Change in the Character of Liposomes as a Drug Carrier by Modifying Various Polyethyleneglycol-Lipids

Abstract

When liposomes, as a superior drug carrier, are injected intravenously, active liposomes as medicines require polyethyleneglycol (PEG) as a modification tool around the liposomal membrane. PEG modification of a liposome forms a fixed aqueous layer, and the trapping by cells of the reticuloendothelial system (RES) is avoided. Hence, PEG-modified liposomes have long circulation in the bloodstream, and passive targeting to tumors has been achieved by PEG modification. We have been studying the passive targeting by liposomes with the expectation of more usefulness. It was proved a correlation between the PEG molecular weight of PEG-modified liposomes and blood circulation time and antitumor effect, too. Liposomes modified by PEG2000 were useful for uptake into tumor cells. We thought that the re-uptake in the liposomal membrane also increased accumulation. Moreover, it was proved that mixing two different PEGs to modify the liposome surface gives a bigger fixed aqueous layer thickness (FALT) around the liposome, giving the liposome strong antitumor activity. Then, we designed a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-PEG (different double arms PEG; DDA-PEG), which had two different PEGs (2000 and 500) in one molecule. One of the innovative characteristics of DDA-PEG-modified liposome (DDAL) is that it heightens the contact ability with tumor cells. DDAL may be an effective DDS carrier for solving various PEG dilemmas. It was observed that passive targeting by PEG-modified liposomes had different characteristics by changing PEG length, anchor type or those combination. Thus, it should be applied to liposomes suitable for various diseases.

1. INTRODUCTION

Polyethyleneglycol (PEG) is a non-antigenic, water-soluble polymer with cyclic ethyleneoxide formation which has few interactions with body composition. PEGs are available at a low price and its molecular weight is easy to control, therefore, it is a highly useful polymer. The compound which coupled alkyl ether or alkyl phenol ether on the end of PEG is amphipathic and has been widely used as a nonionic surfactant. PEG is one of the most famous materials used as a modification tool around liposomes. PEG-modified liposomes have proved useful as drug carriers in drug delivery system (DDS). For example, it is possible to avoid traps by the reticuloendothelial system (RES) such as in the liver and spleen,^1–3) one of the biggest barriers to the application of liposomes as drug carriers in vivo. And prolongation of liposome circulation in the bloodstream and passive targeting to tumor has been achieved by PEG modification of the liposome surface.^4–11) Furthermore, PEG modification around liposomes gives high stability: about a few years at room temperature, high blood circulation, and longer blood half time. In the case of using PEG as a liposome modifying material, PEG should have a hydrophobic anchor for interaction with phospholipids of a liposomal membrane. Studies regarding PEG-modified liposomes containing DOXIL®^12,13) have been performed mainly by using PEG-diacylphosphatidylethanolamine (PEG-PE), which is a lipid derivative of PEG (PEG-lipid) and has indicated change in PEG chain length or anchor length of PEG-PE influenced liposome circulation in the bloodstream. Recently, the best condition for PEG-modified liposomes for passive targeting has been when PEG-lipid with an average molecular weight of 2000 is used. We have been using fixed aqueous layer thickness (FALT) as a factor of circulation time of PEG-modified liposome in the bloodstream. The FALT value is determined by the change of electrostatic potential level around liposome,^14,15) primarily indicated by changes of PEG chain length and the anchor length of 1-monomethoxypolyethyleneglycol-2,3-diacylglycerol (PEG-DAG, Fig. 1) which is a PEG-lipid with no electric charge, which affected the prolonged circulation in the bloodstream. However, it is suggested that there is a danger that modification of the liposome surface with these PEG-lipid prevents the connection of the liposome and tumor cell by the existence of macromolecules on the liposome surface.¹⁶⁾ For PEG-modified liposomes containing an antitumor agent, there are few reports which show the relation between the kind of PEG used and the characteristics of that liposome in vitro and in vivo. Thus, we thought it important to make clear these correlations.

Fig. 1. Chemical Structure of PEG-Diacylglycerol

Miristoyl: R=(CH₂)₁₂CH₃ Palmitoyl: R=(CH₂)₁₄CH₃ Stearoyl: R=(CH₂)₁₆CH₃ PEG2000: n=44, PEG5000: n=112.

2. PEG MODIFICATION AROUND LIPOSOMAL MEMBRANE

2.1. Changing the PEG Chain Length or Anchor Length of Modified PEG around a Liposome

It is expected that modification of the liposome surface with PEG-lipid will prevent the connection of liposome and tumor cell, so the effect of PEG chain length and anchor length on liposome uptake into the tumor cell was examined.¹⁷⁾

We examined doxorubicin (DOX) containing PEG-modified liposomes in this study, using the following six PEG-lipids: 1-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with PEG of average molecular weight 2000 (PEG2000-DMG) and 5000 (PEG5000-DMG), 1-monomethoxypolyethyleneglycol-2,3-dipalmitoylglycerol with PEG of average molecular weight 2000 (PEG2000-DPG) and 5000 (PEG5000-DPG) and 1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol with PEG of average molecular weight 2000 (PEG2000-DSG) and 5000 (PEG5000-DSG).

For determination of DOX uptake into the tumor cell, Ehrlich ascites carcinoma cells (5.0×10⁶ cells/mL) were incubated with liposomes containing 5.0 µg/mL DOX at 37°C. The uptake of DOX by the cells in all groups gradually increased and the ratio was highest in PEG2000-DPG-modified liposomal DOX (PL2000). After 30 min, the uptake of PL2000 was about 2-fold greater than that of PEG-unmodified liposomal DOX. With the same anchor, the increase in the uptake of DOX in PEG5000-modified liposomal DOXs was greater than that in PEG2000-modified liposomes. It was suggested that the length of the PEG chain was more important than the length of the anchor for cell uptake. This is because a longer PEG chain more easily attacked the tumor cell membrane. PL2000 had the greatest uptake, at 2.5-fold and 5.1-fold the level of PEG2000-DMG-modified liposomal DOX (ML2000) and PEG2000-DSG-modified liposomal DOX (SL2000), respectively. The uptake by PL2000 exceeded that of ML5000 and SL5000. Hence, the cell uptake of PEG-modified liposomal DOX did not depend on the length of PEG chain alone.

We have found that the length of the PEG chain and the kinds of anchor of PEG-lipid have an influence on avoidance from RES, as well as the effect of prolonging circulation in the blood in PEG-modified liposomes. If the mechanism of prolonging circulation in the blood was caused by the prevention of opsonin binding in the plasma by the fixed PEG aqueous layer thickness around liposome, the prolonged circulation in the blood by the differences in these PEG-lipids was expected from the difference in the length of the PEG chain and the modified amount of PEG-lipid on the liposome surface. The modified amount of PEG was affected by the percentage of PEG-lipid incorporated on preparing the PEG-liposome. At a standard added amount of PEG-lipid (5.45 mol% lipid), the percentage of PEG-lipids incorporated into the liposomal membrane was 92.9% in ML2000 and 45.3% in SL2000. That of the PEG5000-lipid showed the same trend, namely, the ratio of PEG-lipids with a longer anchor incorporated into the liposomal membrane was lower. The percentage was 63.7% in PEG5000-DMG-modified liposomal DOX (ML5000) and 92.9% in ML2000, that is, less PEG5000-lipid than PEG2000-lipid was incorporated into the liposomal membrane with the same anchor. We examined the correlation between the percentage of PEG-lipids incorporated into the liposome membrane and the effect of the liposome surface. However, there was no correlation, suggesting that the effect of uptake into Ehrlich ascites carcinoma cells was caused by factors other than modification of the amount of PEG.

In PEG-modified liposomes, the percentage of PEG-lipids incorporated into the liposomal membrane did not correlate with the promoting effect on liposome uptake into tumor cells. It was suggested that the promoting effect on liposome uptake was closely related to the stability of PEG-lipids on the liposomal membrane and the re-uptake of PEG-lipids to the liposomal membrane. As to stability, residual PEG-lipid around liposomal membrane decreased with time, and after 24 h the value was 32.2% when the anchor was DSG. By contrast, that for PL2000 which had the best effect on DOX uptake was about 100% after 24 h. The high residual amount of PL2000 in liposomal membranes suggested that stable modification of liposomes with PEG-lipids like this was effective on DOX uptake. At re-uptake, departing PEG-lipids from the liposomal membrane were taken up into the liposomal membrane again. Little re-uptake occurred after PEG-lipid suspension was added to SL2000, and the amount of PEG-lipid taken up was no more than that at the start. On the other hand, the re-uptake of PL2000 into the liposomal membrane increased with time, and after 24 h was about twice the level as when the incubation started. In other words, re-uptake of PEG-lipid by liposomal membranes was small in SL2000 and large in PL2000.

Furthermore, it was necessary to examine the transition of PEG-lipids into the tumor cell for the purpose of investigating the interaction between cell membrane and PEG-lipid. Re-uptake of PEG-lipid by the cell membrane was evaluated which using suspensions prepared by the same method used in the DOX uptake experiment (Fig. 2). After 120 min incubation, the level of PEG2000-DPG transition was maximum, being 2.1-fold that of PEG2000-DMG. The uptake of PEG5000-lipids in cell membrane was extremely small. We expected that PEG2000-lipid may be attacked by the tumor cell membrane. It is speculated that the uptake of PEG-modified liposomes by the tumor cells was increased by this interaction between PEG-lipid in the liposomal membrane and PEG-lipid in cell membrane.

Fig. 2. Cell Uptake of PEG-Lipid at 120 min

The cell suspension with 1.0 µm of PEG-lipid was incubated at 37°C.

Thus, modification of the liposome surface with PEG-lipid did not prevent liposome uptake into cells, but rather, might promote it. And the stability of PEG2000-DPG on the liposomal membrane was caused by removal re-uptake of PEG-lipid on the liposomal membrane. It is expected that these properties of PEG2000-DPG are useful for the uptake of liposomes into tumor cells. Therefore, it was suggested that the liposome adsorption or uptake into a tumor cell membrane was caused by re-uptake of PEG-lipid on the liposomal membrane and induced the remaining PEG-lipid on liposomal membrane.

2.2. Mixing Two Different PEGs to Modify the Liposome Surface

Shimada et al. clarified measurement techniques for the FALT around liposomes, and explained that the FALT of PEG-modified liposomes increased in comparison with PEG-unmodified liposomes.^15,18,19) With the increase of concentration of PEG-lipid on the liposomal membrane, serum protein prevents binding to the liposome membrane because of PEG-lipids overlapping each other.^20–22) As a result, it is considered that PEG-modified liposomes have a long circulation time.^8,9) However, the amount of PEG-lipids inserted into the liposome membranes is limited, and FALT forming around the liposomes reaches a plateau,²³⁾ so it was considered that increasing FALT was difficult. Then, we tried mixing two different PEGs to modify the liposome surface for the purpose of increasing FALT and avoiding trapping by RES.

In single PEG-modified liposomes, the FALT around the liposome increased as the PEG-molecular weight increased.²⁴⁾ The FALTs of PEG-unmodified liposomal DOX, PEG340-DSG-modified liposomal DOX (SL340), PEG500-DSG-modified liposomal DOX (SL500), PEG900-DSG-modified liposomal DOX (SL900) and SL2000 were 0.34±0.05, 0.94±0.03, 1.00±0.01, 2.10±0.02 and 2.52±0.03 nm, respectively. When we mixed PEG2000-DSG and PEG-lipid with a short polyoxyethylene chain, the FALT around the liposome increased in comparison with that of single SL2000. For example, PEG2000-DSG/PEG500-DSG (1/1, mol/mol)-modified liposomal DOX (SL(2000 : 500=1 : 1)) and PEG2000-DSG/PEG500-DSG (2/1, mol/mol)-modified liposomal DOX (SL(2000 : 500=2 : 1)) was 2.95±0.03 nm and 3.58±0.09 nm. No remarkable difference was observed between the size of each liposome.

These incorporated ratios and amounts of PEG-lipid in the liposomal membrane were examined. In single PEG-modified liposomes, the incorporated ratio of PEG2000-DSG in the liposomal membranes was 46.4±2.1% (0.70±0.03 mm, which was lower than that of PEG500-DSG (94.8±1.3%, 1.42±0.02 mm). On the other hand, in mixed PEG-modified liposomes obtained with a mixture of PEG2000-DSG/PEG500-DSG (2/1, mol/mol), the incorporated ratios of PEG2000-DSG and PEG500-DSG were 54.1±2.4% (0.54±0.02 mm) and 100.0±2.4% (0.50±0.01 mm), respectively. Hence, the actual incorporated ratio of PEG2000-DSG : PEG500-DSG was about 1 : 1. With both SL2000 and SL(2000 : 500=2 : 1), FALT increased with increasing incorporated amount of PEG-DSGs, but when this amount was over a certain level, an increase in FALT was not recognized, the FALTs of SL2000 and SL(2000 : 500=2 : 1) being about 2.5 and 3.5 nm, respectively.

Thus, we suggested that FALTs of mixed PEG-modified liposomes obtained with mixture of PEG-DSGs with long and short polyoxyethylene chains were larger than those of single PEG-modified liposomes, but FALTs of mixed PEG-modified liposomes with the same PEG-DSGs with short polyoxyethylene chains did not increase.²⁴⁾ Its values were between the two FALTs of single PEG-modified liposomes. The PEG-DSGs used in this study are nonionic surfactants. The DSG section, which is incorporated in the liposomal membrane, is hydrophobic, as opposed to the polyoxyethylene chain section, which is hydrophilic. Therefore, it was supposed that the longer polyoxyethylene chain, the greater its hydrophilicity. Our results are in accord with this theory. In 10 mm Tris–HCl−150 mm NaCl buffer (pH 7.4), the order of octanol/buffer partitioning of PEG-DSGs was PEG2000≪PEG900<PEG500<PEG340. Hence, this result suggested that PEG-DSG with long polyoxyethylene chains was more hydrophilic than that with short chains. In particular, PEG2000-DSG exhibits remarkable hydrophilicity, as compared with the other three PEG-DSGs. In 9.0% sucrose in 10 mm lactate buffer (pH 4.0), this tendency was similar. On the other hand, when we compared the octanol/buffer partitioning in both buffers, that of each PEG-DSG was pH 4.0 < pH 7.4. These results indicated that in hydrophobicity/hydrophilicity, PEG2000-DSG is conspicuously different from the PEG340-DSG, PEG500-DSG and PEG900-DSG. Because of these results, it is supposed that when a combination of PEG-DSGs whose characteristics are remarkably different is used, an interaction between molecules occurs, leading to increased FALT. Accordingly, it is suggested that we could expect a combination of PEG-lipids that gives increased FALT.

2.3. Effect of Mixed PEG-Modified Liposomal Doxorubicin

However, there has been some concern that the interaction between cells such as tumor cells and liposomes was sterically prevented by the existence of PEG polymers on the surface of liposomes.^16,25,26) Our results in vitro indicated that increasing FALT did not necessarily prevent DOX uptake by Ehrlich ascites carcinoma cells. The intracellular level of DOX was different in the liposomes, the order of DOX uptake by cells after 30 min incubation being PEG-unmodified liposomal DOX=SL2000=SL(2000 : 500=2 : 1)>SL500, thus the level of cell uptake was affected by the kind of PEG modification of liposomes. The DOX uptake in the SL500 group after 30 min incubation was 73.7% (p<0.001) of that of the PEG-unmodified liposomal DOX group. In contrast, the level of cell adsorption at 0 min was not different among the liposomes. We supposed that SL(2000 : 500=2 : 1) that reached the tumor region could be transferred into cells successfully, bringing their ability fully into play.

We next investigated effects on the DOX concentration in tissues of mice after administration of SL(2000 : 500=2 : 1). Tissue distribution after intraperitoneal (i.p.) injection of each liposome was studied.²⁷⁾ In the plasma, at 2 and 6 h after the administration, the order of DOX concentrations was SL(2000 : 500=2 : 1)>SL2000>SL500=PEG-unmodified liposomal DOX>DOX solution. In the DOX solution group, the DOX concentration in plasma was not detected at 2 h after administration. In the PEG-unmodified liposome or SL500 groups, the DOX concentrations in plasma were not detected at 24 h after administration, whereas the concentrations in plasma were detected at 24 h after administration in the SL2000 or SL(2000 : 500=2 : 1) groups. The level at 24 h after SL(2000 : 500=2 : 1) administration was equal to that at 6 h after SL500 administration. The DOX concentration increased with an increase in FALT, the PEG-modified liposomal DOX of large FALT being shown to maintain a high DOX level. In the liver, at 6 and 24 h after the administration of DOX, the order of DOX concentration was PEG-unmodified liposomal DOX > SL500> SL2000 > SL(2000 : 500=2 : 1) = DOX solution. In particular, DOX levels showed the maximum value at 6 h after administration. The avoidance of RES was elevated by an increase in FALT. This phenomenon showed that a fixed aqueous layer was formed on the surface of liposomes by PEG modification, and prevented the attraction of opsonins; as a result, the trapping by cells of the RES was avoided.^28–30) This result supposed that the ability to avoid trapping by RES was caused by an increased FALT. In the tumor, an increase in DOX levels caused by PEG modification was observed at 24 h after administration. DOX levels of SL500, SL2000 and SL(2000 : 500=2 : 1) were 3.3, 5.7 and 9.1 fold higher than that of PEG-unmodified liposomal DOX, respectively. Notably, the DOX level of SL(2000 : 500=2 : 1) was 2.23 µg/g protein at 72 h after administration, being equal to the maximum value of DOX solution. The time course of the DOX concentration showed a relation between an increase in FALT and maintenance of a high DOX level for a long time, namely, the increase in DOX concentration in tumor caused by passive targeting was observed following mixed PEG modification. In the heart, although a remarkable difference was not observed in the DOX concentrations among groups, DOX levels were slightly decreased in SL2000 and SL(2000 : 500=2 : 1) in comparison with DOX solution. As a result, the adverse effects may be reduced for SL2000 and SL(2000 : 500). We also investigated the effects of PEG modification on antitumor activity. Figure 3 shows the tumor weights and DOX concentrations in tumors after administration of DOX in each form. The tumor weight of the control was 3.98±1.48 g. The decrease of tumor weight after SL500 treatment was similar to that in the PEG-unmodified liposomal DOX group. In contrast, the tumor weight after SL2000 treatment and SL(2000 : 500=2 : 1) treatment was, respectively, 1.90±0.99 and 1.10±0.39 g, or a 2.5 and 3.4-fold stronger effect than PEG-unmodified liposomal DOX (Fig. 3A). After SL2000 and SL(2000 : 500=2 : 1) treatments, the DOX concentration in the tumor increased by 1.5-fold (p<0.001) and 2.4-fold (p<0.001) of that in PEG-unmodified liposomal DOX, respectively (Fig. 3B). Hence, the administration of SL(2000 : 500=2 : 1) produced a significant reduction of tumor weight.

Fig. 3. Effect of PEG Modification on the Change of Tumor Weight and DOX Concentration in Tumors of Mice

Ehrlich ascites carcinoma cells were transplanted into the backs of mice, and then each liposome was injected i.p. at a dose of 2.5 mg/kg three times. Each column represents the mean±S.D. (n=4–6). Significant differences from the level of the PEG-unmodified liposomal DOX group are indicated by a) p<0.01 and b) p<0.001.

The reason the antitumor activity of PEG-modified liposomes increased by mixing PEG modification with different PEGs was considered to be as follows. Modeling of PEG-lipids, which modified the surface of liposomes, has shown that at least two regimes can be identified: ‘mushrooms’ (isolated grafts) and ‘brushes’ (extended chain conformations determined by the interaction between neighboring chains).³¹⁾ This phenomenon suggests that because PEG-lipid with a large molecular weight constructed more complete ‘mushroom’ structures than that with a small molecular weight, the increase in FALT was smaller than the increase in PEG molecular weight. We tentatively calculated FALTs of PEG500 and PEG2000 on the basis of bond angle and bond length of polyoxyethylene chains. If PEG2000 formed complete ‘brushes,’ the FALT around the liposome was 6.6 times bigger compared with that with complete ‘mushroom’ structures. The mixture of PEG2000 and PEG500 increased the FALT around liposomes in comparison with that of single SL2000, because, ‘mushroom’ structures of PEG-lipid, which has a large molecular weight, became longer as a result of the insertion of PEG-lipid with PEG500, which has a short polyoxyethylene chain, into the interval of a large one (Fig. 4).

Fig. 4. PEG Modification on Surface of Liposome

(a) Liposomal DOX with PEG500-DSG modification. (b) Liposomal DOX with PEG2000-DSG modification. (c) Liposomal DOX with PEG2000-DSG and PEG500-DSG mixed modification.

2.4. The Novel PEG with Two Different Arms

It was found that mixed PEG-modified liposomal DOX, which has a 2 : 1 mixture of PEG2000 and PEG500, effectively accumulated in tumors based on increased FALT, potentiation of RES cell avoidance, improvement in circulation time into the bloodstream and passive targeting.^24,32,33) It was proved that although PEG2000-DSG and PEG500-DSG were added 2 : 1, the actual incorporated ratio of PEG2000-DSG : PEG500-DSG was about 1 : 1. Based on these results, we designed a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG; DDA-PEG), which had two different PEGs (2000 and 500) in one molecule by NOF Co. (Tokyo, Japan) (Fig. 5). DDA-PEG-modified liposomal DOX (DDAL) was evaluated for its antitumor effect.³⁴⁾ With DDA-PEG, it was expected that PEG2000 and PEG500 was modified at the same ratio all the time on the liposomal membrane. It was important that the PEG-modified liposome could avoid RES cells. The FALT of DDAL was at a maximum level at 3.57 nm when 15 µmol of PEG was added (DDAL(15)). On the other hand, with 7.5 µmol PEG addition (DDAL(7.5)), which was half amount of the maximum case, the FALT was 3.10 nm. In our previous data, the maximum FALT for single PEG-modified liposomal DOX was 2.52±0.03 nm when the liposome was modified with 15 µmol PEG2000-DSG. DDAL(7.5) had the biggest FALT, even if the added amount was half in the single PEG-modified liposome. Moreover, the FALT of DDAL when 7.5 µmol was added was not different that of SL(2000 : 500=2 : 1) when 15 µmol of PEG was added. It caused lift up of PEG2000 by PEG500 in one molecule, and PEG2000 constantly maintained a brush structure around the liposomal membrane, so only a few PEG modifications could increase FALT. It was already confirmed that SL(2000 : 500=2 : 1) had enough circulation time in the bloodstream to display antitumor activity.^24,32) It is thought that although DDAL(7.5) was a single and lower dosage than SL(2000 : 500=2 : 1), it had the same or stronger antitumor activity. Moreover, unlike SL(2000 : 500=2 : 1), DDAL(7.5) had a big advantage in that it was always present in long and short chain PEGs at the same ratio. The PEG-modified ratio around the liposomal membrane is an important factor for cancer therapy.

Fig. 5. Chemical Structure of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-PEG (Different Double Arms PEG; DDA-PEG)

DOX uptake into tumor cells using M5076 ovarian sarcoma cells were evaluated (Fig. 6). The cellular DOX concentration in the DDAL(7.5) group increased compared with liposomes which were modified with 15 µmol of PEG-lipid. This may be because DDAL(7.5) exhibited half the amount of PEG, could swing around the liposomal membrane and thus contact the tumor cells more efficiently compared with 15 µmol PEG-modified liposome. Also, in a study of cell cytotoxicity, the effect tended to increase based on the transit of DOX to tumor cells. DDAL(7.5) is a promising drug carrier since it increased the DOX concentration against M5076 ovarian sarcoma cells. DDAL(7.5) reduced the tumor size and tumor weight. The tumor size of the DDAL(7.5) administration group was half compared with SL2000 group, and the tumor weight following DDAL(7.5) treatment was decreased significantly compared to be control (p<0.05) and SL2000 (p<0.01). Hence, DDAL(7.5) has the strongest antitumor effect as a single PEG-modified liposomal DOX. The heart accumulation of DDAL(7.5) was also confirmed at a significantly low level compared with DOX solution (p<0.05).

Fig. 6. Effects of PEG-Modification on DOX Uptake into M5076 Ovarian Sarcoma Cells

M5076 ovarian sarcoma cells were incubated at 37°C for 30 min with 10 µg DOX/mL of SL2000, SL(2000 : 500=2 : 1), DDAL(7.5) and DDAL(15). Each point represents the mean±S.D. in four samples. Significant differences from the level of SL(2000 : 500=2 : 1) are indicated by a) p<0.001 and b) p<0.01. ◆: SL2000, ■: SL(2000 : 500=2 : 1), ○: DDAL(15), ×: DDAL(7.5).

In conclusion, DDA-PEG had demonstrated new advantages for PEG-modified liposomes as passive targeting carriers. These include (1) increasing the antitumor effect based on long circulation in the bloodstream by large FALT created by small PEG modification amounts, (2) maintaining the FALT around liposomes in vivo because one PEG-lipid has different double PEG chains which is different from PEG mixed modification, (3) heightening its contact ability with tumor cells, since the amount of PEG modification is less than general PEG-modified liposome. DDAL (7.5), which overcame the conflict effect previously caused by PEG modification, may be an effective DDS carrier and a solution to the PEG dilemma.

3. CONCLUSION

We have been studying passive targeting liposomes in search of broader usefulness. We have proven a correlation between the molecular weight of PEG-modified liposome and its blood circulation time and antitumor effect, too. This phenomenon supported an increase of the FALT value around the liposomal membrane by PEG modification. On the other hand, PEG-modified liposomes at larger molecular weight PEG than 2000 had no more effect. In mixed PEG modification (long chain PEG and short chain PEG) aimed at an increase in FALT, the FALT of SL(2000 : 500=2 : 1) was maximum, and the liposome had strong antitumor activity. Thereafter, as PEG2000 and PEG500 was modified at the same ratio all the time on the liposomal membrane, we designed a novel PEG-lipid, DDA-PEG. DDAL(7.5) had the biggest FALT, even if the added amount was half in the single PEG-modified liposome, and it had strong effects. This liposome also had heightened the contact ability with tumor cells, since amount of PEG modification is smaller than general PEG-modified liposomes.

It was observed that passive targeting by PEG-modified liposomes had different characteristics according to changing PEG length, combination and anchor type. Thus, it could be applied to various diseases based on these. And it is suggested that PEG is an important material for the development of DDS formulations in the future.

Acknowledgment

The authors would like to thank NOF Co. (Tokyo, Japan) for their synthesis and gifting of PEG-lipids. This research was supported by the Medical Innovation by Advanced Science and Technology Project (MIAST Project) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

REFERENCES

• 1)  Patel HM. Serum opsonins and liposomes: their interaction and opsonophagocytosis. Crit. Rev. Ther. Drug Carrier Syst., 9, 39–90 (1992).
• 2)  Devine DV, Marjan JM. The role of immunoproteins in the survival of liposomes in the circulation. Crit. Rev. Ther. Drug Carrier Syst., 14, 105–131 (1997).
• 3)  Lopez-Berestein G, Kasi L, Rosenblum MG, Haynie T, Jahns M, Glenn H, Mehta R, Mavligit GM, Hersh EM. Clinical pharmacology of ^99mTc-labeled liposomes in patients with cancer. Cancer Res., 44, 375–378 (1984).
• 4)  Sadzuka Y, Nakai S, Miyagishima A, Nozawa Y, Hirota S. The effect of dose on the distribution of adriamycin encapsulated in polyethyleneglycol-coated liposomes. J. Drug Target., 3, 31–37 (1995).
• 5)  Klibanov AL, Maruyama K, Beckerleg AM, Torchilin VP, Huang L. 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. Biochim. Biophys. Acta, 1062, 142–148 (1991).
• 6)  Yuda T, Pongpaibul Y, Moribe K, Maruyama K, Iwatsuru M. Preparation of thermosensitive liposomes containing amphipathic polyethyleneglycol for macromolecule delivery. J. Pharm. Sci. and Tec., Japan, 57, 74–78 (1997).
• 7)  Senior J, Delgado C, Fisher D, Tilcock C, Gregoriadis G. Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles. Biochim. Biophys. Acta, 1062, 77–82 (1991).
• 8)  Torchilin VP, Omelyanenko VG, Papisov MI, Bogdanov AA Jr, Trubetskoy VS, Herron JN, Gentry CA. Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim. Biophys. Acta, 1195, 11–20 (1994).
• 9)  Kuhl TL, Leckband DE, Lasic DD, Israelachvili JN. Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups. Biophys. J., 66, 1479–1488 (1994).
• 10)  Needham D, McIntosh TJ, Lasic DD. Repulsive interactions and mechanical stability of polymer-grafted lipid membranes. Biochim. Biophys. Acta, 1108, 40–48 (1992).
• 11)  Janzen J, Song X, Brooks DE. Interfacial thickness of liposomes containing poly(ethylene glycol)-cholesterol from electrophoresis. Biophys. J., 70, 313–320 (1996).
• 12)  Coukell AJ, Spencer CM. Polyethylene glycol-liposomal doxorubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in the management of AIDS-related Kaposi’s sarcoma. Drugs, 53, 520–538 (1997).
• 13)  Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of PEGylated liposomal doxorubicin: review of animal and human studies. Clin. Pharmacokinet., 42, 419–436 (2003).
• 14)  Hirota S. Physicochemical specification of drug carrying liposomes for the quality control in the industrial production. Int. J. Pharm., 162, 185–194 (1998).
• 15)  Shimada K, Miyagishima A, Sadzuka Y, Nozawa Y, Mochizuki Y, Ohshima H, Hirota S. Determination of the thickness of the fixed aqueous layer around polyethyleneglycol-coated liposomes. J. Drug Target., 3, 283–289 (1995).
• 16)  Hong RL, Huang CJ, Tseng YL, Pang VF, Chen ST, Liu JJ, Chang FH. Direct comparison of liposomal doxorubicin with or without polyethylene glycol coating in C-26 tumor-bearing mice: is surface coating with polyethylene glycol beneficial? Clin. Cancer Res., 5, 3645–3652 (1999).
• 17)  Sadzuka Y, Kishi K, Hirota S, Sonobe T. Effect of polyethyleneglycol (PEG) chain on cell uptake of PEG-modified liposomes. J. Liposome Res., 13, 157–172 (2003).
• 18)  Zeisig R, Shimada K, Hirota S, Arndt D. Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines. Biochim. Biophys. Acta, 1285, 237–245 (1996).
• 19)  Sugiyama I, Sadzuka Y. Correlation of fixed aqueous layer thickness around PEG-modified liposomes with in vivo efficacy of antitumor agent-containing liposomes. Curr. Drug Discov. Technol., 8, 357–366 (2011).
• 20)  Kenworthy AK, Hristova K, Needham D, McIntosh TJ. Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J., 68, 1921–1936 (1995).
• 21)  Nikolova AN, Jones MN. Phospholipid free thin liquid films with grafted poly(ethylene glycol)-2000: Formation, interaction forces and phase states. Biochim. Biophys. Acta, 1372, 237–243 (1998).
• 22)  Du H, Chandaroy P, Hui SW. Grafted poly(ethylene glycol) on lipid surfaces inhibits protein adsorption and cell adhesion. Biochim. Biophys. Acta, 1326, 236–248 (1997).
• 23)  Matsuo S, Shimada K, Miyagishima A, Sadzuka Y, Nozawa Y, Hirota S. Determination of fixed aqueous layer thickness (FALT) around the liposomes modified with poly(ethylene glycol). Prog. Drug Deliv. Syst., VI, 1–10 (1997).
• 24)  Sadzuka Y, Nakade A, Hirama R, Miyagishima A, Nozawa Y, Hirota S, Sonobe T. Effects of mixed polyethyleneglycol modification on fixed aqueous layer thickness and antitumor activity of doxorubicin containing liposome. Int. J. Pharm., 238, 171–180 (2002).
• 25)  Johnstone SA, Masin D, Mayer L, Bally MB. Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages. Biochim. Biophys. Acta, 1513, 25–37 (2001).
• 26)  Honda K, Maeda Y, Sasakawa S, Ohno H, Tsuchida E. Activities of cell fusion and lysis of the hybrid type of chemical fusogens. (I). Structure and function of the promotor of cell fusion. Biochem. Biophys. Res. Commun., 100, 442–448 (1981).
• 27)  Sadzuka Y, Hirota S. Physical properties and tissue distribution of adriamycin encapsulated in polyethyleneglycol-coated liposomes. Advanced Drug Delivery Reviews, 24, 257–263 (1997).
• 28)  Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue. Cancer Res., 53, 3765–3770 (1993).
• 29)  Schmitz S, Allen TM, Jimbow K. Polyethyleneglycol-mediated delivery of liposome-entrapped pigments into fibroblasts: experimental pigment cells as models for mutator phenotypes. Cancer Res., 52, 6638–6645 (1992).
• 30)  Papahadjopoulos D, Allen TM, Gabizon A, Mayhew E, Matthay K, Huang SK, Lee KD, Woodle MC, Lasic DD, Redemann C, Martin FJ. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U.S.A., 88, 11460–11464 (1991).
• 31)  Needham D, Stoicheva N, Zhelev DV. Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol)-lipid. Biophys. J., 73, 2615–2629 (1997).
• 32)  Sadzuka Y, Nakade A, Tsuruda T, Sonobe T. Study on the characterization of mixed polyethyleneglycol modified liposomes containing doxorubicin. J. Control. Release, 91, 271–280 (2003).
• 33)  Sadzuka Y, Tsuruda T, Sonobe T. The characterization and cytotoxicity of mixed PEG-DSG modified liposomes. Yakugaku Zasshi, 125, 149–157 (2005).
• 34)  Sugiyama I, Sadzuka Y. Enhanced antitumor activity of different double arms polyethyleneglycol-modified liposomal doxorubicin. Int. J. Pharm., 441, 279–284 (2013).