Motoharu Iwatsuru

Size-dependent extravasation and interstitial localization of polyethyleneglycol liposomes in solid tumor-bearing mice

We have examined the size dependence of extravasation and interstitial localization of polyethyleneglycol-coated liposomes (PEG-liposomes) in the solid tumor tissue by means of electron microscopic observation. Liposomes composed of distearoyl phosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared in various size ranges. PEG-liposomes with an average diameter of 100-200 nm showed the most prolonged circulation time and the greatest tumor accumulation in all the solid tumors employed in this experiment. Although large PEG-liposomes with a diameter of 400 nm showed a short circulation time in normal mice, the results in splenectomized mice indicated that they do have an intrinsic prolonged circulation character in vivo. However, large PEG-liposomes could not extravasate into solid tumor tissue. These results indicate that the size of liposomes is critical for extravasation. The electron microscopic observations revealed the almost exclusive engulfment of extravasated liposomes by tumor-associated macrophages; very few were taken up by tumor cells.

Targetability of novel immunoliposomes modified with amphipathic poly(ethylene glycol) s conjugated at their distal terminals to monoclonal antibodies

Distearoyl-N-(3-carboxypropionoyl poly(ethylene glycol) succinyl)phosphatidylethanolamine (DSPE-PEG-COOH) was newly synthesized and used to prepare novel immunoliposomes carrying monoclonal antibodies at the distal ends of the PEG chains (Type C). Liposomes were prepared from egg phosphatidylcholine (ePC) and cholesterol (CH) (2;1, m/m) containing 6 mol% of DSPE-PEG-COOH, and a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, was conjugated to the carboxyl groups of DSPE-PEG-COOH to give various amounts of antibody molecules per liposome. Other immunoliposomes with PEG coating (Type B) or without PEG coating (an earlier type of immunoliposome, Type A) were prepared for comparison. The average molecular weight of PEG in Type B or C immunoliposomes was 2000. Type B and Type C liposomes without antibodies showed prolonged circulation time and reduced reticulo-endothelial system (RES) uptake owing to the presence of PEG. These three different types of 34A-immunoliposomes with 30-35 antibody molecules per vesicle were injected into mice to test the immunotargetability to the lung. The efficiency of lung binding of 34A-Type B was one-half of that of 34A-Type A, though a large amount of 34A-Type B remained in the blood circulation for a long time, suggesting that the steric hindrance of PEG chains reduced not only the immunospecific antibody-antigen binding, but also the RES uptake. The degree of lung binding of 34A-Type C was about 1.3-fold higher than that of 34A-Type A, indicating that recognition by the antibodies attached to the PEG terminal was not sterically hindered and that the free PEG (i.e., that not carrying antibody) was effective in increasing the blood concentration of immunoliposomes by enabling them to evade RES uptake. The latter phenomenon was confirmed by using nonspecific antibody-Type C immunoliposomes (14-Type C), which showed a high blood level for a long time. Our approach provides a simple means of conjugating antibodies directly to the distal end of PEG which is already bound to the liposome membrane, and should contribute to the development of superior targetable drug delivery vehicles for use in diagnostics and therapy.

Liposomes bearing polyethyleneglycol-coupled transferrin with intracellular targeting property to the solid tumors in vivo

AbstractPurpose. The purpose of this study was to determine the usefulness of transferrin (TF)-pendant-type polyethyleneglycol (PEG)-liposomes (TF-PEG-liposomes), in which TF was covalently linked to the distal terminal of PEG chains on the external surface of PEG-liposomes as a carrier for in vivo cytoplasmic targeting to tumor cells. Methods. Small unilamellar TF-PEG-liposomes (100-140 nm in diameter) were prepared from DSPC, CH, DSPE-PEG, and DSPE-PEG-COOH (2:1:0.11:0.021, molar ratio), and were conjugated to TF via the carboxyl residue of DSPE-PEG-COOH. The intracellular targeting ability of TF-PEG-liposomes to tumor cells was examined in vitro and in Colon 26 tumor-bearing mice. Results. TF-PEG-liposomes, bearing approximately 25 TF molecules per liposome, readily bound to mouse Colon 26 cells in vitro and were internalized by receptor-mediated endocytosis. TF-PEG-liposomes showed a prolonged residence time in the circulation and low RES uptake in Colon 26 tumor-bearing mice, resulting in enhanced extravasation of the liposomes into the solid tumor tissue. Electron microscopic studies in Colon 26 tumor-bearing mice revealed that the extravasated TF-PEG-liposomes were internalized into tumor cells by receptor-mediated endocytosis. Conclusion. TF-PEG-liposomes had the capabilities of specific receptor binding and receptor-mediated endocytosis to target cells after extravasation into solid tumors in vivo. Such liposomes should be useful for in vivo cytoplasmic targeting of chemotherapeutic agents or plasmid DNAs to target cells.

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol)

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.

Immunoliposomes bearing polyethyleneglycol-coupled Fab' fragment show prolonged circulation time and high extravasation into targeted solid tumors in vivo.

We have developed a new type of long‐circulating immunoliposome (Fab′–PEG immunoliposomes) which is efficiently extravasated into the targeted solid tumor in vivo. Small unilamellar liposomes (100–130 nm in diameter) were prepared from distearoylphosphatidylcholine (DSPC), cholesterol (CHOL) and a dipalmitoylphosphatidylethanolamine derivative of PEG with a terminal maleimidyl group (DPPE‐PEG‐Mal), and conjugated Fab′ fragment of antibody. Inclusion of DPPE‐PEG‐Mal and linkage of the Fab′ fragment instead of intact antibody to PEG terminals allowed the liposomes to evade RES uptake and remain in the circulation for a long time, resulting in enhanced accumulation of the liposomes in the solid tumor. Because of the ability of such Fab′–PEG immunoliposomes to target solid tumors, they appear highly attractive as carriers of not only chemotherapeutic agents, but also of macromolecular drugs.

Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy

Abstract The extravasation of liposomes of different sizes into solid tumors after i.v. injection was visualized by in vivo fluorescence microscopy in mouse neuroblastoma C-1300-bearing mice. Liposomes composed of distearoylphosphatidylcholine/cholesterol (1/1 molar ratio) and 6 mol% distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared. The PEG-coated liposomes were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a liposome marker or with doxorubicin (DXR) as an aqueous-phase marker. Liposomes with an average diameter of 100–200 nm showed the greatest tumor accumulation. With time after injection of DiI-labeled liposomes, the tumor interstitial fluorescence intensity increased. Most fluorescent spots were located outside and around the vessel wall, indicating extravasation of intact liposomes. The perivascular distribution was heterogeneous. We also obtained the same fluorescence localization pattern with DXR released from extravasated liposomes after injection of DXR-encapsulated liposomes. No fluorescence from extravasated liposomes was detected in normal s.c. tissue; the fluorescent spots were observed only in the vessel wall. Our results indicate that small-size long-circulating liposomes are able to traverse the endothelium of blood vessels in tumors and extravasate into interstitial spaces. Moreover, encapsulated drug was released from extravasated liposomes in the tumor.

Enhanced delivery of doxorubicin to tumor by long-circulating thermosensitive liposomes and local hyperthermia.

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (180-200 nm), prepared from dipalmitoylphosphatidylcholine (DPPC)/distearoylphosphatidylcholine (DSPC) (9:1 (m/m)) and 6 mol% of ganglioside GM1 (GM1), with 95-98% entrapping efficiency by the pH-gradient method. 45% of the entrapped DXR was released from these GM1/DPPC/DSPC liposomes by incubation at 42 degrees C for 5 min in 20% serum or saline (this degree of release was lower than that of hydrophilic drugs such as cisplatin, due to the basic and amphiphilic nature of DXR). Inclusion of GM1 (6 mol%) endowed DPPC/DSPC liposomes with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system uptake over 6 h after injection. Concomitantly, DXR levels in blood remained high for long time. Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR loaded, long-circulating, thermosensitive (DXR-GM1/DPPC/DSPC) liposomes was 2.5-times or 6-times higher than that after treatment with DXR-DPPC/DSPC liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the treatment with DXR-GM1/DPPC/DSPC liposomes and hyperthermia resulted in effective tumor-growth retardation and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.

Phosphatidyl polyglycerols prolong liposome circulation in vivo

Abstract To obtain liposomes with longer circulation times in vivo, we newly synthetized dipalmitoylphosphatidylpolyglycerol (DPP-PG). A series of DPP-PGs of different chain lengths was used in this study. The individual derivatives were incorporated into distearoylphosphatidylcholine/cholesterol liposomes (1:1, molar ratio). The effectiveness of DPP-PG derivatives was dependent on the amount and degree of polymerization. Low-polymerized PGs such as diglycerol and tetraglycerol needed a high incorporation rate of 8 mol%, while high-polymerized PGs such as octaglycerol required 4 mol%. The incorporation of 6 mol% of DPP-hexaglycerol was most effective in prolonging the circulation time of liposomes.

Targeting efficiency of PEG-immunoliposome-conjugated antibodies at PEG terminals

Abstract We have developed a new type of PEG-immunoliposome carrying monoclonal antibodies or their fragments (F(ab′)2, Fab′) at the distal ends of the PEG chains (Type C). Distearoylphosphatidylethanolamine derivatives of PEG with car☐yl group (DSPE-PEG-COOH) or dipalmitoyl phosphatidylethanolamine derivatives of PEG with maleimidyl group (DPPE-PEG-Mal) at the PEG terminal were newly synthesized. Small unilamellar liposomes (90–130 nm in diameter) were prepared from distearoyl phosphatidylcholine and cholesterol (2:1, m/m) containing 6 mol% of DSPE-PEG-COOH or DPPE-PEG-Mal. To target to the vascular endothelial lung surface as a model accessible site, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposome (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mice was significantly higher than that of the 34A-Type A which is an ordinary type immunoliposome (without PEG derivatives). To target to the solid tumor tissue as a model of the less accessible site, 21B2 antibody which is anti-human CEA and its Fab′ fragment were used. The targeting ability of Fab′-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab′-Type C showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. The small Fab′-Type C could predominantly pass through the leaky tumor endothelium by passive convective transport.