liposomes is a promising strategy for further improving the therapeutic efficacy of liposomal antineoplastic drugs targeted selectively to cancer cells by surface-attached ligands that bind to internalizing epitopes.


Anti-MUC-1 immunoliposomal doxorubicin in the treatment of murine models of metastatic breast cancer

The fate of breast cancer patients is dependent upon elimination or control of metastases. We studied the effect of antibody-targeted liposomes containing entrapped doxorubicin (DXR) on development of tumours in two models of breast cancer, pseudometastatic and metastatic, in mice. The former used the mouse mammary carcinoma cell line GZHI, which expresses the human MUC-1 gene (L. Ding, E.N. Lalani, M. Reddish, R. Koganty, T. Wong, J. Samuel, M.B. Yacyshyn, A. Meikle, P.Y.S. Fung, J. Taylor-Papadimitriou, B.M. Longenecker, Cancer Immunol. Immunother. 36 (1993) 9--17). GZHI cells seed into the lungs of Balb/c mice following intravenous injection. The latter used the 4T1-MUC1 cell line, a MUC-1 transfectant of the mouse mammary carcinoma cell line 4T1, which metastasizes from a primary mammary fatpad (mfp) implant to the lungs (C.J. Aslakson, F.R. Miller, Cancer Res. 52 (1992) 1399--1405). B27.29, a monoclonal antibody against the MUC-1 antigen, was used to target sterically stabilized immunoliposomes (SIL[B27.29]) to tumour cells. In vitro, SIL[B27.29] showed high specific binding to both GZHI and 4T1-MUC1 cells. The IC(50) of DXR-loaded SIL[B27.29] was similar to that of free drug for GZHI cells. In the pseudometastatic model, mice treated with a single injection of 6 mg DXR/kg in DXR-SIL[B27.29] at 24 h after cell implantation had longer survival times than those injected with non-targeted liposomal drug. In the metastatic model, severe combined immune deficiency mice given weekly injectionsx3 of 2.5 mg DXR/kg encapsulated in either targeted or non-targeted liposomes were almost equally effective in slowing growth of the primary tumour and reducing development of lung tumours. Surgical removal of the primary tumour from mfp, followed by various chemotherapy regimens, was attempted, but removal of the primary tumour was generally incomplete; tumour regrowth occurred and metastases developed in the lungs in all treatment groups. DXR-SL reduced the occurrence of regrowth of the primary tumour, whereas neither targeted liposomal drug or free drug prevented regrowth. We conclude that monoclonal antibody-targeted liposomal DXR is effective in treating early lesions in both the pseudometastatic and metastatic models, but limitations to the access of the targeted liposomes to tumour cells in the primary tumour compromised their therapeutic efficacy in treating the more advanced lesions.


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.


A combinatorial approach to producing sterically stabilized(Stealth)immunoliposomal drugs


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


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


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