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At least 19 recordsLinked to original sources
PubMed1998

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes.

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.

Animals↗
PubMed1999

Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein.

Major histocompatibility complex (MHC) class I molecules found on antigen-presenting cells present peptides derived from cytoplasmic proteins to T cells. In contrast, peptides from exogenous proteins are mostly presented by class II molecules. It has been well established that liposomes can serve as an efficient delivery system for entry of exogenous protein antigens into the MHC class I pathway. Our previous studies utilizing fluorophore-labeled proteins encapsulated in liposomes demonstrated that after phagocytosis of the liposomes by bone marrow-derived macrophages (BMs), the processed peptides were subsequently visualized in the trans-Golgi, while free conalbumin was excluded from the trans-Golgi area. In the present study, we investigated whether liposomal lipids follow the same intracellular route as the liposomal proteins after phagocytosis by BMs. Multilamellar liposomes with different lipid compositions that also contained fluorescent phospholipids (empty liposomes) were incubated with murine BMs. Our results indicate that although empty liposomes were avidly phagocytosed by macrophages, the fluorescent liposomal lipids did not localize to any particular area of the cell but were distributed throughout the cell. In contrast, when a protein was encapsulated in the liposomes, the liposomal lipids were no longer dispersed throughout the cell, but were concentrated and localized in the trans-Golgi area. Furthermore, when the liposomes contained a fluorescent-labeled protein, the fluorescent peptides also localized to the trans-Golgi. These results demonstrate that the combination of both liposomal lipids and liposomal protein is required for Golgi-specific targeting of liposomal antigens. Transport of both liposomal lipids and liposomal proteins to the Golgi complex, a major subcellular organelle in the passage of MHC class I molecules, might explain why antigens encapsulated in liposomes readily induce cytotoxic T lymphocytes.

Animals↗
PubMed1997

Application of liposomes for development of oral vaccines: study of in vitro stability of liposomes and antibody response to antigen associated with liposomes after oral immunization.

In order to evaluate the usefulness of liposomes as oral vaccines, the stability of liposomes and serum IgA antibody response to antigen associated with liposomes after oral administration were examined. Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), and cholesterol (Chol) (1:1:2, molar ratio), distearoylphosphatidylcholine (DSPC) and Chol (7:2, molar ratio), and DSPC, DPPS, and Chol (7:3:2 or 1:1:2, molar ratio) were stable in acidic solution (pH 2.0), bile, and pancreatin solution, whereas liposomes composed of DPPC and Chol (7:2, molar ratio) and DPPC, DPPS, and Chol (7:3:2, molar ratio) were unstable in pH 2.0 and/or bile solutions. After the oral immunization of antigen (ganglioside GM1)-containing liposomes composed of DPPC, DPPS, and Chol (1:1:2, molar ratio) to mice, the serum IgA antibody responses against ganglioside GM1 were found. Furthermore, when monophosphoryl lipid A was incorporated into liposomes containing ganglioside GM1, further augmentation of IgA responses to ganglioside GM1 was observed. On the other hand, the oral administration with liposomes composed of DPPC, Chol, and ganglioside GM1 (unstable liposomes), ganglioside GM1 mixed with liposomes composed of DPPC, DPPS and Chol, and ganglioside GM1 alone was unable to induce any detectable anti-ganglioside GM1 IgA antibody responses. These results suggest that liposomes which showed the stability to acidic solution, bile, and pancreatin solution would serve effectively as an oral delivery vehicle for inducing mucosal immune responses.

Administration, Oral↗
PubMed1992

Liposomal modification with uronate, which endows liposomes with long circulation in vivo, reduces the uptake of liposomes by J774 cells in vitro.

For overcoming rapid removal of liposomes from the bloodstream, we developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). In this current study, we examined the in vitro interaction of PGlcUA-liposomes with J774 cells derived from mouse macrophages. Liposomal association with J774 cells at 37 degrees C did not increase compared with the binding at 4 degrees C when liposomes were modified with PGlcUA. RES-avoiding ability was not specifically endowed by glucuronate but by uronates in general, since palmityl-D-galacturonide showed a similar effect on liposomal clearance in vivo and liposomal uptake in vitro. These facts indicate that modification of the liposomal surface with uronic acid derivatives endows liposomes with a long circulation time in the bloodstream by reducing their uptake by macrophages.

Animals↗
PubMed2005

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.

ATP-Binding Cassette Transporters↗
PubMed2000

Delivery of cytokines by liposomes: hematopoietic and immunomodulatory activity of interleukin-2 encapsulated in conventional liposomes and in long-circulating liposomes.

Although liposomal delivery of interleukin-2 (IL-2) and other cytokines improves their pharmacokinetics and biologic activity in vivo, there are no comparative functional studies of various liposomal formulations as cytokine carriers. In the present investigation, recombinant human IL-2 was encapsulated in two formulations of large (mean diameter 0.75-1.5 microns) multilamellar vesicles (MLV, referred to as conventional liposomes) or in small (mean diameter, 60 nm), unilamellar, long-circulating liposomes (referred to as sterically stabilized liposomes, SSL). The biologic activity of the liposomal formulations and of free IL-2 was tested in parallel in vitro and in mice. The main observations were as follows: (a) All the liposomal IL-2 (Lip-IL-2) formulations were more efficient than soluble IL-2 in stimulating spleen cell proliferation and lymphokine-activated killer (LAK) cell activation in vitro, particularly at low cytokine doses (1-100 CU/mL). (b) After i.v. injection, the circulation time of MLV-IL-2 and SSL-IL-2 was 7 and 17 times greater, respectively, than that of soluble IL-2. (c) In comparison with IL-2, all Lip-IL-2 formulations caused a marked increase in the leukocyte levels in blood, spleen, and peritoneal exudate, especially in those of myeloid origin (neutrophils, eosinophils, immature granulocytes, and macrophages). (d) Although SSL-IL-2 exhibited the longest circulation time, MLV-IL-2 was more potent in elevating leukocyte levels and in triggering LAK cell activity in vivo. (e) The route of Lip-IL-2 administration greatly affected the immunomodulatory activity in the various compartments. (f) MLV-IL-2 proved to be a much more efficient immunoadjuvant than free IL-2 for influenza subunit vaccines as well as for tumor cell vaccines. These findings lend support to our previous studies in which we demonstrated the superior immunomodulatory activity of liposomal IL-2, and suggest that cytokine pharmacokinetics, biodistribution, and pharmacodynamics are markedly influence both by liposomal formulation and route of administration.

Adjuvants, Immunologic↗
PubMedDate not supplied

Cascade liposomal triggering: light-induced Ca2+ release from diplasmenylcholine liposomes triggers PLA2-catalyzed hydrolysis and contents leakage from DPPC liposomes.

We have previously reported a direct triggering approach [Thompson, D. H., et al. (1996) Biochim. Biophys. Acta 1279, 25-34; Gerasimov, O. V., et al. (1997) Biochim. Biophys. Acta 1324, 200-214] based on the facile degradation of plasmenylcholine and diplasmenylcholine vinyl ether linkages by either photooxidation or low-pH environments. This report describes a novel, cascade-type triggering technique that utilizes liposome photooxidation and contents release to activate an enzyme capable of destabilizing conventional phosphatidylcholine liposomes. Our application of this concept employs a mixture of two different liposome populations, one composed of synthetic diplasmenylcholine (1, 2-dihexadec-1'-enyl-sn-glycero-3-phosphocholine, DPPlsCho) containing Ca2+ as a signaling agent for phospholipase A2 (PLA2) and the second composed of 1, 2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) with encapsulated calcein as the reporter molecule. Bacteriochlorophyll (BChl)-sensitized photorelease of Ca2+ from PLA2-resistant DPPlsCho liposomes activates extravesicular PLA2, thereby promoting catalyzed DPPC hydrolysis in a secondary triggering reaction, leading to calcein release. BChl/DPPlsCho/DHC/DPPE-PEG5000/Ca2+IN (0.5:85:10:5) liposomes can be phototriggered using 800 nm excitation, resulting in Ca2+ release (t50% release = 15 min) that cocatalyzes the release of calcein (t50% release = 40 min) from DPPC liposomes (1.5 mM total lipid in DPPlsCho liposomes, 0.18 mM DPPC, 210 micro M final Ca2+ concentration, 90 units of PLA2/ml, 50 mM calcein, and 36 micro M EDTA). No appreciable calcein release occurs in the absence of either PLA2 or BChl/DPPlsCho/DHC/DPPE-PEG5000/CaIN liposomes. The implications of this cascade triggering technique on drug delivery approaches are briefly discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Animals↗
PubMed1993

Modification of liposomes by addition of HCO60. I. Targeting of liposomes to liver by addition of HCO60 to liposomes.

The influence of HCO60 on the blood clearance and tissue distribution of soybean phosphatidylcholine (PC) liposomes, encapsulating alpha-tocopherol as a marker, was studied in rats. The liposomes were prepared by the hydration method from a lipid film containing different amounts of HCO60, and by extrusion through a 0.1 micron polycarbonate membrane filter. The blood clearance and liver uptake of alpha-tocopherol after i.v. administration increased with increasing the amount of HCO60 the liposome contained. With 80 wt% HCO60 liposomes, the accumulation of alpha-tocopherol in the liver was approximately three-fold that of the 100% PC liposomes. The uptake by lungs, spleen and kidneys did not change with the addition of HCO60. The findings obtained in a gel-filtration study suggested that alpha-tocopherol is not removed from the liposomes, with or without HCO60, by rat plasma proteins and the HCO60 micelle may form a complex with rat plasma proteins. Our findings suggest that liposomes containing large amounts of HCO60 (more than 60 wt%) will be useful for delivering drug to the liver.

Animals↗
PubMed1994

Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes.

Liposomes containing dioleoyl-N-(monomethoxypoly(ethylene glycol)succinyl)- phosphatidylethanolamine (PEG-PE), and of three characteristic sizes (d > 300 nm, d approximately 150-200 nm, and d < 70 nm), were prepared, injected into mice, and their biodistributions examined following a radioactive lipid phase marker. The large and small liposomes accumulated to elevated levels in spleen and liver, respectively. The intermediate sized liposomes were found to be the longest circulating. Furthermore, when injected into mice bearing murine MC-38 colon carcinoma tumor, an approximate 2-fold increase in the % injected dose per g tumor was observed for the long-circulating liposomes compared to liposomes without PEG-PE. The distribution of the injected liposomes within the tumor was examined by fluorescence microscopy, where the liposomes were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The liposomes were found surrounding blood vessels in the tumor, with some degree of extravasation into the tumor mass. A previous explanation for the reduced circulation time of small liposomes has been that they have an ability to pass through the fenestrated liver endothelium and thereby reach the parenchymal cells. DiI-labeled liposomes were therefore used to examine the intrahepatic distribution of the injected liposomes. Liposomes accumulated in liver were localized to Kupffer cells, regardless of liposome size. The small liposomes were not detectable in areas comprised of parenchymal cells when using this fluorescence technique. The reason for reduced long-circulating behavior for the small liposomes may be more directly related to the activity of PEG-PE. Therefore the steric barrier activity of the liposomes was examined by a serum protein binding assay and by streptavidin binding to biotinylated liposomes. The steric barrier was liposome size dependent, with the small liposomes revealing increased protein binding. This decreased steric barrier of the small liposomes may result in increased susceptibility to opsonization and thus explain their more rapid clearance from the circulation. The large liposomes accumulated in spleen were localized in the red pulp and marginal zone. Uptake of the large liposomes may occur by means of a filtration mechanism. These results establish the significance of liposome size in determining liposome circulation time and biodistribution, and are relevant for the optimal design of liposomes for drug delivery.

Animals↗
PubMed2004

An increased adjuvanticity of liposomes by the inclusion of phosphatidylserine in immunization with surface-coupled liposomal antigen.

BACKGROUND: Exposure of phosphatidylserine (PS) on apoptotic cells is known to result in the enhanced recognition of apoptotic cells by phagocytes. By the inclusion of PS in the lipid component of liposomes, increased liposome immune adjuvant activity was expected. METHODS: In the present study, two different liposome preparations containing either PS, i.e. PS-liposome, or phosphatidylcholine (PC), i.e. PC-liposome, were made, and macrophage recognition, processing, and antigen presentation of surface-coupled liposomal antigen were compared. RESULTS: When ovalbumin-liposome conjugates were added to a culture of macrophages, enhanced recognition and processing of ovalbumin by the macrophages were observed by the inclusion of PS in the liposomes. The results correlated well with those regarding macrophage antigen presentation of liposome-coupled ovalbumin. Furthermore, in vivo immunization in mice with ovalbumin-liposome conjugates made with PS-liposomes induced a significantly higher level of anti-ovalbumin IgG antibody production than was induced by ovalbumin-liposome conjugates made with PC-liposomes. IgE-selective unresponsiveness was induced by ovalbumin-liposome conjugates regardless of the lipid components of liposomes. CONCLUSIONS: These results suggest that the inclusion of PS in liposomes enhances recognition and processing of surface-coupled liposomal antigen by macrophages, and increases liposome immune adjuvant activity.

Adjuvants, Immunologic↗
PubMed1990

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay.

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target.

Dioleoyl-N-(monomethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PEG-PE) (mol. wt. of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome. As little as 3.7 mol% of PEG-PE in liposome resulted in maximal enhancement of liposome circulation time. However, this activity of PEG-PE was only seen with relatively small liposomes (d less than or equal to 200 nm); larger liposomes containing PEG-PE showed an unusually high level (approx. 35% injected dose) of accumulation in the spleen. We have tested whether the small, PEG-PE containing liposomes are suitable for immuno targeting by incorporating a lung-specific monoclonal antibody on the liposome surface. While another amphiphile, ganglioside GM1, which is well known for its activity to prolong the liposome circulation time, significantly enhanced the lung binding of the immunoliposomes, PEG-PE incorporation of immunoliposomes resulted in a low level of target binding. To test if the reduced target binding is due to a steric barrier effect of the surface PEG polymer, we have incorporated a small amount of N-biotinaminocaproylphosphatidylethanolamine into the PEG-PE containing liposomes and examined the liposome agglutination induced by the addition of streptavidin. As little as 0.72 mol% PEG-PE in these liposomes completely abolished agglutination. In contrast, incorporation of GM1 in liposomes only reduced the rate, but not the extent, of liposome agglutination. These results strongly support the hypothesis that PEG-PE prolongs liposome circulation time by providing a strong steric barrier which prevents close contact with another liposome or cell. Since GM1 provides only a weak steric barrier effect, its activity to prolong the liposome circulation time must involve another yet unknown mechanism.

Agglutination↗
PubMed1992

Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes.

We have examined the effect of liposome size on liposome circulation time in the blood. Liposomes composed of phosphatidylcholine, cholesterol and ganglioside GM1 were prepared in the various size range. Optimal circulation activity (55% injected dose at 4 h post injection) of GM1-containing liposomes, which correlated with a relatively high uptake of liposomes by EMT6 tumor in mouse, was obtained with a size range of 70 to 200 nm in diameter. Increasing the diameter of liposome to greater than 200 nm resulted in an enhancement of the spleen uptake and decrease of the blood level. For liposomes with a diameter of less than 70 nm, 70% of the injected dose were taken up by the liver, presumably by the parenchymal cells. In contrast, the biodistribution of phosphatidylserine-containing liposomes was relatively insensitive to changes in liposome size; most of the injected dose was found in the liver. The effect of RES blockade on the circulation time of large (d greater than 300 nm), GM1-containing liposomes was also studied. Dextran sulfate 500, a commonly used blockade reagent for Kupffer cells, had no effect. On the other hand, preinjection of a large dose of liposomes with a diameter greater than 500 nm showed variable results depending on the lipid composition of the blocking liposomes. Preinjection of liposomes containing GM1, phosphatidylinositol or (N-polyethyleneglycol) phosphatidylethanolamine effectively reduced the spleen uptake of the large GM1-containing liposomes, whereas liposomes containing phosphatidic acid showed no effect. These results indicate that only spleen homing liposomes can be used as a blocking reagent to prolong the circulation time of the large GM1-containing liposomes.

Animals↗
PubMed2006

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.

Animals↗
PubMed1997

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin.

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals 'immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-(P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 microg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (> 9 microg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.

Animals↗
PubMed2000

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.

Animals↗
PubMed1999

Application of surface-coated liposomes for oral delivery of peptide: effects of coating the liposome's surface on the GI transit of insulin.

We prepared two kinds of surface-coated liposomes and investigated their potencies as oral dosage forms for peptide drugs by focusing on their effects on the gastrointestinal (GI) transit of drugs. The surface of the liposomes was coated with poly(ethylene glycol) 2000 (PEG-Lip) or the sugar chain of mucin (Mucin-Lip). As a model peptide drug, insulin was encapsulated in these liposomes. Coating the surface with poly(ethylene glycol) was found to reduce the transit rate of liposomes in the small intestine after oral administration to rats in vivo. Mucin-Lip was retained in the stomach longer than PEG-Lip or uncoated liposomes. The effect of surface coating on the intestinal transit of liposomes was determined by means of in situ single pass perfusion in the rat small intestine. Statistical moment analysis was applied to the outflow pattern of both liposomes and encapsulated insulin. The mean transit time (MTT) and deviation of transit time (DTT) in the intestinal tract were calculated. The MTT of PEG-Lip was much longer than those of uncoated liposomes and Mucin-Lip and was significantly shortened after removal of the intestinal mucous layer. These results indicated that PEG-Lip interacts strongly with the intestinal mucous layer, leading to its slow transit in the intestine. In contrast, coating the liposome's surface with mucin did not affect either the MTT or DTT of liposomes in the intestine. This result is in accordance with the in vivo observation that Mucin-Lip was highly retained in the stomach, but not in any region of the small intestine in vivo. Both the MTT and DTT values of insulin encapsulated in PEG-Lip and Mucin-Lip were almost the same as those of liposomes themselves, suggesting that surface-coated liposomes retained insulin in the intestinal tract. However, MTT and DTT of insulin were significantly shorter than those of uncoated liposomes because these liposomes degraded and released significant amounts of insulin during single pass perfusion. The ability of surface-coated liposomes, especially of PEG-Lip, to interact with the mucus layer and slow the transit rate in the GI tract is considered desirable for oral delivery of peptide drugs. Modification of the liposomal surface with appropriate materials, therefore, should be an effective method by which to achieve the oral delivery of peptide drugs.

Animals↗